US20200024312A1 - Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8+ t-cell epitopes - Google Patents

Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8+ t-cell epitopes Download PDF

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US20200024312A1
US20200024312A1 US16/480,591 US201816480591A US2020024312A1 US 20200024312 A1 US20200024312 A1 US 20200024312A1 US 201816480591 A US201816480591 A US 201816480591A US 2020024312 A1 US2020024312 A1 US 2020024312A1
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cell
targeting molecule
shiga toxin
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Eric POMA
Erin WILLERT
Sangeetha RAJAGGPALAN
Garrett Lee Robinson
Brigitte Brieschke
Jason Kim
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Molecular Templates Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • 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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/25—Shigella (G)
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to cell-targeting molecules which each comprise (1) a binding region for cell-targeting, (2) a Shiga toxin A Subunit effector polypeptide for subcellular delivery, and (3) one or more, CD8+ T-cell epitopes which is heterologous to the Shiga toxin A Subunit effector polypeptide; wherein the cell-targeting molecule is capable of delivering at least one, heterologous, CD8+ T-cell epitope to the MHC class I presentation pathway of a target cell, such as, e.g. a malignant cell.
  • a target cell such as, e.g. a malignant cell.
  • the Shiga toxin effector polypeptide components of the cell-targeting molecules of the present invention comprise a combination of mutations relative to a wild-type Shiga toxin sequence providing (1) de-immunization. (2) a reduction in protease sensitivity, and/or (3) an embedded, T-cell epitope(s); wherein each Shiga toxin effector polypeptide retains one or more Shiga toxin function, such as, e.g., stimulating cellular internalization, directing intracellular routing, and/or potent cytotoxicity.
  • the cell-targeting molecules of the present invention are useful for administration to chordates, such as, e.g., when it is desirable to (1) reduce or eliminate a certain immune response(s) resulting from the administered molecule, (2) reduce or eliminate non-specific toxicities resulting from the administered molecule, (3) specifically kill a target cell(s) in vivo, and/or (4) target a beneficial immune response(s) to a target cell-type, a tumor mass comprising a target cell-type, and/or a tissue locus comprising a target cell-type, such as via stimulating intercellular engagement of a CD8+ T-cell(s) of the chordate with the target cell-type.
  • the cell-targeting molecules of the present invention have numerous uses, e.g., for the delivery of a specific CD8+ T-cell epitope from an extracellular location to the MHC class I presentation pathway of a target cell; the cell-surface labeling of a target cell with a MHC class I displayed CD8+ T-cell epitope; the selective killing of specific cell-types in the presence of other cells; the stimulation of beneficial immune responses in vivo; the elicitation of a cytotoxic T lymphocyte cell response(s) to a target cell; the repression of a detrimental immune response(s) in vivo; the creation of memory immune cells, and the diagnosis and treatment of a variety of diseases, disorders, and conditions, such as, e.g., cancers, tumors, other growth abnormalities, immune disorders, and microbial infections.
  • diseases, disorders, and conditions such as, e.g., cancers, tumors, other growth abnormalities, immune disorders, and microbial infections.
  • Targeted therapies may be used to target cancer cells and/or cancer tissue loci within a patient for the receipt of highly immunogenic, foreign epitopes (e.g., from an infectious agent) in order to locally activate a variety of beneficial immune responses and to specifically mark cancer cells as being foreign using epitopes which are more immunogenic than any already displayed by the cancer cells in the patient.
  • highly immunogenic, foreign epitopes e.g., from an infectious agent
  • the patient's immune system may become systemically stimulated such as, e.g., including an increase in global immunosurveillance and immune cell activity, but theffy specificity of the immune system may limit or focus immune responses to a certain tissue location, cell-type(s), or even a single foreign epitope(s).
  • This approach may activate the immune system generally in a systemic way (such as shown with Coley's toxins, cytokine therapies, immune-checkpoint inhibitors, and cancer vaccines) while focusing beneficial immune responses to certain tissues and cells in a localized way (such as shown with adoptive chimeric antigen receptor-engineered T cell (CAR-T) and tumor-targeted monoclonal antibody therapies).
  • CAR-T adoptive chimeric antigen receptor-engineered T cell
  • MHC class I plays an essential role in the immune system by providing epitope presentation of intracellular antigens ( Cellular and Molecular Immunology (Abbas A, ed., Saunders, 8 th ed., 2014)). This process is thought to be an important part of the adaptive immune system, a system which evolved in chordates primarily to protect against intracellular pathogens as well as malignant cells expressing intracellular antigens, such as, e.g., cancer cells. For example, human infections involving intracellular pathogens may only be overcome by the combined actions of both the MHC class I and class II systems (see e.g. Chiu C, Openshaw P, Nat Immunol 16: 18-26 (2015)). The MHC class 1 system's contribution is to identify and kill malignant cells based on the identification of intracellular antigens.
  • the MHC class I system functions in any nucleated cell of a vertebrate to present intracellular (or endogenous) antigens, whereas the MHC class II pathway functions in professional antigen-presenting cells (APCs) to present extracellular (or exogenous) antigens (Neefjes J et al., Nat Rev Immunol 11; 823-36 (2011)).
  • APCs professional antigen-presenting cells
  • Intracellular or “endogenous” epitopes recognized by the MHC class I system are typically fragments of molecules encountered in the cytosol or lumen of the endoplasmic reticulum (ER) of a cell, and these molecules are typically proteolytically processed by the proteasome and/or another protease(s) in the cytosol.
  • MHC class I molecules When present in the ER, these endogenous epitopes are loaded onto MHC class I molecules and presented on the surface of the cell as peptide-MHC class I molecule complexes (pMHC Is).
  • pMHC Is peptide-MHC class I molecule complexes
  • the MHC class II system functions only in specialized cells to recognize exogenous epitopes derived from extracellularly encountered molecules processed only in specific endosomal compartments, such as, e.g., late endosomes, lysosomes, phagosomes, and phagolysosomes, and including intracellular pathogens residing in endocytotic organelles.
  • T-cell Intercellular CD8+ T lymphocyte (T-cell) engagement of a cell presenting a specific epitope-MHC class I complex by a CD8+ T-cell initiates protective immune responses that can result in the rejection of the presenting cell, i.e. death of the presenting cell due to the cytotoxic activity of one or more cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • CD8+ T-cells recognize pMHC Is on the cell surface of another cell via their TCRs
  • CD8+ T-cells express different T-cell receptors (TCRs) with differing binding specificities to different cognate pMHC Is
  • CD8+ T-cell specificity depends on each individual T-cell's specific TCR and that TCR's binding affinity to the presented epitope-MHC complex as well as the overall TCR binding occupancy to the presenting cell.
  • TCRs T-cell receptors
  • CD8+ T-cell specificity depends on each individual T-cell's specific TCR and that TCR's binding affinity to the presented epitope-MHC complex as well as the overall TCR binding occupancy to the presenting cell.
  • MHC class I molecules that influence intercellular CD8+ T-cell recognition in at least in two ways; by affecting the specificity of peptides loaded and displayed (i.e. the pMHC I repertoire) and by affecting the contact regions between TCRs and pMHC Is involved
  • CTL killing of pMHC I-presenting cells occurs primarily via cytolytic activities mediated by the delivery of perform and/or granzyme into the presenting cell via cytotoxic granules (see e.g. Russell J, Ley T, Annu Rev Immunol 20; 323-70 (2002); Cullen S. Martin S, Cell Death Dif 15; 251-62 (2008)).
  • CTL-mediated target cell killing mechanisms involve inducing apoptosis in the presenting cell via TNF signaling, such as, e.g., via FasL/Fas and TRAIL/TRAIL-DR signaling (see e.g. Topham D et al., J Immunol 159; 5197-200 (1997); Ishikawa E et al., J Virol 79; 7658-63 (2005); Brincks E et al., J Immunol 181; 4918-25 (2008); Cullen S, Martin S, Cell Death Diff 15; 251-62 (2008)).
  • TNF signaling such as, e.g., via FasL/Fas and TRAIL/TRAIL-DR signaling (see e.g. Topham D et al., J Immunol 159; 5197-200 (1997); Ishikawa E et al., J Virol 79; 7658-63 (2005); Brincks E et al.,
  • activated CTLs can indiscriminately kill other cells in proximity to the recognized, pMHC I-presenting cell regardless of the peptide-MHC class I complex repertoires being presented by the other proximal cells (Wiedemann A et al., Proc Natl Acad Sci USA 103; 10985-90 (2006)).
  • activated CTLs can release immuno-stimulatory cytokines, interleukins, and other molecules to influence the immuno-activation of the microenvironment.
  • This MHC class I and CTL immunosurveillance system could conceivably be harnessed by certain therapies to guide a subject's adaptive immune system into rejecting and specifically killing certain cell types.
  • the MHC class I presentation pathway may be exploited by various therapeutic molecules to force certain targeted cells to display certain epitopes on cell surfaces in order to induce desired immune responses including the increasing immuno-detection and killing of specifically targeted cells by immune cells.
  • Therapeutic molecules might be designed which specifically deliver CD8+ T-cell epitopes to the MHC class I pathway for presentation by malignant cells (e.g. tumor or infected cells) to signal their own destruction and, perhaps, in the aggregate to educe a more wide-spread stimulation of the immune system.
  • therapeutic molecules might be designed which also stimulate or increase MHC presentation activity in target cells.
  • cell-targeting molecules capable, when exogenously administered, of delivering a CD8+ T-cell epitope to the MHC class I presentation pathway of a chosen target cell, where the epitope may be chosen form a wide variety of epitopes, such as, e.g., from common infectious agents, and the target cell may be chosen from a wide variety of cells, such as, e.g., malignant and/or infected cells, particularly cells other than professional APCs like dendritic cells.
  • Such cell-targeting molecules which preferentially target malignant cells over healthy cells, may be administered to a chordate for in vivo delivery of a CD8+ T-cell epitope for MHC class I presentation by target cells, such as, e.g., infected, neoplastic, or otherwise malignant cells.
  • target cells such as, e.g., infected, neoplastic, or otherwise malignant cells.
  • each cell-targeting molecule comprises 1) at least one Shiga toxin A Subunit effector polypeptide derived from the A Subunit of at least one member of the Shiga toxin family, 2) at least one binding region capable of specifically binding at least one extracellular target biomolecule, and 3) at least one CD8+ T-cell epitope-peptide cargo; and wherein each cell-targeting molecule is capable of delivering, from an extracellular location, the at least one CD8+ T-cell epitope-peptide to the MHC class I presentation pathway of a cell.
  • the at least one binding region is heterologous to the Shiga toxin A Subunit effector polypeptide.
  • the at least one Shiga toxin effector polypeptide (i) has a Shiga toxin A1 fragment derived region having a carboxy-terminus, (ii) comprises a disruption of at least one, endogenous, B-cell and/or CD4+ T-cell epitope region, and (iii) comprises a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment derived region.
  • the at least one CD8+ T-cell epitope-peptide cargo is (i) heterologous to the Shiga toxin A Subunit effector polypeptide and (ii) not embedded or inserted in the Shiga toxin A1 fragment region and/or the Shiga toxin A Subunit effector polypeptide (see e.g. FIG. 1 , depicting illustrative examples of exemplary embodiments of cell-targeting molecules of the present invention).
  • the cell-targeting molecule of the present invention upon administration of the cell-targeting molecule to a cell results in (i) the internalization of the cell-targeting molecule by the cell and (ii) the cell presenting on a cellular surface the CD8+ T-cell epitope-peptide cargo complexed with a MHC class I molecule.
  • the cell-targeting molecule of the present invention upon administration of the cell-targeting molecule to a cell, which is physically coupled with extracellular target biomolecule bound by the binding region of the cell-targeting molecule, results in the cell presenting on a cellular surface the CD8+ T-cell epitope-peptide cargo complexed with a MHC class I molecule.
  • having or placing the cell in the presence of an immune cell(s) further results in an immune cell response in trans, an inter-cellular engagement of the cell by an immune cell (e.g. a cytotoxic T lymphocyte), and/or death of the cell induced via an inter-cellular action(s) of an immune cell.
  • an immune cell e.g. a cytotoxic T lymphocyte
  • the cell-targeting molecule of the present invention upon administration of the cell-targeting molecule to a chordate, which comprises cells physically coupled with extracellular target biomolecule bound by the binding region of the cell-targeting molecule, results in at least some of said cells presenting on a cellular surface the CD8+ T-cell epitope-peptide cargo complexed with a MHC class I molecule.
  • the results further include an immune cell response in trans, such as, e.g., the inter-cellular engagement of at least some of said cells by an immune cell and/or death of the cell induced via an inter-cellular action(s) of an immune cell (e.g. a cytotoxic T lymphocyte).
  • Cell-targeting molecules of the present invention may be used for targeted delivery of various CD8+ T-cell epitopes to any nucleated, target cell within a chordate in order to cause the delivered CD8+ T-cell epitope-peptide cargo to be presented on the target cell surface complexed with a MHC class I molecule.
  • the target cells can be of various types, such as, e.g., neoplastic cells, infected cells, cells harboring intracellular pathogens, and other undesirable cells, and the target cell can be targeted by cell-targeting molecules of the invention either in vitro or in vivo.
  • the present invention provides various cell-targeted molecules comprising protease-cleavage resistant, Shiga toxin effector polypeptides capable of intracellular delivery of heterologous, CD8+ T-cell epitopes to the MHC class I presentation pathways of target cells while simultaneously improving extracellular, in vivo tolerability of these cell-targeting molecules.
  • Certain cell-targeting molecules of the present invention have improved usefulness for administration to chordates as either a therapeutic and/or diagnostic agent because of the reduced likelihood of producing nonspecific toxicities at a given dosage.
  • the CD8+ T-cell epitope-peptide cargo is fused, either directly or indirectly, to the Shiga toxin A Subunit effector polypeptide and/or the binding region.
  • the CD8+ T-cell epitope-peptide cargo is fused via a peptide bond, either directly or indirectly, to the Shiga toxin A Subunit effector polypeptide and/or the binding region.
  • the CD8+ T-cell epitope-peptide cargo is fused via a peptide bond, either directly or indirectly, to the Shiga toxin A Subunit effector polypeptide and/or the binding region as a genetic fusion.
  • the cell-targeting molecule of the present invention comprises a polypeptide comprising the binding region, the Shiga toxin effector polypeptide, and the CD8+ T-cell epitope-peptide cargo.
  • the cell-targeting molecule of the present invention comprises the binding region comprising two or more polypeptide chains and the CD8+ T-cell epitope-peptide cargo is fused to a polypeptide comprising the Shiga toxin effector polypeptide and one of the two or more polypeptide chains.
  • the CD8+ T-cell epitope-peptide cargo is positioned carboxy-terminal to the carboxy terminus of the Shiga toxin A1 fragment derived region.
  • the cell-targeting molecule of the present invention comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin A Subunit effector polypeptide.
  • the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin A Subunit effector polypeptide wherein the molecular moiety is cytotoxic.
  • the cell-targeting molecule of the present invention comprises a molecular moiety which comprises at least one amino acid and the Shiga toxin A Subunit effector polypeptide is linked to at least one amino acid residue of the molecular moiety.
  • the molecular moiety and the Shiga toxin A Subunit effector polypeptide are fused forming a continuous polypeptide.
  • the Shiga toxin A Subunit effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions in addition to delivery of the CD8+ T-cell epitope-peptide cargo to a MHC class I molecule of the cell.
  • the Shiga toxin A Subunit effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions in addition to delivery of the CD8+ T-cell epitope-peptide cargo from an early endosomal compartment of a cell in which the Shiga toxin effector polypeptide is present to a MHC class I molecule of the cell.
  • the Shiga toxin A Subunit effector polypeptide is capable of exhibiting a ribosome inhibition activity with a half-maximal inhibitory concentration (IC 50 ) value of 10,000 picomolar or less.
  • the Shiga toxin A Subunit effector polypeptide comprises one or more mutations relative to a naturally occurring A Subunit of a member of the Shiga toxin family which changes an enzymatic activity of the Shiga toxin A Subunit effector polypeptide, the mutation selected from at least one amino acid residue deletion, insertion, or substitution.
  • the mutation, relative to the naturally occurring A Subunit which changes an enzymatic activity of the Shiga toxin A Subunit effector polypeptide reduces or eliminates a cytotoxicity exhibited by the Shiga toxin A Subunit effector polypeptide without the mutation(s).
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule and (ii) a de-immunized.
  • Shiga toxin effector polypeptide of Embodiment Set #1 see e.g. FIG. 1 , depicting illustrative examples of four, exemplary embodiments of the cell-targeting molecules of this embodiment set #1).
  • certain embodiments of set #1 is the cell-targeting molecule comprising (i) a binding region capable of specifically binding an extracellular target biomolecule and (ii) a de-immunized, Shiga toxin effector polypeptide comprising at least one inserted or embedded, heterologous epitope (a) and at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope region (b), wherein the heterologous epitope does not overlap with the embedded or inserted, heterologous, T-cell epitope.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous, T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the cell-targeting molecule of the present invention is capable when introduced to a chordate of exhibiting improved in vivo tolerability and/or stability compared to a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the Shiga toxin effector polypeptide is not cytotoxic and the molecular moiety is cytotoxic.
  • the binding region and Shiga toxin effector polypeptide are linked together, either directly or indirectly.
  • the binding region comprises a polypeptide comprising an immunoglobulin-type binding region.
  • the binding region comprising a polypeptide selected from the group consisting of: an autonomous V H domain, single-domain antibody fragment (sdAb), nanobody, heavy chain-antibody domain derived from a camelid (V H H or V H domain fragment), heavy-chain antibody domain derived from a cartilaginous fish (V H H or V H domain fragment), immunoglobulin new antigen receptor (IgNAR), V NAR fragment, single-chain variable fragment (scFv), antibody variable fragment (Fv), complementary determining region 3 fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, small modular immunopharmaceutical (SMIP) domain, antigen-binding fragment (Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10 th fibronectin type III domain (10F
  • Protein-A-derived Z domain Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain (affilin), Sac7d-derived polypeptide (affitin), Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality, such as, e.g., wherein the relative orientation or order of the heavy and light chains is reversed or flipped.
  • the cell-targeting molecule of the present invention is capable of exhibiting (i) a catalytic activity level comparable to a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosome inhibition activity with a half-maximal inhibitory concentration (IC 50 ) value of 10,000 picomolar or less, and/or (iii) a significant level of Shiga toxin catalytic activity.
  • IC 50 half-maximal inhibitory concentration
  • the cell-targeting molecule of the present invention and/or its Shiga toxin effector polypeptide is capable of exhibiting subcellular routing efficiency comparable to a reference cell-targeting molecule comprising a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide and/or capable of exhibiting a significant level of intracellular routing activity to the endoplasmic reticulum and/or cytosol from an endosomal starting location of a cell.
  • Embodiment Set #1 whereby administration of the cell-targeting molecule of the present invention to a cell physically coupled with the extracellular target biomolecule of the cell-targeting molecule's binding region, the cell-targeting molecule is capable of causing death of the cell.
  • the cell-targeting molecule of the invention is capable of causing cell death to the cell-types physically coupled with an extracellular target biomolecule of the cytotoxic cell-targeting molecule's binding region at a CD 50 at least three times or less than the CD 50 to cell types which are not physically coupled with an extracellular target biomolecule of the cell-targeting molecule's binding region.
  • the cytotoxic effect of the cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cytotoxic effect of the cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cytotoxic effect of the cell-targeting molecule to members of the first population of cells relative to members of the second population of cells is at least 3-fold greater.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting a cytotoxicity with a half-maximal inhibitory concentration (CD 50 ) value of 300 nM or less and/or capable of exhibiting a significant level of Shiga toxin cytotoxicity.
  • CD 50 half-maximal inhibitory concentration
  • the cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the cell-targeting molecule comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide.
  • the molecular moiety comprises or consists of the binding region.
  • the molecular moiety comprises at least one amino acid and the Shiga toxin effector polypeptide is linked to at least one amino acid residue of the molecular moiety.
  • the molecular moiety and the Shiga toxin effector polypeptide are fused forming a continuous polypeptide.
  • the cell-targeting molecule further comprises a cytotoxic molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide.
  • the cytotoxic molecular moiety is a cytotoxic agent, such as, e.g., a small molecule chemotherapeutic agent, anti-neoplastic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor known to the skilled worker and/or described herein.
  • the cytotoxic molecular moiety is cytotoxic at concentrations of less than 10,000, 5,000, 1.000, 500, or 200 pM.
  • the binding region is capable of binding to an extracellular target biomolecule selected from the group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen.
  • CDCP1 endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72, tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1), carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha V beta3, AlphaSbeta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE
  • MHC class I molecule ILT-3, galectin-3, CD11a-c, GITRL, MHC class II molecule.
  • MHC class II molecule (optionally complexed with a peptide), CD284 (TLR4), CD107-Mac3, CD195 (CCR5), HLA-DR, CD16/32, CD282 (TLR2), CD11c, and any immunogenic fragment of any of the foregoing.
  • the binding region is linked, either directly or indirectly, to the Shiga toxin effector polypeptide by at least one covalent bond which is not a disulfide bond.
  • the binding region is fused, either directly or indirectly, to the carboxy-terminus of the Shiga toxin effector polypeptide to form a single, continuous polypeptide.
  • the binding region is an immunoglobulin-type binding region.
  • the disrupted furin-cleavage motif comprises one or more mutations in the minimal, furin-cleavage site relative to a wild-type Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif is not an amino-terminal truncation of sequences that overlap with part or all of at least one amino acid residue of the minimal furin-cleavage site.
  • the mutation in the minimal, furin-cleavage site is an amino acid deletion, insertion, and/or substitution of at least one amino acid residue in the R/Y-x-x-R furin cleavage motif.
  • the disrupted furin-cleavage motif comprises at least one mutation relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in the region natively positioned 1) at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or 2) at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent amino acid sequence position in any Shiga toxin A Subunit.
  • the mutation is an amino acid residue substitution of an arginine residue with a non-positively charged, amino acid residue.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity comparable to a cytotoxicity of a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment.
  • the binding region comprises the peptide or polypeptide shown in any one of SEQ ID NOs: 39-245.
  • the cell-targeting molecule of the present invention comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 252-255 and 288-748, and optionally the cell-targeting molecule comprises an amino-terminal methionine residue.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the binding region is not located proximally to the amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a third cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide which is not positioned at or proximal to the amino-terminus of the third cell-targeting molecule.
  • the cell-targeting molecule of the present invention exhibits cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of the third cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the third cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the third cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the binding region is not located proximally to the amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a third cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide which is not positioned at or proximal to the amino-terminus of the third cell-targeting molecule.
  • the third cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the binding region is not located proximally to the amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the cell-targeting molecule of the present invention exhibits low cytotoxic potency (i.e.
  • the third cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention comprises a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL, TDEL, SKEL, STEL, and EDEL.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fourth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to a reference molecule, such as, e.g., the fourth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fourth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous, epitope, and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif.
  • the cell-targeting molecule of the present invention comprises (a) a binding region capable of specifically binding at least one extracellular target biomolecule; (b) a Shiga toxin effector polypeptide comprising an embedded or inserted, heterologous epitope; and (c) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous. T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL. WDEL. YDEL, HEEF, HEEL, KEEL.
  • the embedded or inserted, heterologous, T-cell epitope disrupts the endogenous, B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof; (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:
  • the heterologous epitope is a CD8+ T-cell epitope capable of being presented by a MHC class I molecule of a cell.
  • the heterologous epitope in is embedded and replaces an equivalent number of amino acid residues in a wild-type Shiga toxin polypeptide region such that the Shiga toxin effector polypeptide has the same total number of amino acid residues as does the wild-type Shiga toxin polypeptide region from which it is derived.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function selected from: directing intracellular routing to a cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, and cytotoxicity.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fifth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the fifth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fifth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the cell-targeting molecule is de-immunized due to the embedded or inserted, heterologous, epitope.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference molecule, such as, e.g., a sixth cell-targeting molecule consisting of the cell-targeting molecule except for it lacks one or more embedded or inserted epitopes present in the cell targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the fifth cell-targeting molecule.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous, epitope; and (iii) a disrupted furin-cleavage motif.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising (a) an inserted or embedded, heterologous, epitope; (b) a Shiga toxin A1 fragment derived region having a carboxy terminus; and (c) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous. T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the embedded or inserted, heterologous, T-cell epitope disrupts the endogenous, B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof; (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18); or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting cytotoxicity comparable to the cytotoxicity of a seventh cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicity exhibited by the seventh cell-targeting molecule.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of the seventh cell-targeting molecule.
  • the cell-targeting molecule is de-immunized due to the embedded or inserted, heterologous, epitope.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference molecule, such as, e.g., an eighth cell-targeting molecule consisting of the cell-targeting molecule except for it lacks one or more embedded or inserted epitopes present in the cell targeting molecule.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a ninth cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous, epitope; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus of a polypeptide.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule, (ii) a polypeptide component, and (iii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous, epitope; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus of the polypeptide component of the cell-targeting molecule.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide. In certain further embodiments, the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous. T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous, T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a tenth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the tenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the tenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the tenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the cell-targeting molecule is de-immunized due to the embedded or inserted, heterologous, epitope.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference molecule, such as, e.g., an eleventh cell-targeting molecule consisting of the cell-targeting molecule except for it lacks one or more embedded or inserted epitopes present in the cell targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the tenth cell-targeting molecule.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule and (ii) a de-immunized, Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule and (ii) a de-immunized, Shiga toxin effector polypeptide comprising (a) a Shiga toxin A1 fragment derived region having a carboxy terminus, (b) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region, and (c) at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting cytotoxicity comparable to the cytotoxicity of a reference molecule, such as, e.g., a twelfth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a twelfth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicity exhibited by the twelfth cell-targeting molecule.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of the twelfth cell-targeting molecule.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment, such as, e.g., the twelfth cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278 and 288-748, and optionally the cell-targeting molecule comprises an amino-terminal methionine residue.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a de-immunized, Shiga toxin effector polypeptide, and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a de-immunized.
  • Shiga toxin effector polypeptide comprising at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region, and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL. WDEL. YDEL, HEEF, HEEL, KEEL. REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL. KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL.
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a thirteenth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the thirteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the thirteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the thirteenth cell-targeting molecule.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule. (ii) a de-immunized, Shiga toxin effector polypeptide; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule: (ii) polypeptide component; and (iii) a de-immunized, Shiga toxin effector polypeptide comprising at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus of the polypeptide component of the cell-targeting molecule.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: 11-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fourteenth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the fourteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the fourteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fourteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is not cylotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the fourteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278 and 288-748, and optionally the cell-targeting molecule comprises an amino-terminal methionine residue.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif; and (iii) a carboxy-terminal endoplasmic reticulum retention/retrieval signal motif.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif; and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fifteenth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the fifteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fifteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of a sixteenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment, such as, e.g., the sixteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the fifteenth cell-targeting molecule.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule and (ii) a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif at the carboxy-terminus of its Shiga toxin A1 fragment region; wherein the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule, (ii) a Shiga toxin effector polypeptide having an amino-terminus and a Shiga toxin A1 fragment derived region having a carboxy terminus, and (iii) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region; wherein the binding region is not located proximally to the amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a seventeenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a seventeenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of an eighteenth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the eighteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the eighteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the eighteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of a nineteenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment, such as, e.g., the nineteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the nineteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278 and 288-748, and optionally the cell-targeting molecule comprises an amino-terminal methionine residue.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule, (ii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif, and (iii) a Shiga toxin effector polypeptide; wherein the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises a (i) binding region capable of specifically binding an extracellular target biomolecule, (ii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family, (iii) a polypeptide component, and (iv) a Shiga toxin effector polypeptide; wherein the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL. WDEL. YDEL, HEEF, HEEL, KEEL, REEL. KAEL, KCEL, KFEL, KGEL KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL. KDEF, KKEL, HADL HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL. QDEL, REDL, RNEL, RTDL. RTEL, SDEL, TDEL, SKEL, STEL, and EDEL.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a twentieth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the twentieth cell-targeting molecule and/or greater than that of a twenty-first cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the twentieth cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to a reference molecule, such as, e.g., the twentieth and/or twenty-first cell-targeting molecules.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the twentieth and/or twenty-first cell-targeting molecules to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the twentieth and/or twenty-first cell-targeting molecules.
  • the cell-targeting molecule of the present invention is multivalent.
  • the multivalent cell-targeting molecule of the present invention comprises two or more binding regions, wherein each binding region is capable of specifically binding an extracellular part of the same extracellular target biomolecule.
  • a cytotoxic effect which is greater than a cytotoxic effect resulting from administration of an equivalent amount, mass, or molarity of a monovalent target-binding molecule component of the multivalent cell-targeting molecule to a population of the same target-positive cells under same conditions by a factor of 2, 2.5, 3, 4, 5, 7.5, 10, or greater than the fold-change in target-binding between the monovalent target-binding molecule component and the multivalent cell-targeting molecule as measured by dissociation constant (K D ).
  • K D dissociation constant
  • the present invention provides multivalent cell-targeting molecules comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule, and (ii) at least one de-immunized. Shiga toxin effector polypeptide.
  • certain Embodiments of Set #11 is the multivalent cell-targeting molecule comprising (i) two or more binding regions each of which is capable of specifically binding the same extracellular target biomolecule and (ii) at least one, de-immunized, Shiga toxin effector polypeptide comprising at least one inserted or embedded, heterologous epitope (a) and at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope region (b), wherein the heterologous epitope does not overlap with the embedded or inserted, heterologous, T-cell epitope.
  • the at least one, de-immunized is the multivalent cell-targeting molecule comprising (i) two or more binding regions each of which is capable of specifically binding the same extracellular target biomolecule and (ii) at least one, de-immunized, Shiga toxin effector polypeptide comprising at least one inserted or embedded, heterologous epitope (a) and at least one disrupted, end
  • Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • Shiga toxin effector function such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the multivalent cell-targeting molecule upon administration of the multivalent cell-targeting molecule to a plurality of cells physically coupled with an extracellular target biomolecule of the two or more binding regions, which have the extracellular part bound by two or more binding regions, at a concentration of multivalent cell-targeting molecule equivalent to five percent, ten percent, twenty percent, thirty-five percent, fifty percent, sixty-five percent, seventy-five percent, and/or eighty percent cell-surface occupancy, the majority of the multivalent cell-targeting molecule internalizes into the plurality of cells in about fifteen hours, ten hours, five hours, four hours, three hours, two hours, one hour, thirty minutes, or less at a physiological temperature appropriate for the cell and/or at about 37 degrees Celsius.
  • the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the multivalent cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing caspase activation, causing cell death, and/or delivering the embedded or inserted, heterologous, T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the multivalent cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or greater than the cytotoxicity of a reference molecule introduced to the same type of cells.
  • references molecules include a second cell-targeting molecule, such as, e.g., (1) a monovalent second cell-targeting molecule comprising only one of the two or more binding regions of the multivalent cell-targeting molecule of interest and one or more of the same Shiga toxin effector polypeptide component(s) of the multivalent cell-targeting molecule, and/or (2) a multivalent third cell-targeting molecule consisting of the multivalent cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprises a wild-type Shiga toxin A1 fragment.
  • Each of the at least one, de-immunized, Shiga toxin effector polypeptides has an amino terminus, whether with regard to a polypeptide regional boundary and/or a physical polypeptide terminus.
  • the at least one, de-immunized. Shiga toxin effector polypeptide comprises a Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region having a carboxy-terminus. In certain embodiments, the at least one, de-immunized, Shiga toxin effector polypeptide comprises a Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region having a carboxy-terminus.
  • all of the Shiga toxin effector polypeptide components of the multivalent cell-targeting molecule each comprises a Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region having a carboxy-terminus.
  • the multivalent cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region of at least one, de-immunized, Shiga toxin effector polypeptide.
  • the multivalent cell-targeting molecule of the present invention is capable when introduced to a chordate of exhibiting improved in vivo tolerability and/or stability compared to a reference molecule, such as, e.g., a fourth cell-targeting molecule consisting of the multivalent cell-targeting molecule except for at least one of the fourth cell-targeting molecule's Shiga toxin effector polypeptide component(s) comprises a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region or Shiga toxin A1 fragment derived region.
  • a reference molecule such as, e.g., a fourth cell-targeting molecule consisting of the multivalent cell-targeting molecule except for at least one of the fourth cell-targeting molecule's Shiga toxin effector polypeptide component(s) comprises a wild-type Shiga toxin A1 fragment and/or wild
  • At least one of the two or more binding regions and the at least one, de-immunized, Shiga toxin effector polypeptide are linked together, either directly or indirectly, such as, e.g., being fused to form a continuous polypeptide (see e.g. FIG. 1 ).
  • all of the two or more binding regions are each linked, either directly or indirectly, to a de-immunized Shiga toxin effector polypeptide component of the multivalent cell-targeting molecule (see e.g. FIG. 1 ).
  • all the two or more binding regions and all the de-immunized, Shiga toxin effector polypeptides are linked together, either directly or indirectly, such as, e.g., being fused to form a continuous polypeptide (see e.g. FIG. 1 ).
  • At least one of the two or more binding regions comprises a polypeptide comprising an immunoglobulin-type binding region.
  • at least one of the two or more binding regions comprises a polypeptide selected from the group consisting of: an autonomous V H domain, single-domain antibody fragment (sdAb), nanobody, heavy chain-antibody domain derived from a camelid (V 11 H or V 11 domain fragment), heavy-chain antibody domain derived from a cartilaginous fish (V H H or V H domain fragment), immunoglobulin new antigen receptor (IgNAR), V NAR fragment, single-chain variable fragment (scFv), antibody variable fragment (Fv), complementary determining region 3 fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, small modular immunopharmaceutical (SMIP) domain, antigen-binding fragment (Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-
  • SMIP small modular immunopharmaceutical
  • each of the two or more binding regions of the multivalent cell-targeting molecule comprises a polypeptide comprising an immunoglobulin-type binding region and/or a polypeptide selected from the group consisting of: an autonomous V H domain, single-domain antibody fragment (sdAb), nanobody, heavy chain-antibody domain derived from a camelid (V H H or V H domain fragment), heavy-chain antibody domain derived from a cartilaginous fish (V H H or V H domain fragment), immunoglobulin new antigen receptor (IgNAR), V NAR fragment, single-chain variable fragment (scFv), antibody variable fragment (Fv), complementary determining region 3 fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4).
  • sdAb single-domain antibody fragment
  • nanobody heavy chain-antibody domain derived from a camelid
  • V H H or V H domain fragment heavy-chain antibody domain derived from a cartilaginous fish
  • Fd fragment small modular immunopharmaceutical (SMIP) domain, antigen-binding fragment (Fab), Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10 th fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3), ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain (LDLR-A), lipocalin (anticalin).
  • SMIP small modular immunopharmaceutical domain
  • Fab antigen-binding fragment
  • ArmRP Armadillo repeat polypeptide
  • Fn3 fibronectin-derived 10 th fibronectin type III domain
  • TNfn3 tenascin type III domain
  • ankyrin repeat motif domain low-density-lipoprotein-receptor-derived A-domain
  • LLR-A low-density-lipoprotein-receptor-derived A-domain
  • Kunitz domain Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide (affitin), Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality.
  • the multivalent cell-targeting molecule of the present invention comprises two or more proteinaceous components (e.g. protein subunits), wherein each proteinaceous component comprises (a) at least one of the two or more binding regions, and, optionally, (b) one or more of the at least one, de-immunized, Shiga toxin effector polypeptide.
  • each proteinaceous component comprises (1) only one of the two or more binding regions and (2) only one, de-immunized. Shiga toxin effector polypeptide.
  • the multivalent cell-targeting molecule of the present invention comprises exactly two proteinaceous components.
  • the multivalent cell-targeting molecule of the present invention comprises two or more components, wherein at least one component is associated with the multivalent cell-targeting molecule through one or more non-covalent interactions.
  • at least one of the components is proteinaceous.
  • the multivalent cell-targeting molecule of the present invention comprises two or more proteinaceous components associated with each other, either directly or indirectly, through one or more non-covalent interactions.
  • each proteinaceous component comprises (1) at least one of the two or more binding regions and (2) at least one of the at least one, de-immunized, Shiga toxin effector polypeptide.
  • the multivalent cell-targeting molecule of the present invention comprises two or more Shiga toxin effector polypeptides, whether de-immunized or not de-immunized.
  • the multivalent cell-targeting molecule of the present invention comprises two or more proteinaceous components (e.g. protein subunits), wherein each proteinaceous component comprises (a) at least one of the two or more binding regions, and, optionally, (b) one or more Shiga toxin effector polypeptides.
  • each proteinaceous component comprises (1) only one of the two or more binding regions and (2) only one Shiga toxin effector polypeptide.
  • the multivalent cell-targeting molecule of the present invention comprises two or more components (e.g. a proteinaceous component), wherein at least one component is associated with the multivalent cell-targeting molecule through one or more non-covalent interactions.
  • each proteinaceous component comprises (1) at least one of the two or more binding regions and (2) at least one Shiga toxin effector polypeptide.
  • a Shiga toxin effector polypeptide component of the multivalent cell-targeting molecule of the present invention comprises a Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region having a carboxy-terminus.
  • all of the Shiga toxin effector polypeptide components of the multivalent cell-targeting molecule each comprises a Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region having a carboxy-terminus.
  • the multivalent cell-targeting molecule of the present invention is capable of exhibiting (i) a catalytic activity level comparable to a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosome inhibition activity with a half-maximal inhibitory concentration (IC 50 ) value of 10,000 picomolar or less, and/or (iii) a significant level of Shiga toxin catalytic activity.
  • IC 50 half-maximal inhibitory concentration
  • the multivalent cell-targeting molecule of the present invention and/or its at least one, de-immunized, Shiga toxin effector polypeptide is capable of exhibiting subcellular routing efficiency comparable to a reference cell-targeting molecule comprising a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide and/or capable of exhibiting a significant level of intracellular routing activity to the endoplasmic reticulum and/or cytosol from an endosomal starting location of a cell.
  • Embodiment Set #11 whereby administration of the multivalent cell-targeting molecule of the present invention to a cell physically coupled with extracellular target biomolecule of the multivalent cell-targeting molecule's two or more binding regions, the multivalent cell-targeting molecule is capable of causing death of the cell.
  • the multivalent cell-targeting molecule of the invention is capable of causing cell death to the cell-types physically coupled with an extracellular target biomolecule of the multivalent cell-targeting molecule's two or more binding regions at a CD 50 at least three times or less than the CD 50 to cell types which are not physically coupled with an extracellular target biomolecule of the multivalent cell-targeting molecule's two or more binding regions.
  • the cytotoxic effect of the multivalent cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cytotoxic effect of the multivalent cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cytotoxic effect of the multivalent cell-targeting molecule to members of the first population of cells relative to members of the second population of cells is at least 3-fold greater.
  • the multivalent cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting a cytotoxicity with a half-maximal inhibitory concentration (CD 50 ) value of 300 nanomolar (nM) or less and/or capable of exhibiting a significant level of Shiga toxin cytotoxicity.
  • CD 50 half-maximal inhibitory concentration
  • the multivalent cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the multivalent cell-targeting molecule comprises a molecular moiety associated with the carboxy-terminus of the at least one, de-immunized. Shiga toxin effector polypeptide.
  • the molecular moiety comprises or consists of the one or more the two or more binding regions.
  • the molecular moiety comprises at least one amino acid and the at least one, de-immunized, Shiga toxin effector polypeptide is linked, either directly or indirectly, to at least one amino acid residue of the molecular moiety.
  • the molecular moiety and the at least one, de-immunized, Shiga toxin effector polypeptide are fused, either directly or indirectly, forming a continuous polypeptide.
  • the molecular moiety(ies) is each fused to the at least one, de-immunized, Shiga toxin effector polypeptide, either directly or indirectly, to form a continuous polypeptide.
  • the multivalent cell-targeting molecule further comprises a cytotoxic molecular moiety associated with the carboxy-terminus of the at least one, de-immunized, Shiga toxin effector polypeptide.
  • the cytotoxic molecular moiety is a cytotoxic agent, such as, e.g., a small molecule chemotherapeutic agent, anti-neoplastic agent, cytotoxic antibiotic, cytotoxic anti-infective, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor known to the skilled worker and/or described herein.
  • the cytotoxic molecular moiety is cytotoxic at concentrations of less than 10,000, 5,000, 1,000, 500, or 200 picomolar (pM).
  • the two or more binding regions are each capable of binding to an extracellular target biomolecule selected from the group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin.
  • an extracellular target biomolecule selected from the group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, muc
  • TAG-72 tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1), carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha V beta3, AlphaSbeta1, ErbB1/EGFR, Erb3, c-MET.
  • IGF1R IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen, prostate stem cell antigen, human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen,
  • one or more of the two or more binding regions is linked, either directly or indirectly, to at least one of the at least one, de-immunized, Shiga toxin effector polypeptide by at least one covalent bond which is not a disulfide bond.
  • one or more of the multivalent cell-targeting molecule's two or more binding regions is fused, either directly or indirectly, to the carboxy-terminus of the at least one, de-immunized, Shiga toxin effector polypeptide to form a single, continuous polypeptide.
  • at least one of the two or more binding regions comprises an immunoglobulin-type binding region.
  • all of the multivalent cell-targeting molecule's two or more binding regions each comprises an immunoglobulin-type binding region.
  • the disrupted furin-cleavage motif comprises one or more mutations in the minimal, furin-cleavage site relative to a wild-type Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif is not an amino-terminal truncation of sequences that overlap with part or all of at least one amino acid residue of the minimal furin-cleavage site.
  • the mutation in the minimal, furin-cleavage site is an amino acid deletion, insertion, and/or substitution of at least one amino acid residue in the R/Y-x-x-R furin cleavage motif.
  • the disrupted furin-cleavage motif comprises at least one mutation relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in the region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent amino acid sequence position in any Shiga toxin A Subunit.
  • the mutation is an amino acid residue substitution of an arginine residue with a non-positively charged, amino acid residue.
  • At least one of the two or more binding regions comprises the peptide or polypeptide shown in any one of SEQ ID NOs: 39-245.
  • the multivalent cell-targeting molecule of the present invention comprises the polypeptide shown in any one of SEQ ID NOs: 252-255 and 288-748. In certain further embodiments, the multivalent cell-targeting molecule of the present invention comprises or consists essentially of two proteins, each protein selected from any one of the polypeptides shown in SEQ ID NOs: 252-255 and 288-748, and optionally, each protein further comprises an amino-terminal methionine residue.
  • At least one of the two or more binding regions sterically covers the carboxy-terminus of the A1 fragment region or Shiga toxin A1 fragment derived region of at least one of the at least one, de-immunized, Shiga toxin effector polypeptide(s).
  • the at least one of the two or more binding regions sterically cover the carboxy-terminals of the A1 fragment region or A1 fragment derived region of all the Shiga toxin effector polypeptide component(s) present in the multivalent cell-targeting molecule.
  • each of the carboxy-terminals of the A1 fragment region or A1 fragment derived region of each of the Shiga toxin effector polypeptide components present in the multivalent cell-targeting molecule is sterically covered by at least one of the two or more binding regions.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region and/or Shiga toxin A1 fragment derived region of at least one of the at least one, de-immunized, Shiga toxin effector polypeptide(s). In certain further embodiments, the molecular moiety comprises at least one of the two or more binding regions.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region and/or Shiga toxin A1 fragment derived region of at least one of the at least one, de-immunized, Shiga toxin effector polypeptide(s). In certain further embodiments, the molecular moiety(ies) sterically cover the carboxy-terminals of the A1 fragment region or A1 fragment derived region of all the Shiga toxin effector polypeptide component(s) present in the multivalent cell-targeting molecule.
  • each of the carboxy-terminals of the A1 fragment region or A1 fragment derived region of each of the Shiga toxin effector polypeptide components present in the multivalent cell-targeting molecule is sterically covered by at least one of the molecular moiety(ies).
  • each of the molecular moieties present in the multivalent cell-targeting molecule comprises at least one of the two or more binding regions.
  • the multivalent cell-targeting molecule of the present invention comprises at least one of the two or more binding regions and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region of the at least one, de-immunized, Shiga toxin effector polypeptide.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the multivalent cell-targeting molecule of the present invention comprises the two or more binding regions and/or the molecular moiety located within the multivalent cell-targeting molecule at a position carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region of the at least one, de-immunized, Shiga toxin effector polypeptide.
  • the multivalent cell-targeting molecule comprises the binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the multivalent cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity, intracellular routing, and/or cellular internalization kinetic parameter(s)).
  • Shiga toxin biological activity e.g., cytotoxicity, intracellular routing, and/or cellular internalization kinetic parameter(s)
  • the two or more binding regions have a combined mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the multivalent cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity, intracellular routing, and/or cellular internalization kinetic parameter(s)).
  • Shiga toxin biological activity e.g., cytotoxicity, intracellular routing, and/or cellular internalization kinetic parameter(s)
  • each of the two or more binding regions of the multivalent cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the multivalent cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity, intracellular routing, and/or cellular internalization kinetics parameter(s)).
  • Shiga toxin biological activity e.g., cytotoxicity, intracellular routing, and/or cellular internalization kinetics parameter(s)
  • At least one of the two or more binding regions is comprised within a relatively large, molecular moiety such as, e.g., the molecular moiety having a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the multivalent cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety such as, e.g., the molecular moiety having a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,
  • At least one, de-immunized, Shiga toxin effector polypeptide is more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions.
  • none of the two or more binding regions comprised within the same polypeptide chain of a component of the multivalent cell-targeting molecule comprising at least one, de-immunized, Shiga toxin effector polypeptide are located proximal to an amino-terminus of that polypeptide chain relative to at least one, de-immunized, Shiga toxin effector polypeptide comprised within that polypeptide chain.
  • the amino-terminus of at least one, de-immunized, Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • the amino-terminus of all the Shiga toxin effector polypeptides present in the multivalent cell-targeting molecule is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • the two or more binding regions and the least one, de-immunized, Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of at least one, de-immunized. Shiga toxin effector polypeptide relative to that Shiga toxin effector polypeptide component's carboxy-terminus. In certain further embodiments, none of the two or more binding regions are located proximal to any amino-terminus of the multivalent cell-targeting molecule relative to at least one Shiga toxin effector polypeptide component.
  • the two or more binding regions are linked within the multivalent cell-targeting molecule more proximal to the carboxy-terminus of the at least one, de-immunized. Shiga toxin effector polypeptide than to the amino-terminus of that de-immunized, Shiga toxin effector polypeptide. In certain embodiments, all the de-immunized. Shiga toxin effector polypeptide components are more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions comprised within the same polypeptide chain of that polypeptide component.
  • the two or more binding regions and the least one, de-immunized, Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of any de-immunized, Shiga toxin effector polypeptide component relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • all the Shiga toxin effector polypeptide components are more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions comprised within the same polypeptide chain of that polypeptide component.
  • the two or more binding regions are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of any Shiga toxin effector polypeptide component relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • the multivalent cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a reference molecule, such as, e.g., a fifth cell-targeting molecule comprising a polypeptide component having an amino-terminus and comprising the same two or more binding regions and the same de-immunized, Shiga toxin effector polypeptide(s) which is not positioned at or proximal to a physical amino-terminus of a polypeptide component of the fifth cell-targeting molecule or a sixth cell-targeting molecule comprising the same two or more binding regions and the same Shiga toxin effector polypeptide component(s) as the multivalent cell-targeting molecule wherein at least one of the two or more binding regions is more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to all the Shiga toxin effector polypeptide components.
  • a reference molecule such as,
  • the cell-targeting molecule of the present invention exhibits cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of the fourth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fifth and/or sixth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the multivalent cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the subcellular routing efficiency of the fifth and/or sixth cell-targeting molecule.
  • the fifth and/or sixth cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the amino-terminus of the at least one, de-immunized, Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • the amino-terminus of all the Shiga toxin effector polypeptides present in the multivalent cell-targeting molecule is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • the at least one, de-immunized, Shiga toxin effector polypeptide is more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions.
  • none of the two or more binding regions comprised within the same polypeptide chain of a polypeptide component of the multivalent cell-targeting molecule comprising at least one, de-immunized, Shiga toxin effector polypeptide are located proximal to an amino-terminus of that polypeptide chain relative to at least one, de-immunized, Shiga toxin effector polypeptide comprised within that polypeptide chain.
  • the two or more binding regions and the least one, de-immunized. Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of at least one, de-immunized, Shiga toxin effector polypeptide relative to that Shiga toxin effector polypeptide component's carboxy-terminus. In certain further embodiments, none of the two or more binding regions are located proximal to any amino-terminus of the multivalent cell-targeting molecule relative to at least one Shiga toxin effector polypeptide component.
  • the two or more binding regions are linked within the multivalent cell-targeting molecule more proximal to the carboxy-terminus of the at least one, de-immunized, Shiga toxin effector polypeptide than to the amino-terminus of that de-immunized, Shiga toxin effector polypeptide. In certain further embodiments, none of the two or more binding regions are located proximal to any amino-terminus of the multivalent cell-targeting molecule relative to at least one Shiga toxin effector polypeptide component.
  • the two or more binding regions are linked within the multivalent cell-targeting molecule more proximal to the carboxy-terminus of the at least one Shiga toxin effector polypeptide than to the amino-terminus of that Shiga toxin effector polypeptide.
  • all the de-immunized, Shiga toxin effector polypeptide components are more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions comprised within the same polypeptide chain of that polypeptide component.
  • the two or more binding regions and the least one, de-immunized, Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of any de-immunized, Shiga toxin effector polypeptide component relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • all the Shiga toxin effector polypeptide components are more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions comprised within the same polypeptide chain of that polypeptide component.
  • the two or more binding regions are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of any Shiga toxin effector polypeptide component relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • the cell-targeting molecule of the present invention exhibits cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of a reference molecule, such as, e.g., a seventh multivalent cell-targeting molecule having an amino-terminus and comprising the same two or more binding regions and the same Shiga toxin effector polypeptide component(s) as the multivalent cell-targeting molecule wherein none of the Shiga toxin effector polypeptide components is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • cytotoxic potency such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of a reference molecule, such as, e.g., a seventh multivalent cell-targeting molecule having an amino
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the seventh cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the multivalent cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the subcellular routing efficiency of the seventh cell-targeting molecule.
  • the seventh cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the two or more binding regions are not located proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to at least one, de-immunized, Shiga toxin effector polypeptide.
  • the two or more binding regions and Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that the two or more binding region are not located proximal to the amino-terminus of at least one, de-immunized, Shiga toxin effector polypeptide.
  • none of the two or more binding regions comprised within the same polypeptide chain of a component of the multivalent cell-targeting molecule comprising at least one, de-immunized, Shiga toxin effector polypeptide are located proximal to an amino-terminus of that polypeptide chain relative to at least one, de-immunized, Shiga toxin effector polypeptide comprised within that polypeptide chain.
  • the amino-terminus of at least one, de-immunized, Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • the amino-terminus of all the Shiga toxin effector polypeptides present in the multivalent cell-targeting molecule is at and/or proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule.
  • the two or more binding regions and the least one, de-immunized, Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of at least one, de-immunized, Shiga toxin effector polypeptide relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • none of the two or more binding regions are located proximal to any amino-terminus of the multivalent cell-targeting molecule relative to at least one Shiga toxin effector polypeptide component.
  • the two or more binding regions are linked within the multivalent cell-targeting molecule more proximal to the carboxy-terminus of the at least one, de-immunized, Shiga toxin effector polypeptide than to the amino-terminus of that de-immunized. Shiga toxin effector polypeptide.
  • all the de-immunized, Shiga toxin effector polypeptide components are more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions comprised within the same polypeptide chain of that polypeptide component.
  • the two or more binding regions and the least one, de-immunized, Shiga toxin effector polypeptide are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of any de-immunized, Shiga toxin effector polypeptide component relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • all the Shiga toxin effector polypeptide components are more proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to any of the two or more binding regions comprised within the same polypeptide chain of that polypeptide component.
  • the two or more binding regions are physically arranged or oriented within the multivalent cell-targeting molecule such that none of the two or more binding regions are located proximal to the amino-terminus of any Shiga toxin effector polypeptide component relative to that Shiga toxin effector polypeptide component's carboxy-terminus.
  • the cell-targeting molecule of the present invention exhibits cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of a reference molecule, such as, e.g., an eighth multivalent cell-targeting molecule having polypeptide component having an amino-terminus and comprising the same two or more binding regions and the same Shiga toxin effector polypeptide component(s) as the multivalent cell-targeting molecule wherein at least one of the two or more binding regions is located proximal to an amino-terminus of a polypeptide component of the multivalent cell-targeting molecule relative to each of all the Shiga toxin effector polypeptide component(s).
  • cytotoxic potency such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of a reference molecule, such as, e.g
  • the multivalent cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the subcellular routing efficiency of the eighth cell-targeting molecule.
  • the eighth cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the multivalent cell-targeting molecule of the present invention, and/or a polypeptide component thereof comprises a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of KDEL. HDEF, HDEL. RDEF.
  • the multivalent cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a reference molecule, such as, e.g., a ninth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • a reference molecule such as, e.g., a ninth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to a reference molecule, such as, e.g., the ninth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the ninth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the multivalent cell-targeting molecule of the present invention exhibits low cytotoxic potency (i.e. is not capable when introduced to certain positive target cell types of exhibiting a cytotoxicity greater than 1% cell death of a cell population at a multivalent cell-targeting molecule concentration of 1000 nM, 500 nM, 100 nM, 75 nM, or 50 nM) and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the subcellular routing efficiency of the second, third, fourth, fifth, sixth, seventh, eighth, and/or ninth cell-targeting molecule.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous epitope; and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif.
  • the cell-targeting molecule of the present invention comprises (i) two or more binding regions, each capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an embedded or inserted, heterologous epitope; and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, CD8+ T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL, TDEL, SKEL, STEL, and EDEL.
  • the inserted or embedded, heterologous, T-cell epitope disrupts the endogenous, B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof; (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:
  • the heterologous epitope is a CD8+ T-cell epitope capable of being presented by a MHC class I molecule of a cell.
  • the heterologous epitope in is embedded and replaces an equivalent number of amino acid residues in a wild-type Shiga toxin polypeptide region such that the Shiga toxin effector polypeptide has the same total number of amino acid residues as does the wild-type Shiga toxin polypeptide region from which it is derived.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function selected from directing intracellular routing to a cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, and cytotoxicity.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fifth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the fifth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fifth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the cell-targeting molecule is de-immunized due to the embedded or inserted, heterologous epitope.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference molecule, such as, e.g., a sixth cell-targeting molecule consisting of the cell-targeting molecule except for it lacks one or more embedded or inserted epitopes present in one or more Shiga toxin effector polypeptide components of the cell targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the subcellular routing efficiency of a reference molecule, such as, e.g., the fifth cell-targeting molecule.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous epitope; and (iii) a disrupted furin-cleavage motif.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising (a) an inserted or embedded, heterologous epitope; (b) a Shiga toxin A1 fragment derived region having a carboxy terminus; and (c) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment derived region.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising (a) an inserted or embedded, heterologous epitope; (b) a Shiga toxin A1 fragment region having a carboxy terminus, and (c) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous, T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for at least one of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for at least one of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the inserted or embedded, heterologous, epitope disrupts the endogenous, B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof; (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region or Shiga toxin A1 fragment derived region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region or Shiga toxin A1 fragment derived region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region or Shiga toxin A1 fragment derived region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region or Shiga toxin A1 fragment derived region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting cytotoxicity comparable to the cytotoxicity of a seventh cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region and/or Shiga toxin A1 fragment derived region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicity exhibited by the seventh cell-targeting molecule.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of the seventh cell-targeting molecule.
  • the molecular moiety is not toxic and the molecular moiety comprises the binding region
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of the seventh cell-targeting molecule only when all the binding regions of the cell-targeting molecule are associated or linked, either directly or indirectly, to a Shiga toxin effector polypeptide at a position carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region and/or Shiga toxin A1 fragment derived region of that Shiga toxin effector polypeptide.
  • the cell-targeting molecule is de-immunized due to the embedded or inserted, heterologous epitope.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference molecule, such as, e.g., an eighth cell-targeting molecule consisting of the cell-targeting molecule except for it lacks one or more embedded or inserted epitopes present in the cell targeting molecule.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a ninth cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule; (ii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous epitope; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus of a polypeptide.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule, (ii) a polypeptide component, and (iii) a Shiga toxin effector polypeptide comprising an inserted or embedded, heterologous epitope: wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus of the polypeptide component of the cell-targeting molecule.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide. In certain further embodiments, the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as, e.g., with regard to a human immune system.
  • the heterologous, T-cell epitope is capable of being presented by a MHC class I molecule of a cell.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, causing cell death, and/or delivering the embedded or inserted, heterologous, T-cell epitope to a MHC class I molecule for presentation on a cellular surface.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a tenth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the tenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the tenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the tenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the cell-targeting molecule is de-immunized due to the embedded or inserted, heterologous epitope.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference molecule, such as, e.g., an eleventh cell-targeting molecule consisting of the cell-targeting molecule except for it lacks one or more embedded or inserted epitopes present in the cell targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the tenth cell-targeting molecule.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule, and (ii) a de-immunized. Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule and (ii) a de-immunized, Shiga toxin effector polypeptide comprising (a) a Shiga toxin A1 fragment derived region having a carboxy terminus, (b) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region, and (c) at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting cytotoxicity comparable to the cytotoxicity of a reference molecule, such as, e.g., a twelfth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a twelfth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicity exhibited by the twelfth cell-targeting molecule.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of the twelfth cell-targeting molecule.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment, such as, e.g., the twelfth cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises two proteins selected from any one of the polypeptides shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278 and 288-748, and which optionally comprises an amino-terminal methionine residue.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule; (ii) a de-immunized, Shiga toxin effector polypeptide, and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a de-immunized, Shiga toxin effector polypeptide comprising at least one disrupted, endogenous.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF. HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL. KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL. KWEL. KYEL, KEDL, KIEL, DKEL, FDEL, KDEF.
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 1-18;
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a thirteenth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the thirteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the thirteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the thirteenth cell-targeting molecule.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule, (ii) a de-immunized, Shiga toxin effector polypeptide; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) polypeptide component; and (iii) a de-immunized, Shiga toxin effector polypeptide comprising at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region; wherein the Shiga toxin effector polypeptide is at or proximal to an amino-terminus of the polypeptide component of the cell-targeting molecule.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fourteenth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the fourteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the fourteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fourteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the fourteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises two proteins selected from any one of the polypeptides shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278 and 288-748, and which optionally comprises an amino-terminal methionine residue.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule; (ii) a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif; and (iii) a carboxy-terminal endoplasmic reticulum retention/retrieval signal motif.
  • the present invention provides cell-targeting molecules, each comprising (i) a binding region capable of specifically binding an extracellular target biomolecule; (ii) a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif; and (iii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a second cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a fifteenth cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the fifteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the fifteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of a sixteenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment, such as, e.g., the sixteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the fifteenth cell-targeting molecule.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule, and (ii) a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif at the carboxy-terminus of its Shiga toxin A1 fragment region; wherein the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises (i) a binding region capable of specifically binding an extracellular target biomolecule, (ii) a Shiga toxin effector polypeptide having an amino-terminus and a Shiga toxin A1 fragment derived region having a carboxy terminus, and (iii) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region; wherein the binding region is not located proximally to the amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the cell-targeting molecule is capable when introduced to cells of exhibiting a cytotoxicity comparable or better than a reference molecule, such as, e.g., a seventeenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • a reference molecule such as, e.g., a seventeenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide components comprise a wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the disrupted furin-cleavage motif comprises one or more mutations, relative to a wild-type Shiga toxin A Subunit, in a minimal furin cleavage site of the furin-cleavage motif.
  • the minimal furin cleavage site is represented by the consensus amino acid sequence R/Y-x-x-R and/or R-x-x-R.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of: alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.
  • the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a histidine.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of an eighteenth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the eighteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the eighteenth cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the eighteenth cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule is capable when introduced to a chordate of exhibiting improved, in vivo tolerability compared to in vivo tolerability of a nineteenth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the cell-targeting molecule is de-immunized due to the furin-cleavage motif disruption.
  • the cell-targeting molecule is capable of exhibiting less relative antigenicity and/or relative immunogenicity as compared to a reference cell-targeting molecule consisting of the cell-targeting molecule except for the furin-cleavage motif is wild-type and/or all the Shiga toxin effector polypeptide components consist of a wild-type Shiga toxin A1 fragment, such as, e.g., the nineteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the nineteenth cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises two proteins selected from any one of the polypeptides shown in any one of SEQ ID NOs: 252-255, 259-271, 274-278 and 288-748, and which optionally comprises an amino-terminal methionine residue.
  • the present invention provides multivalent cell-targeting molecules, each comprising (i) two or more binding regions, each capable of specifically binding an extracellular part of the same target biomolecule, (ii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif, and (iii) a Shiga toxin effector polypeptide; wherein the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the cell-targeting molecule of the present invention comprises a (i) binding region capable of specifically binding an extracellular target biomolecule, (ii) a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family, (iii) a polypeptide component, and (iv) a Shiga toxin effector polypeptide; wherein the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is not located proximally to an amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the Shiga toxin effector polypeptide is capable of exhibiting at least one Shiga toxin effector function, such as, e.g., directing intracellular routing to the endoplasmic reticulum and/or cytosol of a cell in which the polypeptide is present, inhibiting a ribosome function, enzymatically inactivating a ribosome, causing cytostasis, and/or causing cytotoxicity.
  • the cell-targeting molecule of the present invention is capable of one or more the following: entering a cell, inhibiting a ribosome function, causing cytostasis, and/or causing cell death.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL, TDEL, SKEL, STEL, and EDEL.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a twentieth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide region which is not positioned at or proximal to the amino-terminus of the twentieth cell-targeting molecule and/or greater than that of a twenty-first cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the twentieth cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to a reference molecule, such as, e.g., the twentieth and/or twenty-first cell-targeting molecules.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the twentieth and/or twenty-first cell-targeting molecules to a second population of target positive cells as assayed by CD 50 values.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., the twentieth and/or twenty-first cell-targeting molecules.
  • the heterologous, CD8+ T-cell epitope-peptide cargo is fused, either directly or indirectly, to the Shiga toxin effector polypeptide and/or the binding region.
  • the cell-targeting molecule comprises a single-chain polypeptide comprising the binding region, the Shiga toxin effector polypeptide, and the heterologous, CD8+ T-cell epitope-peptide cargo.
  • the binding region comprises two or more polypeptide chains and the heterologous, CD8+ T-cell epitope-peptide cargo is fused either directly or indirectly, to a polypeptide comprising the Shiga toxin effector polypeptide and one of the two or more polypeptide chains of the binding region.
  • the cell-targeting molecule of the present invention comprises a heterologous, CD8+ T-cell epitope-peptide cargo which is positioned within the cell-targeting molecule carboxy-terminal to the Shiga toxin effector polypeptide and/or binding region.
  • the cell-targeting molecule comprises two, three, four, five, or more heterologous, CD8+ T-cell epitope-peptide cargos positioned within the cell-targeting molecule carboxy-terminal to the Shiga toxin effector polypeptide and/or binding region.
  • the cell-targeting molecule comprises a carboxy-terminal, heterologous, CD8+ T-cell epitope-peptide cargo.
  • the cell-targeting molecule upon administration of the cell-targeting molecule to a target cell physically coupled with an extracellular target biomolecule of the binding region, the cell-targeting molecule is capable of causing intercellular engagement of the target cell by a CD8+ immune cell.
  • the cell-targeting molecule of the present invention upon administration of the cell-targeting molecule of the present invention to a target cell physically coupled with an extracellular target biomolecule of the binding region, the cell-targeting molecule is capable of causing intercellular engagement of the target cell by a CD8+ immune cell.
  • the cell-targeting molecule upon administration of the cell-targeting molecule of the present invention to a target cell physically coupled with an extracellular target biomolecule of the binding region, the cell-targeting molecule is capable of causing death of the target cell.
  • the cytotoxic effect of the cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cell-targeting molecule comprises or consists essentially of the polypeptide of any one of SEQ ID NOs: 252-255, 259-278, and 288-748.
  • the Shiga toxin effector polypeptide comprises a mutation relative to a naturally occurring A Subunit of a member of the Shiga toxin family which changes the enzymatic activity of the Shiga toxin effector polypeptide, the mutation selected from at least one amino acid residue deletion, insertion, or substitution. In certain further embodiments, the mutation is selected from at least one amino acid residue deletion, insertion, or substitution that reduces or eliminates cytotoxicity of the toxin effector polypeptide.
  • the binding region comprises the heterologous, CD8+ T-cell epitope cargo, whether the CD8+ epitope-peptide is autogenous or heterologous with respect to the binding region.
  • the Shiga toxin effector polypeptide is fused to a binding region, either directly or indirectly, such as, e.g., via a linker known to the skilled worker.
  • the cell-targeting molecule comprises a molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the Shiga toxin effector polypeptide has a Shiga toxin A1 fragment derived region having a carboxy terminus and further comprises a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region.
  • the cell-targeting molecule of the present invention is capable of exhibiting (i) a catalytic activity level comparable to a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosome inhibition activity with a half-maximal inhibitory concentration (IC 50 ) value of 10,000 picomolar or less, and/or (iii) a significant level of Shiga toxin catalytic activity.
  • IC 50 half-maximal inhibitory concentration
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting a cytotoxicity with a half-maximal inhibitory concentration (CD 50 ) value of 300 nM or less and/or capable of exhibiting a significant level of Shiga toxin cytotoxicity.
  • CD 50 half-maximal inhibitory concentration
  • the cell-targeting molecule of the present invention exhibits low cytotoxic potency (i.e. is not capable when introduced to certain positive target cell types of exhibiting a cytotoxicity greater than 1% cell death of a cell population at a cell-targeting molecule concentration of 100 nM, 500 nM, 100 nM, 75 nM, or 50 nM).
  • the cell-targeting molecule of the present invention comprises a carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of a member of the KDEL family.
  • the carboxy-terminal endoplasmic reticulum retention/retrieval signal motif is selected from the group consisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL, KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL, KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL, HNEL.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity that is greater than that of a reference molecule, such as, e.g., a twenty-second cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • a reference molecule such as, e.g., a twenty-second cell-targeting molecule consisting of the cell-targeting molecule except for it does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the cell-targeting molecule of the present invention is capable of exhibiting a cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to a reference molecule, such as, e.g., the twenty-second cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the twenty-second cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the Shiga toxin effector polypeptide further comprises at least one inserted or embedded, heterologous epitope.
  • the Shiga toxin effector polypeptide further comprises at least one, two, or three disrupted, endogenous, B-cell and/or CD4+ T-cell epitope regions. In certain further embodiments, the Shiga toxin effector polypeptide comprises a disruption of at least one, two, or three endogenous, B-cell and/or T-cell epitopes and/or epitope regions. In certain further embodiments, the Shiga toxin effector polypeptide further comprises at least one disrupted, endogenous, B-cell and/or CD4+ T-cell epitope region which does not overlap with at least one inserted or embedded, heterologous epitope.
  • the amino-terminus of the Shiga toxin effector polypeptide is at and/or proximal to an amino-terminus of a polypeptide component of the cell-targeting molecule.
  • the binding region is not located proximally to the amino-terminus of the cell-targeting molecule relative to the Shiga toxin effector polypeptide.
  • the binding region and Shiga toxin effector polypeptide are physically arranged or oriented within the cell-targeting molecule such that the binding region is not located proximally to the amino-terminus of the Shiga toxin effector polypeptide.
  • the binding region is located within the cell-targeting molecule more proximal to the carboxy-terminus of the Shiga toxin effector polypeptide than to the amino-terminus of the Shiga toxin effector polypeptide.
  • the cell-targeting molecule of the present invention is not cytotoxic and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., a twenty-third cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide which is not positioned at or proximal to the amino-terminus of the third cell-targeting molecule.
  • a reference molecule such as, e.g., a twenty-third cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide which is not positioned at or proximal to the amino-terminus of the third cell-targeting molecule.
  • the cell-targeting molecule of the present invention exhibits cytotoxicity with better optimized, cytotoxic potency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to the cytotoxicity of the twenty-third cell-targeting molecule.
  • the cytotoxicity of the cell-targeting molecule of the present invention to a population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of the twenty-third cell-targeting molecule to a second population of target positive cells as assayed by CD 50 values.
  • the twenty-third cell-targeting molecule does not comprise any carboxy-terminal, endoplasmic reticulum retention/retrieval signal motif of the KDEL family.
  • the Shiga toxin effector polypeptide further comprises a disruption in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18; 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-
  • there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the Shiga toxin effector polypeptide further comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell immunogenic, amino acid residue selected from the group of natively positioned Shiga toxin A Subunit amino acid residues: L49, D197, D198. R204, and R205.
  • the embedded or inserted, heterologous, T-cell epitope disrupts the endogenous, B-cell and/or T-cell epitope region is selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof; (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs:
  • the Shiga toxin effector polypeptide comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof, wherein there is no disruption which is an amino-terminal truncation
  • the Shiga toxin effector polypeptide comprises a disruption of at least one endogenous epitope region selected from the group of natively positioned Shiga toxin A Subunits consisting of: (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the Shiga toxin effector polypeptide does not comprise a heterologous, MHC class I-restricted, T-cell epitope.
  • MHC class I-restricted, T-cell epitopes are known in the art or can be predicted by the skilled worker.
  • heterologous refers to MHC class I-restricted, T-cell epitopes which are not natively present in wild-type Shiga toxin A Subunits, such as, e.g., the wild-type Shiga toxin A Subunit which is most closely related to the Shiga toxin effector polypeptide of interest.
  • the Shiga toxin effector polypeptide comprises disruptions of at least four, five, six, seven, eight, or more endogenous, B-cell and/or T-cell epitope regions.
  • one or more disruptions comprises an amino acid residue substitution relative to a wild-type Shiga toxin A Subunit.
  • one or more endogenous, B-cell and/or T-cell epitope regions comprises a plurality of amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit.
  • At least one, two, three, or four disruptions comprise a plurality of amino acid residue substitutions in the endogenous, B-cell and/or T-cell epitope region relative to a wild-type Shiga toxin A Subunit.
  • At least one disruption comprises at least one, two, three, four, five, six, seven, eight, or more amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit, and optionally wherein at least one substitution occurs at the natively positioned Shiga toxin A Subunit amino acid residue selected form the group consisting of: 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1
  • At least two disruptions each comprise at least one amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit selected form the group consisting of 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO: 1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO: 1 or SEQ ID NO:2; 48 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID
  • the Shiga toxin effector polypeptide comprises disruption of at least three, endogenous.
  • B-cell and/or T-cell epitope regions selected from the group of consisting of: (i) 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof, wherein there is no disruption which is an amino-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous.
  • B-cell and/or T-cell epitope region (ii) 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6; 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs: 3 and 7-18; 204 of SEQ ID NO:3; 205 of any one of SEQ ID NOs: 1-2 and 4-6; and 210-218 of any one of SEQ ID NOs: 3 and 7-18, or the equivalent region in a Shiga toxin A Subunit or derivative thereof, wherein there is no disruption which is a carboxy-terminal truncation of amino acid residues that overlap with part or all of at least one disrupted, endogenous, B-cell and/or T-cell epitope and/or epitope region.
  • the Shiga toxin effector polypeptide comprises disruptions of at least two, endogenous. B-cell and/or T-cell epitope regions, wherein each disruption comprises one or more amino acid residue substitutions, and wherein the endogenous, B-cell and/or T-cell epitope regions are selected from the group of natively positioned Shiga toxin A Subunit regions consisting of: 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18; 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6; 42-48 of any one of SEQ ID NOs: 3 and 7-18; 53-66 of any one of SEQ ID NOs: 1-18; or the equivalent region in a Shiga toxin A Subunit or derivative thereof.
  • the embedded or inserted, heterologous, T-cell epitope does not disrupt any endogenous, B-cell and/or CD4+ T-cell epitope region described herein.
  • At least one disruption comprises one or more amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit is selected from the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, D to M.
  • the one or more amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit is selected from the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, G to A, H to A, H to G, H to V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q
  • At least one of the disruption(s) comprises one or more amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit selected from the group consisting of: K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F M, and S; S9 to A, G, V, L, I, F, and M; K11 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43 to
  • the cell-targeting molecule of the present invention is capable when introduced to a chordate of exhibiting improved in vivo tolerability and/or stability compared to a reference molecule, such as, e.g., a twenty-fourth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxy terminus of its A1 fragment region.
  • the Shiga toxin effector polypeptide is not cytotoxic and the molecular moiety is cytotoxic.
  • the binding region and Shiga toxin effector polypeptide are linked together, either directly or indirectly.
  • the binding region comprises at least one peptide and/or polypeptide.
  • the binding region is or comprises an immunoglobulin-type binding region.
  • the binding region comprising a polypeptide selected from the group consisting of an autonomous V H domain, single-domain antibody fragment (sdAb), nanobody, heavy chain-antibody domain derived from a camelid (V H H or V H domain fragment), heavy-chain antibody domain derived from a cartilaginous fish (V H H or V H domain fragment), immunoglobulin new antigen receptor (IgNAR), V NAR fragment, single-chain variable fragment (scFv), antibody variable fragment (Fv), complementary determining region 3 fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fd fragment, small modular immunopharmaceutical (SMIP) domain, antigen-binding fragment (Fab), Armadillo repeat polypeptide (SMIP) domain, antigen-binding fragment (Fab), Armadillo repeat polypeptide
  • the cell-targeting molecule of the present invention is capable of exhibiting (i) a catalytic activity level comparable to a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosome inhibition activity with a half-maximal inhibitory concentration (IC 50 ) value of 10,000 picomolar or less, and/or (iii) a significant level of Shiga toxin catalytic activity.
  • IC 50 half-maximal inhibitory concentration
  • the cell-targeting molecule of the present invention and/or its Shiga toxin effector polypeptide is capable of exhibiting subcellular routing efficiency comparable to a reference cell-targeting molecule comprising a wild-type Shiga toxin A1 fragment or wild-type Shiga toxin effector polypeptide and/or capable of exhibiting a significant level of intracellular routing activity to the endoplasmic reticulum and/or cytosol from an endosomal starting location of a cell.
  • Embodiment Sets #1 to #20 whereby administration of the cell-targeting molecule of the present invention to a cell physically coupled with the extracellular target biomolecule of the cell-targeting molecule's binding region, the cell-targeting molecule is capable of causing death of the cell.
  • the cell-targeting molecule of the invention is capable of causing cell death to the cell-types physically coupled with an extracellular target biomolecule of the cytotoxic cell-targeting molecule's binding region at a CD 50 at least three times or less than the CD 50 to cell types which are not physically coupled with an extracellular target biomolecule of the cell-targeting molecule's binding region.
  • the cytotoxic effect of the cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cytotoxic effect of the cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater.
  • the cytotoxic effect of the cell-targeting molecule to members of the first population of cells relative to members of the second population of cells is at least 3-fold greater.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting a cytotoxicity with a half-maximal inhibitory concentration (CD5) value of 300 nM or less and/or capable of exhibiting a significant level of Shiga toxin cytotoxicity.
  • CD5 half-maximal inhibitory concentration
  • the cell-targeting molecule of the present invention is capable of delivering an embedded or inserted, heterologous, CD8+ T-cell epitope to a MHC class I presentation pathway of a cell for cell-surface presentation of the epitope bound by a MHC class I molecule.
  • the cell-targeting molecule comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide.
  • the molecular moiety comprises or consists of the binding region.
  • the molecular moiety comprises at least one amino acid and the Shiga toxin effector polypeptide is linked to at least one amino acid residue of the molecular moiety.
  • the molecular moiety and the Shiga toxin effector polypeptide are fused forming a continuous polypeptide.
  • the cell-targeting molecule further comprises a cytotoxic molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide.
  • the cytotoxic molecular moiety is a cytotoxic agent, such as, e.g., a small molecule chemotherapeutic agent, anti-neoplastic agent, cytotoxic antibiotic, alkylating agent, antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor known to the skilled worker and/or described herein.
  • the cytotoxic molecular moiety is cytotoxic at concentrations of less than 10,000, 5,000, 1,000, 500, or 200 pM.
  • the binding region is capable of binding to an extracellular target biomolecule selected from the group consisting of CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72, tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1), carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR.
  • an extracellular target biomolecule selected from the group consisting of CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu
  • HPV-E7 Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, SAIL, CD38, CD15, CD23, CD45 (protein tyrosine phosphatase receptor type C), CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, IL-1R (interleukin-1 receptor), mrp-14, NKG2D ligand, programmed death-ligand 1 (PD-L1).
  • P-L1 programmed death-ligand 1
  • the binding region is linked, either directly or indirectly, to the Shiga toxin effector polypeptide by at least one covalent bond which is not a disulfide bond.
  • the binding region is fused, either directly or indirectly, to the carboxy-terminus of the Shiga toxin effector polypeptide to form a single, continuous polypeptide.
  • the binding region is an immunoglobulin-type binding region.
  • the disrupted furin-cleavage motif comprises one or more mutations in the minimal, furin-cleavage site relative to a wild-type Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif is not an amino-terminal truncation of sequences that overlap with part or all of at least one amino acid residue of the minimal furin-cleavage site.
  • the mutation in the minimal, furin-cleavage site is an amino acid deletion, insertion, and/or substitution of at least one amino acid residue in the R/Y-x-x-R furin cleavage motif.
  • the disrupted furin-cleavage motif comprises at least one mutation relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in the region natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), or at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or the equivalent amino acid sequence position in any Shiga toxin A Subunit.
  • the mutation is an amino acid residue substitution of an arginine residue with a non-positively charged, amino acid residue.
  • the cell-targeting molecule of the present invention is capable when introduced to cells of exhibiting cytotoxicity comparable to a cytotoxicity of a reference molecule, such as, e.g., a twenty-fifth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment.
  • a reference molecule such as, e.g., a twenty-fifth cell-targeting molecule consisting of the cell-targeting molecule except for all of its Shiga toxin effector polypeptide component(s) each comprise a wild-type Shiga toxin A1 fragment.
  • one or more binding region(s) comprises the peptide or polypeptide shown in any one of SEQ ID NOs: 39-245.
  • two or more binding regions comprise the peptide or polypeptide shown in any one of SEQ ID NOs: 39-245. In certain further embodiments of Embodiment Sets #11 to #20, two or more binding regions each comprise the same peptide or polypeptide shown in any one of SEQ ID NOs: 39-245.
  • Certain embodiments of the cell-targeting molecule of the present invention comprises any one of SEQ ID NOs: 19-255, 259-278, and 288-748.
  • cell-targeting molecule of the present invention comprise or consist essentially of the polypeptide represented by the amino acid sequence shown in any one of SEQ ID NOs: 252-255, 259-278, and 288-748.
  • At least one binding region sterically covers the carboxy-terminus of the A1 fragment region.
  • the molecular moiety sterically covers the carboxy-terminus of the A1 fragment region. In certain further embodiments, the molecular moiety comprises the binding region.
  • the cell-targeting molecule of the present invention comprises a binding region and/or molecular moiety located carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment region.
  • the mass of the binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater.
  • the cell-targeting molecule comprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein (e.g., cytotoxicity and/or intracellular routing).
  • the binding region is comprised within a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 kDa, or greater, as long as the cell-targeting molecule retains the appropriate level of the Shiga toxin biological activity noted herein.
  • a relatively large, molecular moiety comprising such as, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100 k
  • the cell-targeting molecule of the present invention exhibits low cytotoxic potency (i.e. is not capable when introduced to certain positive target cell types of exhibiting a cytotoxicity greater than 1% cell death of a cell population at a cell-targeting molecule concentration of 100 nM, 500 nM, 100 nM, 75 nM, or 50 nM) and is capable when introduced to cells of exhibiting a greater subcellular routing efficiency from an extracellular space to a subcellular compartment of an endoplasmic reticulum and/or cytosol as compared to the cytotoxicity of a reference molecule, such as, e.g., a twenty-sixth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shiga toxin effector polypeptide which is not positioned at or proximal to the amino-terminus of the third cell-targeting molecule.
  • the twenty-sixth cell-targeting molecule having an amino-terminus and comprising the binding region and the Shig
  • the molecular moiety comprises a peptide and/or polypeptide derived from the Shiga toxin A2 fragment of a naturally occurring Shiga toxin.
  • the embodiments of the present invention are not intended to cover any naturally-occurring Shiga holotoxin or Shiga toxin A Subunit.
  • the cell-targeting molecule of the present invention does not comprise a naturally occurring Shiga toxin B Subunit.
  • the cell-targeting molecule of the invention does not comprise any polypeptide comprising or consisting essentially of a functional binding domain of a native Shiga toxin B subunit. Rather, in certain embodiments of the cell-targeting molecules of the invention, the Shiga toxin A Subunit derived regions are functionally associated with heterologous binding regions to effectuate cell-targeting.
  • the cell-targeting molecule of the present invention is limited by the proviso that the heterologous, CD8+ T-cell epitope-peptide cargo does not comprise or consist of the polypeptide shown in SEQ ID NO:25 for all variations described above of Embodiment Sets #1 to #20.
  • the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise the Shiga toxin effector polypeptide comprising the CD8+ T-cell epitope-peptide GILGFVFTL (SEQ ID NO:25) embedded at native position 53 in SLT-1A (SEQ ID NO: 1) for all variations described above of Embodiment Sets #1 to #20.
  • Shiga toxin effector polypeptide comprising the CD8+ T-cell epitope-peptide GILGFVFTL (SEQ ID NO:25) embedded at native position 53 in SLT-1A (SEQ ID NO: 1) for all variations described above of Embodiment Sets #1 to #20.
  • the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise the polypeptide shown in SEQ ID NO:25 for all variations described above of Embodiment Sets #1 to #20. In certain embodiments of Embodiment Sets #1 to #20, the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise any Shiga toxin effector polypeptide comprising any embedded or inserted, CD8+ T-cell epitope for all variations described above of Embodiment Sets #1 to #20.
  • the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise the linker shown in SEQ ID NO:247 wherein the linker is fused, either directly or indirectly, between a binding region and a Shiga toxin effector polypeptide and wherein the binding region is positioned amino-terminal to the Shiga toxin effector polypeptide for all variations described above of Embodiment Sets #1 to #20.
  • the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise the linker shown in SEQ ID NO:247 wherein the linker is fused between a binding region and a Shiga toxin effector polypeptide for all variations described above of Embodiment Sets #1 to #20.
  • the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise or consist essentially of the polypeptide shown in any one of SEQ ID NOs: 259-278 and 287 for all variations described above of Embodiment Sets #1 to #20.
  • the cell-targeting molecule of the present invention does not comprise any heterologous, CD8+ T-cell epitope-peptide cargo fused between a binding region and a Shiga toxin effector polypeptide wherein the binding region is positioned amino-terminal to the Shiga toxin effector. In certain embodiments, the cell-targeting molecule of the present invention does not comprise any heterologous, CD8+ T-cell epitope-peptide cargo fused between a binding region and a Shiga toxin effector polypeptide.
  • the target cell is not a professional antigen presenting cell, such as a dendritic cell type.
  • the extracellular target biomolecule of the binding region is not expressed by a professional antigen presenting cell.
  • the extracellular target biomolecule of the binding region is not physically associated in significant quantities with a professional antigen presenting cell.
  • the extracellular target biomolecule of the binding region is not physically associated with a professional antigen presenting cell.
  • the target biomolecule of the binding region is not expressed in significant amounts on the cellular surface of any professional antigen presenting cell within the chordate subject to be treated.
  • the heterologous, CD8+ T-cell epitope-peptide cargo is not directly associated with any amino acid residue of the Shiga toxin A1 fragment derived region of the Shiga toxin effector polypeptide.
  • the heterologous, CD8+ T-cell epitope-peptide cargo is not directly associated with any internal amino acid residue of the Shiga toxin effector polypeptide, meaning either the amino- or carboxy-terminal amino acid residue of the Shiga toxin effector polypeptide may be directly linked to a heterologous, CD8+50 T-cell epitope-peptide cargo.
  • the binding region does not comprise a fragment of human CD4 corresponding to amino acid residues 19-183. In certain further embodiments, the binding region does not comprise a fragment of human CD4, a type-I transmembrane glycoprotein. In certain further embodiments, the binding region does not comprise a fragment of a human, immune cell surface co-receptor.
  • the cell-targeting molecule of the present invention does not comprise a carboxy-terminal, binding region comprising a fragment of an immune cell surface receptor.
  • the Shiga toxin effector polypeptide comprises at least two, embedded or inserted, heterologous epitopes.
  • the Shiga toxin effector polypeptide does not comprise the set of amino acid residue substitutions relative to a wild-type Shiga toxin A Subunit selected from the following sets; (1) R248H and R251H; (2) R248G and R251G; (3) A246G, S247A, A253G, and S254A; and (4) A246G, S247A, R248G, R251G, A253G, and S254A.
  • the Shiga toxin effector polypeptide does not comprise a deletion of the region natively positioned at 247-252 in a wild-type Shiga toxin A Subunit. In certain embodiments of Embodiment Sets #1 to #20, the Shiga toxin effector polypeptide does not comprise deletions of the regions natively positioned at 245-247 and 253-255 in a wild-type Shiga toxin A Subunit.
  • the Shiga toxin effector polypeptide comprises one or more mutations relative to a naturally occurring A Subunit of a member of the Shiga toxin family which changes an enzymatic activity of the Shiga toxin effector polypeptide, the mutation selected from at least one amino acid residue deletion, insertion, or substitution.
  • the mutation relative to the naturally occurring A Subunit reduces of eliminates a cytotoxic activity of the Shiga toxin effector polypeptide but the Shiga toxin effector polypeptide retains at least one other Shiga toxin effector function, such as, e.g., promoting cellular internalization and/or directing intracellular routing to a certain subcellular compartment(s).
  • the mutation relative to the naturally occurring A Subunit is selected from at least one amino acid residue substitution, such as, e.g., A231E, N75A, Y77S, Y114S, E167D, R170A, R176K, W202A, and/or W203A in SEQ ID NO: 1-18.
  • the Shiga toxin effector polypeptide is capable of (i) routing to a subcellular compartment of a cell in which the Shiga toxin effector polypeptide is present selected from the following: cytosol, endoplasmic reticulum, and lysosome; (ii) intracellular delivery of the epitope-cargo from an early endosomal compartment to a proteasome of a cell in which the Shiga toxin effector polypeptide is present; and/or (iii) intracellular delivery of the epitope to a MHC class I molecule from an early endosomal compartment of a cell in which the Shiga toxin effector polypeptide is present.
  • the Shiga toxin effector polypeptide is capable of intracellular delivery of the CD8+ T-cell epitope for presentation by a MHC class I molecule on the surface of a cell in which the Shiga toxin effector polypeptide is present.
  • the molecule of the present invention does not comprise, at a position carboxy-terminal of the Shiga toxin effector polypeptide and/or the carboxy-terminus of the Shiga toxin A1 fragment region, any additional exogenous material representing an antigen and/or heterologous, CD8+, T-cell epitope-peptide cargo.
  • the binding region does not comprise a ligand. In certain embodiments of Embodiment Sets #1 to #20, the binding region does not comprise a chemokine or a TNF-related apoptosis-inducing ligand (TRAIL) nor a receptor binding fragment thereof. In certain embodiments of Embodiment Sets #1 to #20, the binding region does not comprise a human chemokine or human TRAIL nor a receptor binding fragment thereof. In embodiments of Embodiment Sets #1 to #20, the immunoglobulin-type binding region does not comprise a ligand nor a receptor binding fragment thereof.
  • TRAIL TNF-related apoptosis-inducing ligand
  • the immunoglobulin-type binding region does not comprise a chemokine or a TNF-related apoptosis-inducing ligand (TRAIL) nor a receptor binding fragment thereof.
  • the binding region does not comprise a human CC chemokine nor a receptor binding fragment thereof.
  • the binding region does not comprise the human CC chemokine CCL2 (see Bose S et al., Arch Pharm Res 36; 1039-50 (2013)).
  • the binding region does not comprise the human, CC chemokine CCL2, nor a receptor binding fragment thereof and a carboxy-terminal, Shiga toxin effector polypeptide consisting of amino acids 75-247 of StxA.
  • the binding region does not comprise the human, CC chemokine CCL2, nor a receptor binding fragment thereof, fused to a carboxy-terminal, Shiga toxin effector polypeptide consisting of amino acids 75-247 of StxA (SEQ ID NO:2).
  • the binding region does not comprise the human TRAIL nor a receptor binding fragment thereof.
  • the cell-targeting molecule of the present invention comprises or consists essentially of the polypeptide of any one of SEQ ID NOs: 252-255, 259-278, and 288-748. In certain embodiments, the cell-targeting molecule of the present invention does not comprise SEQ ID NO:25 and/or SEQ ID NO:247. In certain embodiments, the cell-targeting molecule of the present invention does not comprise or consist essentially of SEQ ID NO:287.
  • the cell-targeting molecule of the present invention is limited by the proviso that the cell-targeting molecule of the present invention does not comprise or consist essentially of the polypeptide shown in SEQ ID NO:287 for all variations described above of Embodiment Sets #1 to #20.
  • the present invention also provides pharmaceutical compositions comprising a cell-targeting molecule of the present invention and at least one pharmaceutically acceptable excipient or carrier; and the use of such a cell-targeting molecule, or a composition comprising it, in methods of the invention as further described herein.
  • Certain embodiments of the present invention are pharmaceutical compositions comprising any cell-targeting molecule of the present invention; and at least one pharmaceutically acceptable excipient or carrier.
  • a diagnostic composition comprising any one of the above cell-targeting molecules of the present invention and a detection promoting agent for the collection of information, such as diagnostically useful information about a cell-type, tissue, organ, disease, disorder, condition, and/or patient.
  • a detection promoting agent for the collection of information, such as diagnostically useful information about a cell-type, tissue, organ, disease, disorder, condition, and/or patient.
  • cell-targeting molecules of the present invention wherein the detection promoting agent is a heterologous epitope-peptide cargo and the cell-targeting molecule comprises the heterologous epitope-peptide cargo.
  • polynucleotides capable of encoding a cell-targeting molecule of the present invention, or a protein component thereof are within the scope of the present invention, as well as expression vectors which comprise a polynucleotide of the invention and host cells comprising an expression vector of the invention.
  • Host cells comprising an expression vector may be used, e.g., in methods for producing a cell-targeting molecule of the present invention, or a protein component or fragment thereof, by recombinant expression.
  • the present invention also encompasses any composition of matter of the present invention which is immobilized on a solid substrate. Such arrangements of the compositions of matter of the present invention may be utilized, e.g., in methods of screening molecules as described herein.
  • the present invention is a method of delivering into a cell a CD8+ T-cell epitope-peptide cargo capable of being presented by a MHC class I molecule of the cell, the method comprising the step of contacting the cell with the cell-targeting molecule of the present invention and/or a composition thereof (e.g., a pharmaceutical or diagnostic composition of the present invention).
  • the present invention is a method of inducing a cell to present an exogenously administered CD8+ T-cell epitope-peptide cargo complexed to a MHC class I molecule, the method comprising the step of contacting the cell, either in vitro or in vivo, with the cell-targeting molecule of the present invention, which comprises the CD8+ T-cell epitope, and/or a composition thereof (e.g., a pharmaceutical or diagnostic composition of the present invention comprising such a cell-targeting molecule of the present invention).
  • the present invention is a method of inducing an immune cell-mediated response to target cell via a CD8+ T-cell epitope MHC class I molecule complex, the method comprising the step of contacting the target cell either in vitro or in vivo, with the cell-targeting molecule of the present invention, which comprises the CD8+ T-cell epitope as a cargo, and/or a composition thereof (e.g., a pharmaceutical or diagnostic composition of the present invention comprising such a cell-targeting molecule of the present invention).
  • the cell-targeting molecule of the present invention which comprises the CD8+ T-cell epitope as a cargo, and/or a composition thereof (e.g., a pharmaceutical or diagnostic composition of the present invention comprising such a cell-targeting molecule of the present invention).
  • the immune response is selected from the group consisting: CD8+ immune cell secretion of a cytokine(s), cytotoxic T lymphocyte-(CTL) induced growth arrest in the target cell, CTL-induced necrosis of the target cell, CTL-induced apoptosis of the target cell, immune cell-mediated cell killing of a cell other than the target cell.
  • cytokine(s) cytotoxic T lymphocyte-(CTL) induced growth arrest in the target cell
  • CTL-induced necrosis of the target cell CTL-induced apoptosis of the target cell
  • immune cell-mediated cell killing of a cell other than the target cell immune cell-mediated cell killing of a cell other than the target cell.
  • the method comprises the step of contacting the target cell with the cell-targeting molecule of the present invention in the presence of a CD8+ immune cell or with the subsequent step of contacting the target cell with one or more CD8+ immune cells.
  • the contacting step occurs in vitro.
  • the contacting step occurs in vivo, such as, e.g., by administering the cell-targeting molecule to a chordate, vertebrate, and/or mammal.
  • the intercellular engagement occurs in vitro.
  • the intercellular engagement occurs in vivo.
  • composition comprising a cell-targeting molecule of the present invention for “seeding” a tissue locus within a chordate.
  • a method of the present invention is for “seeding” a tissue locus within a chordate, the method comprising the step of: administering to the chordate a cell-targeting molecule of the present invention, a pharmaceutical composition of the present invention, and/or a diagnostic composition of the present invention.
  • the method is for “seeding” a tissue locus within a chordate which comprises a malignant, diseased, and/or inflamed tissue.
  • the method is for “seeding” a tissue locus within a chordate which comprises the tissue selected from the group consisting of: diseased tissue, tumor mass, cancerous growth, tumor, infected tissue, or abnormal cellular mass.
  • the method for “seeding” a tissue locus within a chordate comprises the step of: administering to the chordate a cell-targeting molecule of the present invention comprising the heterologous, CD8+ T-cell epitope-peptide cargo selected from the group consisting of: peptides not natively presented by the target cells of the cell-targeting molecule in MHC class I complexes, peptides not natively present within any protein expressed by the target cell, peptides not natively present within the transcriptome and/or proteome of the target cell, peptides not natively present in the extracellular microenvironment of the site to be seeded, and peptides not natively present in the tumor mass or infected tissue site to be targeted.
  • a cell-targeting molecule of the present invention comprising the heterologous, CD8+ T-cell epitope-peptide cargo selected from the group consisting of: peptides not natively presented by the target cells of the cell-targeting molecule in MHC class I complexes
  • the present invention provides methods of killing a cell(s) comprising the step of contacting a cell(s) with a cell-targeting molecule of the present invention or a pharmaceutical composition comprising a cell-targeting molecule of the invention.
  • the step of contacting the cell(s) occurs in vitro.
  • the step of contacting the cell(s) occurs in vivo.
  • the method is capable of selectively killing cell(s) and/or cell-types preferentially over other cell(s) and/or cell-types when contacting a mixture of cells which differ with respect to the extracellular presence and/or expression level of an extracellular target biomolecule of the binding region of the cell-targeting molecule.
  • the present invention further provides methods of treating diseases, disorders, and/or conditions in patients in need thereof comprising the step of administering to a patient in need thereof a therapeutically effective amount of a composition comprising a cell-targeting molecule or pharmaceutical composition of the present invention.
  • the disease, disorder, or condition to be treated using this method of the invention is selected from: a cancer, tumor, growth abnormality, immune disorder, or microbial infection.
  • the cancer to be treated is selected from the group consisting of, bone cancer, breast cancer, central/peripheral nervous system cancer, gastrointestinal cancer, germ cell cancer, glandular cancer, head-neck cancer, hematological cancer, kidney-urinary tract cancer, liver cancer, lung/pleura cancer, prostate cancer, sarcoma, skin cancer, and uterine cancer.
  • the immune disorder to be treated is an immune disorder associated with a disease selected from the group consisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-versus-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis, polyarteritis nodosa, polyarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjögren's syndrome, ulcerative colitis, and vasculitis.
  • a disease selected from the group consisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes, graft rejection, graft-versus-host disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupus
  • compositions comprising a cell-targeting molecule of the present invention for the treatment or prevention of a cancer, tumor, growth abnormality, immune disorder, or microbial infection.
  • compositions of the present invention for the treatment or prevention of a cancer, tumor, growth abnormality, and/or immune disorder is within the scope of the present invention.
  • Certain embodiments of the present invention include a method of treating cancer in a patient using immunotherapy, the method comprising the step of administering to the patient in need thereof the cell-targeting molecule and/or pharmaceutical composition of the present invention.
  • any composition of matter of the present invention for the treatment or prevention of a cancer, tumor, growth abnormality, and/or immune disorder is within the scope of the present invention.
  • a cell-targeting molecule of the present invention and/or a pharmaceutical composition thereof for the treatment or prevention of a cancer, tumor, growth abnormality, immune disorder, and/or microbial infection is the use of a cell-targeting molecule of the present invention and/or pharmaceutical composition thereof in the manufacture of a medicament for the treatment or prevention of a cancer, tumor, growth abnormality, immune disorder, or microbial infection.
  • compositions comprising a cell-targeting molecule of the present invention for the delivery of one or more additional exogenous materials into a cell physically coupled with an extracellular target biomolecule of the binding region of the cell-targeting molecule of the present invention.
  • Certain embodiments of the cell-targeting molecules of the present invention may be used to deliver one or more additional exogenous materials into a cell physically coupled with an extracellular target biomolecule of the binding region of the cell-targeting molecule of the present invention.
  • the present invention provides a method for delivering exogenous material to the inside of a cell(s) comprising contacting the cell(s), either in vitro or in vivo, with a cell-targeting molecule, pharmaceutical composition, and/or diagnostic composition of the present invention.
  • the present invention further provides a method for delivering exogenous material to the inside of a cell(s) in a patient in need thereof the method comprising the step of administering to the patient a cell-targeting molecule of the present invention, wherein the target cell(s) is physically coupled with an extracellular target biomolecule of the binding region of the cell-targeting molecule of the present invention.
  • compositions of the present invention e.g. a cell-targeting molecule, a pharmaceutical composition, or diagnostic composition
  • diagnosis, prognosis, and/or characterization of a disease, disorder, and/or condition is within the scope of the present invention.
  • the method of detecting a cell using a cell-targeting molecule and/or diagnostic composition of the invention comprising the steps of contacting a cell with said cell-targeting molecule and/or diagnostic composition and detecting the presence of said cell-targeting molecule and/or diagnostic composition.
  • the step of contacting the cell(s) occurs in vitro.
  • the step of contacting the cell(s) occurs in vivo.
  • the step of detecting the cell(s) occurs in vitro.
  • the step of detecting the cell(s) occurs in vivo.
  • a diagnostic composition of the invention may be used to detect a cell in vivo by administering to a chordate subject a composition comprising cell-targeting molecule of the present invention which comprises a detection promoting agent and then detecting the presence of the cell-targeting molecule of the present invention and/or the heterologous, CD8+ T-cell epitope-peptide cargo either in vitro or in vivo.
  • the cell-targeting molecules of the present invention may be utilized for the delivery of additional exogenous material into a cell physically coupled with an extracellular target biomolecule of the cell-targeting molecule of the invention. Additionally, the present invention provides a method for delivering exogenous material to the inside of a cell(s) comprising contacting the cell(s), either in vitro or in vivo, with a cell-targeting molecule, pharmaceutical composition, and/or diagnostic composition of the present invention.
  • the present invention further provides a method for delivering exogenous material to the inside of a cell(s) in a patient, the method comprising the step of administering to the patient a cell-targeting molecule of the present invention (with or without cytotoxic activity), wherein the target cell(s) is physically coupled with an extracellular target biomolecule of the cell-targeting molecule.
  • a method of delivering into a cell a T-cell epitope-peptide cargo capable of being presented by a MHC class I molecule of the cell comprising the step of contacting the cell with the cell-targeting molecule of the present invention which is associated with a heterologous, T-cell epitope-peptide cargo and/or a composition thereof (e.g., a pharmaceutical or diagnostic composition of the present invention).
  • the present invention is a method for “seeding” a tissue locus within a chordate, the method comprising the step of: administering to the chordate a cell-targeting molecule of the present invention, a pharmaceutical composition of the present invention, and/or a diagnostic composition of the present invention.
  • the methods of the invention for “seeding” a tissue locus are for “seeding” a tissue locus which comprises a malignant, diseased, or inflamed tissue.
  • the methods of the invention for “seeding” a tissue locus are for “seeding” a tissue locus which comprises the tissue selected from the group consisting of: diseased tissue, tumor mass, cancerous growth, tumor, infected tissue, or abnormal cellular mass.
  • the methods of the invention for “seeding” a tissue locus comprises administering to the chordate the cell-targeting molecule of the invention, the pharmaceutical composition of the invention, or the diagnostic composition of the invention comprising the heterologous, T-cell epitope-peptide cargo selected from the group consisting of: peptides not natively presented by the target cells of the cell-targeting molecule in MHC class I complexes, peptides not natively present within any protein expressed by the target cell, peptides not natively present within the proteome of the target cell, peptides not natively present in the extracellular microenvironment of the site to be seeded, and peptides not natively present in the tumor mass or infected tissue site to be targeted.
  • composition of matter of the present invention for the diagnosis, prognosis, and/or characterization of a disease, disorder, and/or condition is within the scope of the present invention.
  • a method of using a cell-targeting molecule of the present invention comprising a detection promoting agent and/or composition of the invention (e.g. a diagnostic composition) for the collection of information useful in the diagnosis, prognosis, or characterization of a disease, disorder, or condition.
  • the present invention is the method of detecting a cell (or subcellular compartment thereof) using a cell-targeting molecule and/or diagnostic composition of the present invention, the method comprising the steps of contacting a cell with the cell-targeting molecule and/or diagnostic composition and detecting the presence of said cell-targeting molecule and/or diagnostic composition.
  • the step of contacting the cell(s) occurs in vitro.
  • the step of contacting the cell(s) occurs in vivo.
  • the step of detecting the cell(s) occurs in vitro.
  • the step of detecting the cell(s) occurs in vivo.
  • the method involves the detection of the location of the cell-targeting molecule in an organism using one or more imaging procedures after the administration of the cell-targeting molecule to said organism.
  • cell-targeting molecules of the invention which incorporate detection promoting agents as described herein may be used to characterize diseases as potentially treatable by a related pharmaceutical composition of the present invention.
  • certain cell-targeting molecules of the present invention and compositions thereof e.g. pharmaceutical compositions and diagnostic compositions of the present invention
  • methods of the present invention may be used to determine if a patient belongs to a group that responds to a pharmaceutical composition of the present invention.
  • certain cell-targeting molecules of the present invention and compositions thereof may be used to identify cells which present a delivered heterologous epitope-peptide cargo on a cellular surface and/or to identify subjects containing cells which present a heterologous epitope-peptide cargo delivered by a cell-targeting molecule of the present invention.
  • the present invention is a method of producing a molecule of the present invention, the method comprising the step of purifying the molecule of the invention or a polypeptide component of thereof using a bacterial cell-wall protein domain interaction, such as, e.g., protein L from P. magnus or derivatives and binding domain fragments thereof.
  • the purifying step of the method involves the Shiga toxin effector polypeptide comprising or consisting essentially of any one of the polypeptides shown in SEQ ID NOs: 29-38.
  • kits comprising a composition of matter of the invention, and optionally, instructions for use, additional reagent(s), and/or pharmaceutical delivery device(s).
  • the kit may further comprise reagents and other tools for detecting a cell type (e.g. a tumor cell) in a sample or in a subject.
  • FIG. 1A and FIG. 1B depict exemplary, cell-targeting molecules comprising a heterologous, CD8+ T-cell epitope-peptide cargo (shown as a diamond shape).
  • These exemplary cell-targeting molecules each comprise a Shiga toxin effector polypeptide having a combination of features: de-immunizing mutations (shown as a horizontally striped region), an embedded, heterologous, CD8+ T-cell epitope (shown as a vertically striped region), and sometimes a disrupted or missing protease site (shown with a dashed line).
  • de-immunizing mutations shown as a horizontally striped region
  • an embedded, heterologous, CD8+ T-cell epitope shown as a vertically striped region
  • a disrupted or missing protease site shown with a dashed line.
  • the “N” and “C” denote an amino-terminus and carboxy-terminus, respectively, of a polypeptide component of
  • exemplary cell-targeting molecules sometimes comprise a Shiga toxin effector polypeptide having the third feature of a disrupted furin-cleavage site at the carboxy-terminus of an A1 fragment derived region depicted with a dashed, vertical, gray line.
  • FIG. 1A and FIG. 1B are for illustrative purposes of certain, general arrangements of the structural features of a limited set of embodiments of the present invention. It is to be understood that these exemplary molecules do not intend, nor should any be construed, to be wholly definitive as to the arrangement of any structural features and/or components of a molecule of the present invention.
  • the relative size, location, or number of features shown in the schematics of FIG. 1A and FIG. 1B have been simplified. For example, the relative positions of embedded, heterologous epitopes and disruptions of an endogenous, epitope regions are not fixed. Similarly, the total numbers of embedded, heterologous epitopes and disruptions of an endogenous, epitope regions are not fixed.
  • Certain embodiments of the molecules of the present invention comprise a plurality of disrupted, endogenous, epitope regions in a single, Shiga toxin effector polypeptide, such as, e.g., disruptions of four, five, six, seven, eight, nine, or more regions; wherein these disrupted, endogenous, epitope regions may be distributed throughout the Shiga toxin effector polypeptide, including disruptions which overlap with or are within the furin-cleavage motif of the carboxy-terminus region of a Shiga toxin A1 fragment derived region (see e.g. WO 2016/196344).
  • Certain embodiments of the present invention comprise disruptions of endogenous, epitope regions which are carboxy-terminal to the carboxy-terminus of the Shiga toxin A1 fragment, or a derivative thereof such as, e.g. at a position carboxy-terminal to any disrupted furin-cleavage site motif.
  • the schematics in FIG. 1A and FIG. 1B are not intended to accurately portray any information regarding the relative sizes of molecular structures in any embodiment of the present invention.
  • FIG. 2 graphically shows that the exemplary cell-targeting molecule SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) exhibited cytotoxicity to two, different cell-types comparable to a “control” cell-targeting molecule SLT-1A-DI-FR::scFv-1 (SEQ ID NO:258).
  • the percent viability of target positive cells for each cell type was plotted over the logarithm to base 10 of the cell-targeting molecule concentration administered to the respective cells.
  • FIG. 3 graphically shows cell-surface presentation of a cell-targeting molecule delivered, heterologous, CD8+ T-cell epitope-peptide complexed with MHC class I molecule by a target positive cancer cell as compared to a negative control.
  • FIG. 3 shows overlays of the results of a TCR-STARTM assay, flow cytometric analysis of sets of cells treated either with the exemplary cell-targeting molecule of the present invention SLT-A-DI-FR::scFv1::C2 (SEQ ID NO:252) or a negative control, the cell-targeting molecule SLT-1A-DI-FR::scFv1 (SEQ ID NO:258), which lacks any C2 epitope-peptide cargo.
  • the FACS cell count of target positive cells was plotted over the light signal from PE-STARTM multimer reagent in relative light units (RLU) representing the presence of cell-surface, MHC class I molecule (human HLA-A2) displayed C2 epitope-peptide (SEQ ID NO:21) complexes.
  • RLU relative light units
  • Target positive cells treated with the exemplary cell-targeting molecule of the present invention SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MHC class I molecules on their cell surfaces (upper graph), whereas target positive cells treated with the parental cell-targeting molecule SLT-1A-DI-FR::scFv1 (SEQ ID NO:258) did not display the C2 epitope-peptide (SEQ ID NO:21) on a cell surface (lower graph).
  • FIG. 4 graphically shows the sizes and proportions of molecules present in a sample preparation of SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) analyzed by size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • FIG. 5 shows a Coomassie-stained, sodium dodecyl sulfate, polyacrylamide gel (SDS-PAGE) after electrophoresis of sample preparation of SLT-1A-DI-FR::scFv1:C2 (SEQ ID NO:252) prepared for gel-loading under reducing conditions.
  • FIG. 5 shows that size of reduced SLT-1A-DI-FR::scFv1::C2 (SEQ ID NO:252) was about 55 kiloDaltons (kDa).
  • FIG. 6 graphically shows fusing a heterologous, CD8+ T-cell epitope-peptide to a Shiga toxin A Subunit derived, cell-targeting molecule did not significantly impair the cytotoxic activity of the cell-targeting molecule toward target positive cells.
  • the percent viability of cells was plotted over the logarithm to base 10 of the protein concentration.
  • FIG. 6 graphically shows the results of a cell-kill assay where SLT-1A::scFv1::C2 (SEQ ID NO:278) exhibited cytotoxicity similar to the cytotoxicity of the parental cell-targeting molecule SLT-1A::scFv1 (SEQ ID NO:280), which lacked any heterologous, CD8+ T-cell epitope-peptide.
  • FIG. 7 graphically shows the results of a cell-kill assay where the cytotoxic activity of the exemplary cell-targeting molecule SLT-1A::scFv1::C2 (SEQ ID NO:278) was specific to target positive cells over a certain concentration range. The percent viability of cells was plotted over the logarithm to base 10 of the protein concentration.
  • SLT-1A::scFv1::C2 SLT-1A::scFv1::C2
  • SEQ ID NO:278 SLT-1A::scFv1::C2
  • FIG. 8 graphically shows cell-surface presentation of a cell-targeting molecule delivered, heterologous, CD8+ T-cell epitope-peptide complexed with MHC class I molecule by a target positive cancer cell as compared to a negative control.
  • FIG. 8 shows overlays of the results of a TCR-STARTM assay, flow cytometric analysis of sets of cells treated either with a negative control, the cell-targeting molecule SLT-1A::scFv1::C2 (SEQ ID NO:278), or the cell-targeting molecule SLT-1A::scFv2 (SEQ ID NO:281).
  • FACS fluorescence-activated cell sorting
  • Target positive cells treated with the exemplary cell-targeting molecule of the present invention SLT-1A::scFv1::C2 (SEQ ID NO:278) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MHC class I molecules on their cell surfaces (upper graph), whereas target positive cells treated with the related cell-targeting molecule SLT-1A::scFv2 (SEQ ID NO:281) did not display the C2 epitope-peptide (SEQ ID NO:21) on a cell surface (lower graph).
  • FIG. 9 graphically shows cell-surface presentation of a cell-targeting molecule delivered, heterologous, CD8+ T-cell epitope-peptide complexed with MHC class I molecule by a target positive cancer cell as compared to negative controls.
  • the indexed, mean, fluorescent intensity (“iMFI,” the fluorescence of the positive population multiplied by the percent positive) of the PE-STARTM multimer reagent in RLU corresponding to the sets of cells receiving the different treatments was graphed.
  • iMFI fluorescent intensity
  • the C2 peptide (SEQ ID NO:21) combined with Peptide Loading Enhancer (PLE) treatment provides a positive control where exogenously administered C2 peptide (SEQ ID NO:21) may be loaded onto cell-surface MHC class I molecules without ever entering a cell.
  • PLE Peptide Loading Enhancer
  • Target positive cells treated with the exemplary cell-targeting molecule of the present invention “inactive SLT-1A::scFv2::C2” displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MI-IC class I molecules on their cell surfaces, whereas the same cells treated with only exogenous C2 epitope-peptide (SEQ ID NO:21) or the parental cell-targeting molecule “inactive SLT-1A::scFv2” (SEQ ID NO:282) did not display the C2 epitope-peptide (SEQ ID NO:21) on a cell surface.
  • FIG. 10 graphically shows cell-surface presentation of a cell-targeting molecule delivered, heterologous, CD8+ T-cell epitope-peptide complexed with MHC class I molecule by a target positive cancer cell for different incubation times (4 hours or 16 hours) as compared to a negative control.
  • FIG. 10 shows overlays of the results of a TCR-STARTM assay, flow cytometric analysis of sets of cells treated either with the cell-targeting molecule SLT-1A::scFv1::C2 (SEQ ID NO:278) or a negative control.
  • the FACS cell count of target positive cells was plotted over the light signal from PE-STARTM multimer reagent in relative light units (RLU) representing the presence of cell-surface, MHC class I molecule (human HLA-A2) displayed C2 epitope-peptide (SEQ ID NO:21) complexes.
  • Target positive cells treated with the exemplary cell-targeting molecule of the present invention SLT-1A::scFv1::C2 (SEQ ID NO:278) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MHC class I molecules on their cell surfaces after either a 4-hour (4 hrs) (upper graph) or 16-hour (16 hrs) (lower graph) incubation duration.
  • FIG. 11 graphically shows cell-surface presentation of a cell-targeting molecule delivered, heterologous, CD8+ T-cell epitope-peptide complexed with MHC class I molecule by a target positive cancer cell as compared to a negative control.
  • FIG. 11 shows overlays of the results of a TCR-STARTM assay, flow cytometric analysis of sets of cells treated either with a negative control, the cell-targeting molecule SLT-1A::scFv5::C2 (SEQ ID NO:274), or the cell-targeting molecule SLT-1A::scFv5 (SEQ ID NO:283).
  • the FACS cell count of target positive cells was plotted over the light signal from PE-STARTM multimer reagent in relative light units (RLU) representing the presence of cell-surface, MHC class I molecule (human HLA-A2) displayed C2 epitope-peptide (SEQ ID NO:21) complexes.
  • RLU relative light units
  • Target positive cells treated with the exemplary cell-targeting molecule of the present invention SLT-1A::scFv5::C2 (SEQ ID NO:274) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MHC class I molecules on their cell surfaces (upper graph), whereas target positive cells treated with the parental cell-targeting molecule SLT-1A::scFv5 (SEQ ID NO:283) did not display the C2 epitope-peptide (SEQ ID NO:21) on a cell surface (lower graph).
  • FIG. 12 graphically shows cell-surface presentation of a cell-targeting molecule delivered, heterologous, CD8+ T-cell epitope-peptide complexed with MHC class I molecule by a target positive cancer cell as compared to a negative control.
  • FIG. 12 shows overlays of the results of a TCR-STARTM assay, flow cytometric analysis of sets of cells treated either with a negative control, the cell-targeting molecule SLT-1A::scFv7::C2 (SEQ ID NO:277), or the cell-targeting molecule SLT-1A::scFv7 (SEQ ID NO:286).
  • the FACS cell count of target positive cells was plotted over the light signal from PE-STARTM multimer reagent in relative light units (RLU) representing the presence of cell-surface, MHC class I molecule (human HLA-A2) displayed C2 epitope-peptide (SEQ ID NO:21) complexes.
  • RLU relative light units
  • Target positive cells treated with the exemplary cell-targeting molecule of the present invention SLT-1A::scFv7::C2 (SEQ ID NO:277) displayed the C2 epitope-peptide (SEQ ID NO:21) complexed to MHC class I molecules on their cell surfaces (upper graph), whereas target positive cells treated with the parental cell-targeting molecule SLT-1A::scFv7 (SEQ ID NO:286) did not display the C2 epitope-peptide (SEQ ID NO:21) on a cell surface (lower graph).
  • FIG. 13 graphically shows the results from an Interferon Gamma ELIspot assay with the number of spots, or secreting cells, plotted for each condition tested.
  • target positive human cancer cells were pretreated with a cell-targeting molecule or negative control and, then, incubated with human PBMCs (HLA-A2 serotype) for 24 hours before performing the ELISPOT assay.
  • human PBMCs HLA-A2 serotype
  • FIG. 13 shows that target positive cells treated with the exemplary cell-targeting molecule of the present invention “inactive SLT-1A::scFv2::C2” (SEQ ID NO:270) promoted cytokine secretion by PBMCs, whereas treatment with the parental cell-targeting molecule “inactive SLT-1A::scFv2” (SEQ ID NO:282) resulted only in a background level of interferon-y secretion at about the same level as the negative control treatment of “buffer only.”
  • the notation “Target positive Cell Line G+ PBMCs” indicates that all the samples shown involved a coculture of tumor cells of cell line G with PBMCs.
  • FIG. 14 graphically shows the results from an intercellular T lymphocyte (T-cell) activation assay with luciferase activity plotted in RLU for each condition tested.
  • T-cell intercellular T lymphocyte
  • target positive human cancer cells were pretreated with a cell-targeting molecule or negative control and, then, incubated for 18 hours with human J76 T-cells expressing a human T-cell receptor (TCR) that specifically recognizes cell-surface presented, human MHC class I molecule (HLA-A2) F2 epitope (SEQ ID NO:25) complexes, and comprising a nuclear factor of activated T-cells (NFAT) transcriptional response element driving luciferase expression (luciferase-reporter-transfected).
  • TCR human T-cell receptor
  • HLA-A2 human MHC class I molecule
  • SEQ ID NO:25 nuclear factor of activated T-cells
  • FIG. 14 shows that target positive tumor cells pretreated with the exemplary cell-targeting molecule of the present invention “inactive SLT-1A::scFv6::F2” (SEQ ID NO:276) stimulated an intermolecular response in the form of T-cell activation via TCR recognition and NFAT signaling: whereas, treatment of target positive cells with the parental cell-targeting molecule “inactive SLT-1A::scFv6” (SEQ ID NO:285) resulted only in the background level of light signal at about the same level as the negative control treatment of “buffer only.”
  • the notation “Target positive Cell Line F+ Reporter T cells” indicates that all the samples shown involved a coculture of tumor cells of cell line G with the luciferase-reporter transfected J76 T-cells (SEQ ID NO:276) indicates that all the samples shown involved a coculture of tumor cells of cell line G with the luciferase-reporter transfected J76 T-cells (S
  • FIG. 15 graphically shows the results from an IFN- ⁇ ELISA assay with data from the “buffer only” sample in black and from the cell-targeting molecule treated sample in grey.
  • the cells of the coculture consisted of human PBMCs enriched for C2-restricted PBMCs and human tumor cells.
  • the tumor cells used were of the cell line I and were pretreated with “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253).
  • FIG. 16 graphically shows the results from a CellTiter-Glo® Luminescent Cell Viability assay of a coculture experiment with data from the “buffer only” sample in black and from the cell-targeting molecule treated sample in grey. Along the Y-axis is the quantification of adherent-cell viability expressed as a percentage of the “buffer only” negative control in RLU.
  • the cells of the coculture consisted of human PBMCs enriched for C2-restricted PBMCs and human tumor cells.
  • the tumor cells used were of the cell line I and were pretreated with “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253).
  • FIG. 17 graphically plots the number of immune cells clustered during different time-points of a 140-hour coculture experiment with data from the “buffer only” sample in black and from the cell-targeting molecule treated sample in grey.
  • the cells were counted using the IncuCyte® S3 Live-Cell imaging assay performed as described herein.
  • the cells of the coculture consisted of human PBMCs enriched for C2-restricted PBMCs and human tumor cells.
  • the tumor cells used were of the cell line I and were pretreated with “inactive SLT-1A-DI-4::scFv6::(C2)3” (SEQ ID NO:253).
  • the quantity cells involved and the quantity of PBMC clusters of over 100 microns are indicative of immune cell activation occurring in the coculture.
  • the quantities of cells and clusters over time (the persistency of immune cell clustering behavior) is also indicative immune cell activation occurring in the coculture.
  • FIG. 18 graphically shows the results from IFN- ⁇ ELISA assays with data from three-different tumor cell types treated with three different concentrations of an exemplary cell-targeting molecule of the present invention (SEQ ID NO:254) (shown in grey) and data from a control treatment using a cell-targeting molecule lacking any C2 epitope-peptide cargo for delivery (SEQ ID NO:256) (shown in black).
  • SEQ ID NO:254 an exemplary cell-targeting molecule of the present invention
  • SEQ ID NO:256 shows the quantification of IFN- ⁇ present in the supernatants from coculture experiments reported in pg/mL.
  • the x-axis shows four different experimental conditions: no PBMC-coculture, “buffer only” treatment of tumor cells before coculture, treatment of tumor cells with 100 nM of molecule, and treatment with tumor cells with 500 nM of molecule.
  • the cells of the coculture consisted of human PBMCs enriched for C2-restricted PBMCs and human tumor cells.
  • the tumor cells used were of the cell line I, J, or K and were pretreated with either “inactive SLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) or “inactive SLT-1A-DI-1::scFv8” (SEQ ID NO:256).
  • the concentration of exemplary cell-targeting molecule of the present invention in the treatment goes up from zero to 100, and 500 nM
  • the quantity of secreted human IFN- ⁇ in the coculture goes up (e.g., to above about 400 or 600 pg/mL).
  • the results show that neither “buffer only” or the closely related reference cell-targeting molecule “inactive SLT-1A-DI-1::scFv8” (SEQ ID NO:256) lacking the of C2 epitope-peptide (SEQ ID NO:21) was capable of inducing IFN- ⁇ secretion after 48 hours post-coculture.
  • the notation “+ PBMCs” indicates the sample shown involved a coculture of tumor cells and PBMCs.
  • FIG. 19 graphically shows the results from CellTiter-Glo® Luminescent Cell Viability assays of coculture experiments with data from three-different tumor cell types treated with three different concentrations of an exemplary cell-targeting molecule of the present invention (SEQ ID NO:254) (shown in grey) and data from a control treatment using a cell-targeting molecule lacking any C2 epitope-peptide cargo for delivery (SEQ ID NO:256) (shown in black).
  • SEQ ID NO:254 an exemplary cell-targeting molecule of the present invention
  • SEQ ID NO:256 shows the quantification of adherent-cell viability in RLU.
  • the x-axis shows four different experimental conditions: no PBMC-coculture, “buffer only” treatment of tumor cells before coculture, treatment of tumor cells with 100 nM of molecule, and treatment with tumor cells with 500 nM of molecule.
  • the cells of the coculture consisted of human PBMCs enriched for C2-restricted PBMCs and human tumor cells.
  • the tumor cells used were of the cell line I, J, or K and were pretreated with either “inactive SLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) or “inactive SLT-1A-DI-1::scFv8” (SEQ ID NO:256).
  • the concentration of the exemplary cell-targeting molecule of the present invention “inactive SLT-1A-DI-1::scFv8::C2” (SEQ ID NO:254) in the treatment goes up from zero to 100, and 500 nM, the adherent cell viability drops to below 50 percent.
  • the results show that neither “buffer only” or the closely related reference cell-targeting molecule “inactive SLT-1A-DI-FR::scFv8” (SEQ ID NO:256) lacking the of C2 epitope-peptide (SEQ ID NO:21) was capable of causing significant target cell death during 96 hours of coculture.
  • the notation “+ PBMCs” indicates the sample shown involved a coculture of tumor cells and PBMCs.
  • FIG. 20 graphically shows the results from IFN- ⁇ ELISA and CellTiter-Glo® Luminescent Cell Viability assays of coculture experiments with pretreatment of tumor cells with an exemplary cell-targeting molecule of the present invention (SEQ ID NO:255) shown in grey and data from the control treatment using a catalytically-active cell-targeting molecule lacking any C2 epitope-peptide cargo for delivery shown in black (SEQ ID NO:257).
  • the cells of the coculture consisted of human PBMCs enriched for C2-restricted PBMCs and human tumor cells.
  • the tumor cells used were of the cell line I and were pretreated with the catalytically-active SLT-1A-DI-1::scFv8::C2 (SEQ ID NO:255) or with the catalytically active control molecule SLT-1A-DI-1::scFv8 (SEQ ID NO:257).
  • the Y-axis shows the quantification of IFN- ⁇ present in the supernatants from the coculture experiments reported in pg/mL.
  • the Y-axis shows the quantification of adherent-cell viability in RLU.
  • the x-axis shows four different experimental conditions: no PBMC-coculture, “buffer only” treatment of tumor cells before coculture, treatment of tumor cells with 30 nM of molecule, and treatment of tumor cells with 100 nM of molecule.
  • SLT-1A-DI-1::scFv8::C2 As the concentration of the exemplary cell-targeting molecule of the present invention SLT-1A-DI-1::scFv8::C2 (SEQ ID NO:255) in the treatment goes up from zero to and 100 nM, the quantity of secreted human IFN- ⁇ in the coculture goes up to above 200 pg/mL and the adherent cell viability is reduced more than treatment with a closely related, catalytically-active cell-targeting molecule SLT-1A-DI-1::scFv8 (SEQ ID NO:257) lacking any C2 epitope-peptide (SEQ ID NO:21) cargo.
  • SLT-1A-DI-1::scFv8::C2 As the concentration of the exemplary cell-targeting molecule of the present invention SLT-1A-DI-1::scFv8::C2 (SEQ ID NO:255) in the treatment goes up from zero to and 100 n
  • the control molecule did not induce any human IFN- ⁇ secretion by the PBMCs in coculture and did not reduce tumor cell viability over time as much as the exemplary molecule of the present invention did for a given molecule treatment concentration.
  • the notation “+ PBMCs” indicates the sample shown involved a coculture of tumor cells and PBMCs.
  • the term “and/or” when referring to two species, A and B, means at least one of A and B.
  • the term “and/or” when referring to greater than two species, such as A, B, and C, means at least one of A, B, or C, or at least one of any combination of A, B, or C (with each species in singular or multiple possibility).
  • amino acid residue or “amino acid” includes reference to an amino acid that is incorporated into a protein, polypeptide, and/or peptide.
  • polypeptide includes any polymer of amino acids or amino acid residues.
  • polypeptide sequence refers to a series of amino acids or amino acid residues which physically comprise a polypeptide.
  • a “protein” is a macromolecule comprising one or more polypeptides or polypeptide “chains.”
  • a “peptide” is a small polypeptide of sizes less than about a total of 15 to 20 amino acid residues.
  • amino acid sequence refers to a series of amino acids or amino acid residues which physically comprise a peptide or polypeptide depending on the length. Unless otherwise indicated, polypeptide and protein sequences disclosed herein are written from left to right representing their order from an amino terminus to a carboxy terminus.
  • amino acid amino acid residue
  • amino acid sequence amino acid sequence
  • amino acid sequence amino acid sequence
  • proteins include naturally occurring amino acids (including L and D isosteriomers) and, unless otherwise limited, also include known analogs of natural amino acids that can function in a similar manner as the common natural amino acids, such as selenocysteine, pyrrolysine, N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine, pyroglutamic acid, and selenomethionine (see e.g.
  • a peptide, peptide region, polypeptide region, protein, or molecule refers to a change in the amino acid composition of the peptide, peptide region, polypeptide region, protein, or molecule that does not substantially alter the function and structure of the overall peptide, peptide region, polypeptide region, protein, or molecule (see Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, New York (2nd ed., 1992))).
  • the phrase “derived from” when referring to a polypeptide or polypeptide region means that the polypeptide or polypeptide region comprises highly similar amino acid sequences originally found in a “parental” protein and which may now comprise certain amino acid residue additions, deletions, truncations, rearrangements, or other alterations relative to the original polypeptide or polypeptide region as long as a certain function(s) and a structure(s) of the “parental” molecule are substantially conserved.
  • the skilled worker will be able to identify a parental molecule from which a polypeptide or polypeptide region was derived using techniques known in the art, e.g., protein sequence alignment software.
  • terminals refers to the regional boundaries of that region, regardless of whether additional amino acid residues are linked by peptide bonds outside of that region. In other words, the terminals of the polypeptide region regardless of whether that region is fused to other peptides or polypeptides.
  • a fusion protein comprising two proteinaceous regions, e.g., a binding region comprising a peptide or polypeptide and a Shiga toxin effector polypeptide
  • a fusion protein may have a Shiga toxin effector polypeptide region with a carboxy terminus ending at amino acid residue 251 of the Shiga toxin effector polypeptide region despite a peptide bond involving residue 251 to an amino acid residue at position 252 representing the beginning of another proteinaceous region, e.g., the binding region.
  • the carboxy terminus of the Shiga toxin effector polypeptide region refers to residue 251, which is not a terminus of the fusion protein but rather represents an internal, regional boundary.
  • terminal amino terminus
  • amino terminus amino terminus
  • carboxy terminus are used to refer to the boundaries of polypeptide regions, whether the boundary is a physically terminus or an internal, position embedded within a larger, continuous polypeptide chain.
  • wild-type generally refers to a naturally occurring, Shiga toxin protein sequence(s) found in a living species, such as, e.g., a pathogenic bacterium, wherein that Shiga toxin protein sequence(s) is one of the most frequently occurring variants. This is in contrast to infrequently occurring Shiga toxin protein sequences that, while still naturally occurring, are found in less than one percent of individual organisms of a given species when sampling a statistically powerful number of naturally occurring individual organisms of that species which comprise at least one Shiga toxin protein variant.
  • a clonal expansion of a natural isolate outside its natural environment does not alter the naturally occurring requirement as long as the clonal expansion does not introduce new sequence variety not present in naturally occurring populations of that species and/or does not change the relative proportions of sequence variants to each other.
  • association refers to the state of two or more components of a molecule being joined, attached, connected, or otherwise coupled to form a single molecule or the act of making two molecules associated with each other to form a single molecule by creating an association, linkage, attachment, and/or any other connection between the two molecules.
  • linked may refer to two or more components associated by one or more atomic interactions such that a single molecule is formed and wherein the atomic interactions may be covalent and/or non-covalent.
  • covalent associations between two components include peptide bonds and cysteine-cysteine disulfide bonds.
  • non-covalent associations between two molecular components include ionic bonds.
  • the term “linked” refer to two or more molecular components associated by one or more atomic interactions such that a single molecule is formed and wherein the atomic interactions include at least one covalent bond.
  • the term “linking” refers to the act of creating a linked molecule as described above.
  • the term “fused” refers to two or more proteinaceous components associated by at least one covalent bond which is a peptide bond, regardless of whether the peptide bond involves the participation of a carbon atom of a carboxyl acid group or involves another carbon atom, such as, e.g., the ⁇ -carbon, ⁇ -carbon, ⁇ -carbon, ⁇ -carbon, etc.
  • Non-limiting examples of two proteinaceous components fused together include, e.g., an amino acid, peptide, or polypeptide fused to a polypeptide via a peptide bond such that the resulting molecule is a single, continuous polypeptide.
  • fused refers to the act of creating a fused molecule as described above, such as, e.g., a fusion protein generated from the recombinant fusion of genetic regions which when translated produces a single proteinaceous molecule.
  • bispecific will be understood to include molecules which bind different extracellular target biomolecules or which bind the same extracellular target biomolecule at two or more different epitopes, whether non-overlapping or overlapping epitopes (e.g. a bivalent biparatopic molecule).
  • the terms “expressed,” “expressing,” or “expresses,” and grammatical variants thereof, refer to translation of a polynucleotide or nucleic acid into a protein.
  • the expressed protein may remain intracellular, become a component of the cell surface membrane or be secreted into an extracellular space.
  • cells which express a significant amount of an extracellular target biomolecule at least one cellular surface are “target positive cells” or “target+cells” and are cells physically coupled to the specified, extracellular target biomolecule.
  • target positive cells or “target+cells” and are cells physically coupled to the specified, extracellular target biomolecule.
  • target biomolecule is defined below.
  • the symbol “a” is shorthand for an immunoglobulin-type binding region capable of binding to the biomolecule following the symbol.
  • the symbol “ ⁇ ” is used to refer to the functional characteristic of an immunoglobulin-type binding region based on its ability to bind to the biomolecule following the symbol with a binding affinity described by a dissociation constant (K D ) of 10 ⁇ 5 or less.
  • V H or V L domain respectively refer to any antibody V H or V L domain (e.g. a human V H or V L domain) as well as any derivative thereof retaining at least qualitative antigen binding ability of the corresponding native antibody (e.g. a humanized V H or V L domain derived from a native murine V H or V L domain).
  • a V H or V L domain consists of a “framework” region interrupted by the three CDRs or ABRs. The framework regions serve to align the CDRs or ABRs for specific binding to an epitope of an antigen.
  • both V H and V L domains comprise the following framework (FR) and CDR regions or ABR regions; FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; or, similarly, FR1, ABR1, FR2, ABR2, FR3, ABR3, and FR4.
  • FR framework
  • HCDR1,” “HCDR2,” or “HCDR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V H domain
  • LCDR1,” “LCDR2,” and “LCDR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V L domain.
  • HABR1 As used herein, the terms “HABR1,” “HABR2,” or “HABR3” are used to refer to ABRs 1, 2, or 3, respectively, in a V H domain, and the terms “LABR1,” “LABR2,” or “LABR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V L domain.
  • V H H fragments IgNARs of cartilaginous fish.
  • the terms “HCDR1,” “HCDR2,” or “HCDR3” may be used to refer to CDRs 1, 2, or 3, respectively, in a single heavy chain variable domain.
  • effector means providing a biological activity, such as cytotoxicity, biological signaling, enzymatic catalysis, subcellular routing, and/or intermolecular binding resulting in an allosteric effect(s) and/or the recruitment of one or more factors.
  • a Shiga toxin effector polypeptide provides one or more biological activities present in a Shiga toxin, Shiga toxin component, and/or fragment thereof.
  • the phrases “Shiga toxin effector polypeptide,” “Shiga toxin effector polypeptide region,” and “Shiga toxin effector region” refer to a polypeptide or polypeptide region derived from at least one Shiga toxin A Subunit of a member of the Shiga toxin family wherein the polypeptide or polypeptide region is capable of exhibiting at least one Shiga toxin function.
  • heterologous as describing a binding region means the binding region is from a different source than a naturally occurring Shiga toxin, e.g. a heterologous binding region which is a polypeptide is polypeptide not naturally found as part of any native Shiga toxin.
  • the term “heterologous” as describing a CD8+ T-cell epitope means the CD8+ T-cell epitope is from a different source than (1) an A Subunit of a naturally occurring Shiga toxin, e.g. a heterologous polypeptide is not naturally found as part of any A Subunit of a native Shiga toxin and (2) a prior art Shiga toxin effector polypeptide.
  • the term “heterologous” with regard to a CD8+ T-cell epitope-peptide refers to a peptide sequence which did not initially occur in a cell-targeting molecule to be modified (parental molecule), but which was added to the molecule, whether added via the processes of embedding, fusion, insertion, and/or amino acid substitution as described herein, or by any other engineering means to create a modified cell-targeting molecule.
  • the result is a modified cell-targeting molecule comprising a CD8+ T-cell epitope-peptide which is foreign to the original, unmodified cell-targeting molecule, i.e. the CD8+ T-cell epitope was not present in the unmodified cell-targeting molecule (parental molecule).
  • the ‘heterologous’ CD8+ T-cell epitope may be autogenous or heterologous with respect to the binding region.
  • the heterologous, CD8+ T-cell epitope is also heterologous to the binding region component(s) of the cell-targeting molecule, e.g. a heterologous epitope is one that is not required for the binding activity of the binding region and is not part of the structure of the minimum binding region structure which provides the binding activity of the binding region.
  • a CD8+ T-cell epitope not natively present in an immunoglobulin is heterologous to an immunoglobulin-type binding region derived from that immunoglobulin if it is not required for the binding activity of the immunoglobulin-type binding region and is not part of the structure of the minimum binding region structure which provides the binding activity of the immunoglobulin-type binding region.
  • heterologous as describing a CD8+ T-cell epitope means of a different source than (1) an A Subunit of a naturally occurring Shiga toxin and (2) the binding region of the cell-targeting molecule comprising the heterologous component.
  • a heterologous, CD8+ T-cell epitope-peptide of the cell-targeting molecule of the present invention is an CD8+ T-cell epitope-peptide not already present in a wild-type Shiga toxin A1 fragment; a naturally occurring Shiga toxin A1 fragment: and/or a prior art Shiga toxin effector polypeptide used as a component of the cell-targeting molecule.
  • the phrase “intercellular engagement” by a CD8+ immune cell refers to a CD8+ immune cell responding to different cell (for example, by sensing the other is displaying one or more pMHC Is) in fashion indicative of the activation of an immune response by the CD8+ immune cell, such as, e.g., responses involved in killing the other cell, recruiting and activating other immune cells (e.g. cytokine secretion), maturation of the CD8+ immune cell, activation of the CD8+ immune cell, etc.
  • an immune response by the CD8+ immune cell such as, e.g., responses involved in killing the other cell, recruiting and activating other immune cells (e.g. cytokine secretion), maturation of the CD8+ immune cell, activation of the CD8+ immune cell, etc.
  • CD8+ T-cell epitope delivering when describing a functional activity of a molecule means that a molecule provides the biological activity of localizing within a cell to a subcellular compartment that is competent to result in the proteasomal cleavage of a proteinaceous part of the molecule which comprises a CD8+ T-cell epitope-peptide.
  • the “CD8+ T-cell epitope delivering” function of a molecule can be assayed by observing the MHC presentation of a CD8+ T-cell epitope-peptide cargo of the molecule on a cell surface of a cell exogenously administered the molecule or in which the assay was begun with the cell containing the molecule in one or more of its endosomal compartments.
  • the ability of a molecule to deliver a CD8+ T-cell epitope to a proteasome can be determined where the initial location of the “CD8+ T-cell epitope delivering” molecule is an early endosomal compartment of a cell, and then, the molecule is empirically shown to deliver the epitope-peptide to the proteasome of the cell.
  • a “CD8+ T-cell epitope delivering” ability may also be determined where the molecule starts at an extracellular location and is empirically shown, either directly or indirectly, to deliver the epitope into a cell and to proteasomes of the cell.
  • CD8+ T-cell epitope delivering molecules pass through an endosomal compartment of the cell, such as, e.g. after endocytotic entry into that cell.
  • “CD8+ T-cell epitope delivering” activity may be observed for a molecule starting at an extracellular location whereby the molecule does not enter any endosomal compartment of a cell-instead the “CD8+ T-cell epitope delivering” molecule enters a cell and delivers a T-cell epitope-peptide to proteasomes of the cell, presumably because the “CD8+ T-cell epitope delivering” molecule directed its own routing to a subcellular compartment competent to result in proteasomal cleavage of its CD8+ T-cell epitope-peptide component.
  • a Shiga toxin effector function is a biological activity conferred by a polypeptide region derived from a Shiga toxin A Subunit.
  • Shiga toxin effector functions include promoting cell entry; lipid membrane deformation; promoting cellular internalization; stimulating clathrin-mediated endocytosis; directing intracellular routing to various intracellular compartments such as, e.g., the Golgi, endoplasmic reticulum, and cytosol; directing intracellular routing with a cargo; inhibiting a ribosome function(s); catalytic activities, such as, e.g., N-glycosidase activity and catalytically inhibiting ribosomes; reducing protein synthesis, inducing caspase activation, activating effector caspases, effectuating cytostatic effects, and cytotoxicity.
  • Shiga toxin catalytic activities include, for example, ribosome inactivation, protein synthesis inhibition, N-glycosidase activity, polynucleotide:adenosine glycosidase activity, RNAase activity, and DNAase activity.
  • Shiga toxins are ribosome inactivating proteins (RIPs).
  • RIPs can depurinate nucleic acids, polynucleosides, polynucleotides, rRNA, ssDNA, dsDNA, mRNA (and polyA), and viral nucleic acids (see e.g., Barbieri L et al., Biochem J 286: 1-4 (1992); Barbieri L et al., Nature 372: 624 (1994); Ling J et al., FEBS Lett 345: 143-6 (1994); Barbieri L et al., Biochem J 319: 507-13 (1996); Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20 (1996); Stirpe F et al., FEBSLett 382: 309-12 (1996); Barbieri L et al., Nucleic Acids Res 25: 518-22 (1997); Wang P, Turner N, Nucleic Acids Res 27: 1900-5 (1999); Barbieri L et al., Biochim Biophys Acta 1480
  • Non-limiting examples of assays for Shiga toxin effector activity measure various activities, such as, e.g., protein synthesis inhibitory activity, depurination activity, inhibition of cell growth, cytotoxicity, supercoiled DNA relaxation activity, and nuclease activity.
  • the retention of Shiga toxin effector function refers to being capable of exhibiting a level of Shiga toxin functional activity, as measured by an appropriate quantitative assay with reproducibility, comparable to a wild-type, Shiga toxin effector polypeptide control (e.g. a Shiga toxin A1 fragment) or cell-targeting molecule comprising a wild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin A1 fragment) under the same conditions.
  • Shiga toxin effector polypeptide control e.g. a Shiga toxin A1 fragment
  • cell-targeting molecule comprising a wild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin A1 fragment) under the same conditions.
  • retained Shiga toxin effector function is exhibiting an IC 50 of 10,000 picomolar (pM) or less in an in vitro setting, such as, e.g., by using an assay known to the skilled worker and/or described herein.
  • IC 50 10,000 picomolar (pM) or less in an in vitro setting
  • retained Shiga toxin effector function of cytotoxicity in a target positive cell-kill assay is exhibiting a CD 50 of 1,000 nanomolar (nM) or less, depending on the cell-type and its expression of the appropriate extracellular target biomolecule, as shown, e.g., by using an assay known to the skilled worker and/or described herein.
  • the term “equivalent” with regard to ribosome inhibition means an empirically measured level of ribosome inhibitory activity, as measured by an appropriate quantitative assay with reproducibility, which is reproducibly within 10% or less of the activity of the reference molecule (e.g., the second cell-targeting molecule or third cell-targeting molecule) under the same conditions.
  • the term “equivalent” with regard to cytotoxicity means an empirically measured level of cytotoxicity, as measured by an appropriate quantitative assay with reproducibility, which is reproducibly within 10% or less of the activity of the reference molecule (e.g., the second cell-targeting molecule or third cell-targeting molecule) under the same conditions.
  • the term “attenuated” with regard to cytotoxicity means a molecule exhibits or exhibited a CD 50 between 10-fold to 100-fold of a CD 50 exhibited by a reference molecule under the same conditions.
  • a failure to detect activity in Shiga toxin effector function may be due to improper expression, polypeptide folding, and/or polypeptide stability rather than a lack of cell entry, subcellular routing, and/or enzymatic activity.
  • Assays for Shiga toxin effector functions may not require much cell-targeting molecule of the invention to measure significant amounts of Shiga toxin effector function activity.
  • an underlying cause of low or no effector function is determined empirically to relate to protein expression or stability, one of skill in the art may be able to compensate for such factors using protein chemistry and molecular engineering techniques known in the art, such that a Shiga toxin functional effector activity may be restored and measured.
  • improper cell-based expression may be compensated for by using different expression control sequences; improper polypeptide folding and/or stability may benefit from stabilizing terminal sequences, or compensatory mutations in non-effector regions which stabilize the three-dimensional structure of the protein, etc.
  • Shiga toxin effector regions or polypeptides may be analyzed for any level of those Shiga toxin effector functions, such as for being within a certain-fold activity of a wild-type Shiga toxin effector polypeptide.
  • Examples of meaningful activity differences are, e.g., Shiga toxin effector regions that have 1000-fold or 100-fold or less the activity of a wild-type Shiga toxin effector polypeptide; or that have 3-fold to 30-fold or more activity compared to a functional knock-down or knockout Shiga toxin effector polypeptide.
  • Shiga toxin effector functions are not easily measurable, e.g. subcellular routing functions. For example, there is no routine, quantitative assay to distinguish whether the failure of a Shiga toxin effector polypeptide to be cytotoxic and/or deliver a heterologous, CD8+ T-cell epitope is due to improper subcellular routing, but at a time when tests are available, then Shiga toxin effector polypeptides may be analyzed for any significant level of subcellular routing as compared to the appropriate wild-type Shiga toxin effector polypeptide.
  • a Shiga toxin effector polypeptide component of a cell-targeting molecule of the present invention exhibits cytotoxicity comparable or equivalent to a wild-type Shiga toxin A Subunit construct
  • the subcellular routing activity level is inferred to be comparable or equivalent, respectively, to the subcellular routing activity level of a wild-type Shiga toxin A Subunit construct at least under the conditions tested.
  • Shiga toxin effector polypeptides and/or cell-targeting molecules comprising Shiga toxin effector polypeptides may be analyzed for any level of those Shiga toxin effector functions, such as a being within 1000-fold or 100-fold or less the activity of a wild-type Shiga toxin effector polypeptide or exhibiting 3-fold to 30-fold or greater activity as compared to a functional knockout, Shiga toxin effector polypeptide.
  • Sufficient subcellular routing may be merely deduced by observing a cell-targeting molecule's Shiga toxin cytotoxic activity levels in cytotoxicity assays, such as, e.g., cytotoxicity assays based on T-cell epitope presentation or based on a Shiga toxin effector function involving a cytosolic and/or endoplasmic reticulum-localized, target substrate.
  • cytotoxicity assays such as, e.g., cytotoxicity assays based on T-cell epitope presentation or based on a Shiga toxin effector function involving a cytosolic and/or endoplasmic reticulum-localized, target substrate.
  • the retention of “significant” Shiga toxin effector function refers being capable of exhibiting a level of Shiga toxin functional activity, as measured by an appropriate quantitative assay with reproducibility comparable to a wild-type Shiga toxin effector polypeptide control (e.g. a Shiga toxin A1 fragment).
  • significant Shiga toxin effector function is exhibiting an IC 50 of 300 pM or less depending on the source of the ribosomes used in the assay (e.g. a bacterial, archaeal, or eukaryotic (algal, fungal, plant, or animal) source).
  • the term “reasonable activity” refers to exhibiting at least a moderate level (e.g. within 1-fold to 1,000-fold) of Shiga toxin effector activity as defined herein in relation to a molecule comprising a naturally occurring Shiga toxin, wherein the Shiga toxin effector activity is selected from the group consisting of: internalization efficiency, subcellular routing efficiency to the cytosol, delivered epitope presentation by a target cell(s), ribosome inhibition, and cytotoxicity.
  • a reasonable level of Shiga toxin effector activity includes being within 1,000-fold of a wild-type, Shiga toxin construct, such as, e.g., exhibiting a CD 50 of 500 nM or less when a wild-type Shiga toxin construct exhibits a CD 50 of 0.5 nM (e.g. a cell-targeting molecule comprising a wild-type Shiga toxin A1 fragment).
  • the term “optimal” refers to a level of Shiga toxin catalytic domain mediated cytotoxicity that is within 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold of the cytotoxicity of a molecule comprising wild-type Shiga toxin A1 fragment (e.g. a Shiga toxin A Subunit or certain truncated variants thereof) and/or a naturally occurring Shiga toxin.
  • Shiga toxin effector polypeptides may be equally or more effective than using wild-type Shiga toxin effector polypeptides because the highest potency variants might exhibit undesirable effects which are minimized or reduced in reduced cytotoxic-potency variants.
  • Wild-type Shiga toxins are very potent, being able to kill an intoxicated cell after only one toxin molecule has reached the cytosol of the intoxicated cell or perhaps after only forty toxin molecules have been internalized into the intoxicated cell.
  • Shiga toxin effector polypeptides with even considerably reduced Shiga toxin effector functions, such as, e.g., subcellular routing or cytotoxicity, as compared to wild-type Shiga toxin effector polypeptides may still be potent enough for practical applications, such as, e.g., applications involving targeted cell-killing, heterologous epitope delivery, and/or detection of specific cells and their subcellular compartments.
  • certain reduced-activity Shiga toxin effector polypeptides may be particularly useful for delivering cargos (e.g. an additional exogenous material or T-cell epitope) to certain intracellular locations or subcellular compartments of target cells.
  • selective cytotoxicity with regard to the cytotoxic activity of a molecule refers to the relative level of cytotoxicity between a biomolecule target positive cell population (e.g. a targeted cell-type) and a non-targeted bystander cell population (e.g. a biomolecule target negative cell-type), which can be expressed as a ratio of the half-maximal cytotoxic concentration (CD 50 ) for a targeted cell-type over the CD 50 for an untargeted cell-type to provide a metric of cytotoxic selectivity or indication of the preferentiality of killing of a targeted cell versus an untargeted cell.
  • a biomolecule target positive cell population e.g. a targeted cell-type
  • a non-targeted bystander cell population e.g. a biomolecule target negative cell-type
  • the cell surface representation and/or density of a given extracellular target biomolecule may influence the applications for which certain cell-targeting molecules of the present invention may be most suitably used. Differences in cell surface representation and/or density of a given target biomolecule between cells may alter, both quantitatively and qualitatively, the efficiency of cellular internalization and/or cytotoxicity potency of a given cell-targeting molecule of the present invention.
  • the cell surface representation and/or density of a given target biomolecule can vary greatly among target biomolecule positive cells or even on the same cell at different points in the cell cycle or cell differentiation.
  • the total cell surface representation of a given target biomolecule and/or of certain extracellular epitopes of a given target biomolecule on a particular cell or population of cells may be determined using methods known to the skilled worker, such as methods involving fluorescence-activated cell sorting (FACS) flow cytometry.
  • FACS fluorescence-activated cell sorting
  • the phrase “target biomolecule natively present on the surface of a cell” means a cell expresses the target biomolecule using its own internal machinery and localizes the target biomolecule to a cellular surface using its own internal machinery such that the target biomolecule is physically coupled to said cell and at least a part of the target biomolecule is accessible from an extracellular space, i.e. on the surface of a cell.
  • the terms “disrupted,” “disruption,” or “disrupting,” and grammatical variants thereof, with regard to a polypeptide region or feature within a polypeptide refers to an alteration of at least one amino acid within the region or composing the disrupted feature.
  • Amino acid alterations include various mutations, such as, e.g., a deletion, inversion, insertion, or substitution which alter the amino acid sequence of the polypeptide. Amino acid alterations also include chemical changes, such as, e.g., the alteration one or more atoms in an amino acid functional group or the addition of one or more atoms to an amino acid functional group.
  • de-immunized means reduced antigenic and/or immunogenic potential after administration to a chordate as compared to a reference molecule, such as, e.g., a wild-type peptide region, polypeptide region, or polypeptide. This includes a reduction in overall antigenic and/or immunogenic potential despite the introduction of one or more, de novo, antigenic and/or immunogenic epitopes as compared to a reference molecule.
  • “de-immunized” means a molecule exhibited reduced antigenicity and/or immunogenicity after administration to a mammal as compared to a “parental” molecule from which it was derived, such as, e.g., a wild-type Shiga toxin A1 fragment.
  • the de-immunized, Shiga toxin effector polypeptide of the present invention is capable of exhibiting a relative antigenicity compared to a reference molecule which is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than the antigenicity of the reference molecule under the same conditions measured by the same assay, such as, e.g., an assay known to the skilled worker and/or described herein like a quantitative ELISA or Western blot analysis.
  • the de-immunized, Shiga toxin effector polypeptide of the present invention is capable of exhibiting a relative immunogenicity compared to a reference molecule which is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater than the immunogenicity of the reference molecule under the same conditions measured by the same assay, such as, e.g., an assay known to the skilled worker and/or described herein like a quantitative measurement of anti-molecule antibodies produced in a mammal(s) after receiving parenteral administration of the molecule at a given time-point.
  • the relative immunogenicities of exemplary cell-targeting molecules may be determined using an assay for adaptive immune reactions (e.g. in vivo antibody responses) or innate immune reactions to the cell-targeting molecules after repeat, parenteral administrations over periods of days, weeks, and/or months.
  • the relative immunogenicities of exemplary cell-targeting molecules were determined using an assay for in vivo antibody responses to the cell-targeting molecules after repeat, parenteral administrations over periods of many.
  • CD8+ T-cell hyper-immunized means that the cell-targeting molecule, when present inside a nucleated, chordate cell within a living chordate, has an increased antigenic and/or immunogenic potential regarding CD8+ T-cell antigenicity or immunogenicity when compared to the same molecule that lacks any heterologous, CD8+ T-cell epitope-peptide.
  • T-cell epitope or T-cell epitope-peptide component of a polypeptide refers to the internal replacement of one or more amino acids within a polypeptide region with different amino acids in order to generate a new polypeptide sequence sharing the same total number of amino acid residues with the starting polypeptide region.
  • the term “embedded” does not include any external, terminal fusion of any additional amino acid, peptide, or polypeptide component to the starting polypeptide nor any additional internal insertion of any additional amino acid residues, but rather includes only substitutions for existing amino acids.
  • the internal replacement may be accomplished merely by amino acid residue substitution or by a series of substitutions, deletions, insertions, and/or inversions. If an insertion of one or more amino acids is used, then the equivalent number of proximal amino acids must be deleted next to the insertion to result in an embedded T-cell epitope.
  • inserted refers to the insertion of one or more amino acids internally within a polypeptide resulting in a new polypeptide having an increased number of amino acids residues compared to the starting polypeptide.
  • inserted and grammatical variants thereof with regard to a T-cell epitope contained within a polypeptide refers to the insertion of one or more amino acids within a polypeptide resulting in a new polypeptide sequence having an increased number of amino acids residues compared to the starting polypeptide.
  • proximal to an amino terminus refers to a distance wherein at least one amino acid residue of the Shiga toxin effector polypeptide region is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more, e.g., up to 18-20 amino acid residues, of an amino terminus of the cell-targeting molecule as long as the cell-targeting molecule is capable of exhibiting the appropriate level of Shiga toxin effector functional activity noted herein (e.g., a certain level of cytotoxic potency).
  • any amino acid residue(s) fused amino-terminal to the Shiga toxin effector polypeptide should not reduce any Shiga toxin effector function (e.g., by sterically hindering a structure(s) near the amino terminus of the Shiga toxin effector polypeptide region) such that a functional activity of the Shiga toxin effector polypeptide is reduced below the appropriate activity level required herein.
  • the phrase “more proximal to an amino terminus” with reference to the position of a Shiga toxin effector polypeptide region within a cell-targeting molecule of the present invention as compared to another component refers to a position wherein at least one amino acid residue of the amino terminus of the Shiga toxin effector polypeptide is closer to the amino terminus of a linear, polypeptide component of the cell-targeting molecule of the present invention as compared to the other referenced component.
  • the phrase “active enzymatic domain derived from one A Subunit of a member of the Shiga toxin family” refers to having the ability to inhibit protein synthesis via a catalytic ribosome inactivation mechanism.
  • the enzymatic activities of naturally occurring Shiga toxins may be defined by the ability to inhibit protein translation using assays known to the skilled worker, such as, e.g., in vitro assays involving RNA translation in the absence of living cells or in vivo assays involving RNA translation in a living cell.
  • the potency of a Shiga toxin enzymatic activity may be assessed directly by observing N-glycosidase activity toward ribosomal RNA (rRNA), such as, e.g., a ribosome nicking assay, and/or indirectly by observing inhibition of ribosome function and/or protein synthesis.
  • rRNA ribosomal RNA
  • the term “Shiga toxin A1 fragment region” refers to a polypeptide region consisting essentially of a Shiga toxin A1 fragment and/or derived from a Shiga toxin A1 fragment of a Shiga toxin.
  • terminal refers generally to the last amino acid residue of a polypeptide chain of the cell-targeting molecule (e.g., a single, continuous polypeptide chain).
  • a cell-targeting molecule may comprise more than one polypeptide or protein, and, thus, a cell-targeting molecule of the present invention may comprise multiple amino-terminals and carboxy-terminals.
  • amino terminus of a cell-targeting molecule may be defined by the first amino acid residue ofa polypeptide chain representing the amino-terminal end of the polypeptide, which is generally characterized by a starting, amino acid residue which does not have a peptide bond with any amino acid residue involving the primary amino group of the starting amino acid residue or involving the equivalent nitrogen for starting amino acid residues which are members of the class of N-alkylated alpha amino acid residues.
  • the “carboxy terminus” of a cell-targeting molecule may be defined by the last amino acid residue of a polypeptide chain representing the carboxyl-terminal end of the polypeptide, which is generally characterized by a final, amino acid residue which does not have any amino acid residue linked by a peptide bond to the alpha-carbon of its primary carboxyl group.
  • the phrase “Shiga toxin A1 fragment derived region” refers to all or part of a Shiga toxin effector polypeptide wherein the region consists of a polypeptide homologous to a naturally occurring Shiga toxin A1 fragment or truncation thereof, such as, e.g., a polypeptide consisting of or comprising amino acids 75-239 of SLT-1A (SEQ ID NO: 1), 75-239 of StxA (SEQ ID NO:2), or 77-238 of (SEQ ID NO:3) or the equivalent region in another A Subunit of a member of the Shiga toxin family.
  • the carboxy-terminus of a “Shiga toxin A1 fragment derived region” is defined, relative to a naturally occurring Shiga toxin A1 fragment, (1) as ending with the carboxy-terminal amino acid residue sharing homology with a naturally occurring, Shiga toxin A1 fragment; (2) as ending at the junction of the A1 fragment and the A2 fragment; (3) as ending with a furin-cleavage site or disrupted furin-cleave site; and/or (4) as ending with a carboxy-terminal truncation of a Shiga toxin A1 fragment, i.e. the carboxy-terminal amino acid residue sharing homology with a naturally occurring, Shiga toxin A1 fragment.
  • the phrase “carboxy terminus region of a Shiga toxin A1 fragment” refers to a polypeptide region derived from a naturally occurring Shiga toxin A1 fragment, the region beginning with a hydrophobic residue (e.g., V236 of StxA-A1 and SLT-1A1, and V235 of SLT-2A1) that is followed by a hydrophobic residue and the region ending with the furin-cleavage site conserved among Shiga toxin A1 fragment polypeptides and ending at the junction between the A1 fragment and the A2 fragment in native, Shiga toxin A Subunits.
  • a hydrophobic residue e.g., V236 of StxA-A1 and SLT-1A1, and V235 of SLT-2A1
  • the carboxy-terminal region of a Shiga toxin A1 fragment includes a peptidic region derived from the carboxy terminus of a Shiga toxin A1 fragment polypeptide, such as, e.g., a peptidic region comprising or consisting essentially of the carboxy terminus of a Shiga toxin A1 fragment.
  • Non-limiting examples of peptidic regions derived from the carboxy terminus of a Shiga toxin A1 fragment include the amino acid residue sequences natively positioned from position 236 to position 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, or 251 in Shiga toxin A subunit variants (SEQ ID NOs: 1-2 and 4-6); and from position 235 to position 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 in SLT-2A variants (SEQ ID NOs: 3 and 7-18).
  • proximal to the carboxy terminus of an A1 fragment polypeptide refers to being within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues from the amino acid residue defining the last residue of the Shiga toxin A1 fragment polypeptide.
  • the phrase “sterically covers the carboxy terminus of the A1 fragment-derived region” includes any molecular moiety of a size of 4.5 kDa or greater (e.g., an immunoglobulin-type binding region) linked and/or fused to an amino acid residue in the carboxy terminus of the A1 fragment-derived region, such as, e.g., the amino acid residue derived from the amino acid residue natively positioned at any one of positions 236 to 251 in Shiga toxin variants (SEQ ID NOs: 1-2 and 4-6) or from 235 to 250 in SLT-2A variants (SEQ ID NOs: 3 and 7-18).
  • the phrase “sterically covers the carboxy terminus of the A1 fragment-derived region” also includes any molecular moiety of a size of 4.5 kDa or greater (e.g., an immunoglobulin-type binding region) linked and/or fused to an amino acid residue in the carboxy terminus of the A1 fragment-derived region, such as, e.g., the amino acid residue carboxy-terminal to the last amino acid A1 fragment-derived region and/or the Shiga toxin effector polypeptide.
  • the phrase “sterically covers the carboxy terminus of the A1 fragment-derived region” also includes any molecular moiety of a size of 4.5 kDa or greater (e.g., an immunoglobulin-type binding region) physically preventing cellular recognition of the carboxy terminus of the A1 fragment-derived region, such as, e.g. recognition by the ERAD machinery of a eukaryotic cell.
  • a binding region such as, e.g., an immunoglobulin-type binding region, that comprises a polypeptide comprising at least forty amino acids and that is linked (e.g., fused) to the carboxy terminus of the Shiga toxin effector polypeptide region comprising an A1 fragment-derived region is a molecular moiety which is “sterically covering the carboxy terminus of the A1 fragment-derived region.”
  • a binding region such as, e.g., an immunoglobulin-type binding region, that comprises a polypeptide comprising at least forty amino acids and that is linked (e.g., fused) to the carboxy terminus of the Shiga toxin effector polypeptide region comprising an A1 fragment-derived region is a molecular moiety “encumbering the carboxy terminus of the A1 fragment-derived region.”
  • the term “A fragment of a member of the Shiga toxin family” refers to the remaining amino-terminal fragment of a Shiga toxin A Subunit after proteolysis by furin at the furin-cleavage site conserved among Shiga toxin A Subunits and positioned between the A1 fragment and the A2 fragment in wild-type Shiga toxin A Subunits.
  • furin-cleavage motif at the carboxy terminus of the A1 fragment region refers to a specific, furin-cleavage motif conserved among Shiga toxin A Subunits and bridging the junction between the A1 fragment and the A2 fragment in naturally occurring, Shiga toxin A Subunits.
  • furin-cleavage site proximal to the carboxy terminus of the A1 fragment region refers to any identifiable, furin-cleavage site having an amino acid residue within a distance of less than 1, 2, 3, 4, 5, 6, 7, or more amino acid residues of the amino acid residue defining the last amino acid residue in the A1 fragment region or A1 fragment derived region, including a furin-cleavage motif located carboxy-terminal of an A1 fragment region or A1 fragment derived region, such as, e.g., at a position proximal to the linkage of the A1 fragment-derived region to another component of the molecule, such as, e.g., a molecular moiety of a cell-targeting molecule of the present invention.
  • the phrase “disrupted furin-cleavage motif” refers to (i) a specific furin-cleavage motif as described herein and (ii) which comprises a mutation and/or truncation that can confer a molecule with a reduction in furin-cleavage as compared to a reference molecule, such as, e.g., a reduction in furin-cleavage reproducibly observed to be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or less (including 100% for no cleavage) than the furin-cleavage of a reference molecule observed in the same assay under the same conditions.
  • the percentage of furin-cleavage as compared to a reference molecule can be expressed as a ratio of cleaved/uncleaved material of the molecule of interest divided by the cleaved/uncleaved material of the reference molecule (see e.g. WO 2015/191764; US 2016/0177284).
  • suitable reference molecules include certain molecules comprising a wild-type Shiga toxin furin-cleavage motif and/or furin-cleavage site as described herein and/or molecules used in the Examples below.
  • furin-cleavage resistant means a molecule or specific polypeptide region thereof exhibits reproducibly less furin cleavage than (i) the carboxy terminus of a Shiga toxin A1 fragment in a wild-type Shiga toxin A Subunit or (ii) the carboxy terminus of the Shiga toxin A1 fragment derived region of construct wherein the naturally occurring furin-cleavage site natively positioned at the junction between the A1 and A2 fragments is not disrupted; as assayed by any available means to the skilled worker, including by using a method described herein.
  • the phrase “active enzymatic domain derived form an A Subunit of a member of the Shiga toxin family” refers to a polypeptide structure having the ability to inhibit protein synthesis via catalytic inactivation of a ribosome based on a Shiga toxin enzymatic activity.
  • the ability of a molecular structure to exhibit inhibitory activity of protein synthesis and/or catalytic inactivation of a ribosome may be observed using various assays known to the skilled worker, such as, e.g., in vitro assays involving RNA translation assays in the absence of living cells or in vivo assays involving the ribosomes of living cells.
  • the enzymatic activity of a molecule based on a Shiga toxin enzymatic activity may be assessed directly by observing N-glycosidase activity toward ribosomal RNA (rRNA), such as, e.g., a ribosome nicking assay, and/or indirectly by observing inhibition of ribosome function, RNA translation, and/or protein synthesis.
  • rRNA ribosomal RNA
  • a “combination” describes a Shiga toxin effector polypeptide comprising two or more sub-regions wherein each sub-region comprises at least one of the following: (1) a disruption in an endogenous epitope or epitope region and (2) a disrupted furin-cleavage motif at the carboxy terminus of a Shiga toxin A1 fragment derived region.
  • multivalent with regard to a cell-targeting molecule refers to an individual target-binding molecule or plurality of target-binding molecules comprising two or more high-affinity binding regions, such as, e.g. a protein comprising two or more binding regions wherein each individual binding region has a dissociation constant of 10 ⁇ 5 to 10 ⁇ 12 moles per liter toward an extracellular part of a target biomolecule.
  • the term “monomeric” with regard to describing a protein and/or proteinaceous molecule refers to a molecule comprising only one polypeptide component consisting of a single, continuous polypeptide, regardless of its secondary or tertiary structure, which may be synthesized by a ribosome from a single polynucleotide template, including a continuous linear polypeptide which later forms a cyclic structure.
  • a multimeric molecule comprises two or more polypeptides (e.g. subunits) which together do not form a single, continuous polypeptide that may be synthesized by a ribosome from a single polynucleotide template.
  • multimeric with regard to describing a protein and/or proteinaceous molecule refers to a molecule that comprises two or more, individual, polypeptide components associated together and/or linked together, such as, e.g., a molecule consisting of two components each of which is its own continuous polypeptide.
  • association or linkage between components of a molecule may include 1) one or more non-covalent interactions; 2) one or more post-translational, covalent interactions; 3) one or more, covalent chemical conjugations; and/or 4) one or more covalent interactions resulting in a single molecule comprising a non-linear polypeptide, such as, e.g., a branched or cyclic polypeptide structure, resulting from the arrangement of the two or more polypeptide components.
  • a non-linear polypeptide such as, e.g., a branched or cyclic polypeptide structure, resulting from the arrangement of the two or more polypeptide components.
  • a molecule comprising two, discontinuous polypeptides as a result of the proteolytic cleavage of one or more peptide bonds in a single, continuous polypeptide synthesized by a ribosome from a single polynucleotide templates is “multimeric” and not “monomeric.”
  • malignant cells e.g. tumor cells
  • malignant tissue loci e.g. tumors
  • this approach could use highly immunogenic neoepitopes (derived from either infectious or non-infectious agents) or highly immunogenic, non-self epitopes derived from non-infectious agents, such as, e.g., tumor-specific antigens, tumor-associated antigens, and molecules from plants, fungi, etc.
  • the present invention exploits the abilities of Shiga toxin A Subunit derived polypeptides to drive their own subcellular routing in order to deliver highly immunogenic, CD8+ T-cell antigens, such as e.g. peptide-epitopes, to the MHC class I presentation system of a chordate cell.
  • the present invention provides various exemplary, Shiga toxin A Subunit derived constructs capable of delivering heterologous, CD8+ T-cell epitopes to the MHC class I system of a target cell resulting in cell-surface presentation of the delivered epitope wherein the Shiga toxin effector polypeptide components comprise a combination of mutations providing (1) de-immunization, (2) a reduction in protease sensitivity, and/or (3) an embedded. T-cell epitope(s). Certain peptide-epitopes presented in complexes with MHC class I molecules on a cellular surface can signal CD8+ effector T-cells to kill the presenting cell as well as stimulate other immune responses in the local area.
  • the present invention provides Shiga toxin A Subunit derived, cell-targeting molecules which kill specific target cells, such as, e.g., via presentation of certain CD8+ T-cell epitope-peptides by the MHC class I pathway.
  • the cell-targeting molecules of the present invention may be utilized, e.g., as cell-killing molecules, cytotoxic therapeutics, therapeutic delivery agents, and diagnostic molecules.
  • the cell-targeting molecules of the present invention each comprise 1) a cell-targeting binding region, 2) a Shiga toxin A Subunit effector polypeptide, and 3) a CD8+ T-cell epitope-peptide which is not embedded or inserted in the Shiga toxin A1 fragment region and which is heterologous to Shiga toxin A Subunits and the binding region of the molecule.
  • This system is modular, in that any number of diverse, CD8+ T-cell epitope-peptides may be used as cargos for delivery to the MHC class I presentation pathway of target cells by the cell-targeting molecules of the present invention.
  • Shiga toxin effector polypeptides and cell-targeting molecules of the present invention comprise at least one, Shiga toxin effector polypeptide derived from wild-type Shiga toxin A Subunits but comprise one or more structural modifications, such as, e.g., a mutation like a truncation and/or amino acid residue substitution(s).
  • the present invention involves the engineering of improved, Shiga toxin A Subunit effector polypeptides comprising the combination of two or more of the following Shiga toxin effector polypeptide sub-regions; (1) a de-immunized sub-region, (2) a protease-cleavage resistant sub-region near the carboxy-terminus of a Shiga toxin A1 fragment region, and (3) a T-cell epitope-peptide embedded or inserted sub-region.
  • a Shiga toxin effector polypeptide of a cell-targeting molecule of the present invention is a polypeptide derived from a Shiga toxin A Subunit member of the Shiga toxin family that is capable of exhibiting one or more Shiga toxin functions.
  • Shiga toxin effector polypeptides that are suitable for use in the present invention or to use as parental polypeptides to be modified into a Shiga toxin effector polypeptide of the present invention using techniques known the art (see e.g., Cheung M et al., Mol Cancer 9: 28 (2010); WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452: WO 2015/191764).
  • Shiga toxin functions include, e.g., promoting cell entry, increasing cellular internalization, directing retrograde transport, directing subcellular routing, directing subcellular routing from an endosomal compartment to the cytosol, avoiding intracellular degradation, catalytically inactivating ribosomes, effectuating cytotoxicity, and effectuating cytostatic effects.
  • the Shiga toxin family of protein toxins is composed of various naturally occurring toxins which are structurally and functionally related, e.g., Shiga toxin, Shiga-like toxin I, and Shiga-like toxin 2 (Johannes L, R6mer W, Nat Rev Microbiol 8: 105-16 (2010)).
  • Holotoxin members of the Shiga toxin family contain targeting domains that preferentially bind a specific glycosphingolipid present on the surface of some host cells and an enzymatic domain capable of permanently inactivating ribosomes once inside a cell (Johannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)).
  • Stx, SLT-1 and SLT-2 display indistinguishable enzymatic activity in cell free systems (Head S et al., J Biol Chem 266: 3617-21 (1991); Tesh V et al., Infect Immun 61: 3392-402 (1993); Brigotti M et al., Toxicon 35: 1431-7 (1997)).
  • the Shiga toxin family encompasses true Shiga toxin (Stx) isolated from S. dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stx1 or SLT-1 or Slt-I) isolated from serotypes of enterohemorrhagic E, coli , and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2) isolated from serotypes of enterohemorrhagic E. coli .
  • SLT1 differs by only one amino acid residue from Stx, and both have been referred to as Verocytotoxins or Verotoxins (VTs) (O'Brien A. Curr Top Microbiol Immunol 180: 65-94 (1992)).
  • SLT1 and SLT2 variants are only about 53-60% similar to each other at the primary amino acid sequence level, they share mechanisms of enzymatic activity and cytotoxicity common to the members of the Shiga toxin family (Johannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)). Over 39 different Shiga toxins have been described, such as the defined subtypes Stx1a, Stx1c, Stx1d, Stx1e, Stx2a-g, and Stx2dact (Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012); Probert W et al., J Clin Microbiol 52: 2346-51 (2014)).
  • Shiga toxin Members of the Shiga toxin family are not naturally restricted to any bacterial species because Shiga-toxin-encoding genes can spread among bacterial species via horizontal gene transfer (Strauch E et al., Infect Immun 69: 7588-95 (2001); Bielaszewska M et al., Appl Environ Micrbiol 73: 3144-50 (2007); Zhaxybayeva O, Doolittle W, Curr Biol 21: R242-6 (2011); Kruger A, Lucchesi P, Microbiology 161: 451-62 (2015)).
  • interspecies transfer a Shiga toxin was discovered in a strain of A.
  • Shiga toxin amino acid sequence is presumed to be capable of developing slight sequence variations due to genetic drift and/or selective pressure while still maintaining a mechanism of cytotoxicity common to members of the Shiga toxin family (see Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012)).
  • the Shiga toxin effector polypeptide of the present invention is de-immunized, such as, e.g., as compared to a wild-type Shiga toxin, wild-type Shiga toxin polypeptide, and/or Shiga toxin effector polypeptide comprising only wild-type polypeptide sequences.
  • the de-immunized, Shiga toxin effector polypeptides of the present invention each comprise a disruption of at least one, putative, endogenous, epitope region in order to reduce the antigenic and/or immunogenic potential of the Shiga toxin effector polypeptide after administration of the polypeptide to a chordate.
  • Shiga toxin effector polypeptide and/or Shiga toxin A Subunit polypeptide can be de-immunized by a method described herein, described in WO 2015/113005, WO 2015/113007, and/or WO 2016/196344, and/or known to the skilled worker, wherein the resulting molecule retains one or more Shiga toxin A Subunit functions.
  • Shiga toxin effector polypeptides of the present invention comprise or consist essentially of a polypeptide derived from a Shiga toxin A Subunit dissociated from any form of its native Shiga toxin B Subunit.
  • the Shiga toxin effector polypeptides of the present invention do not comprise the cell-targeting domain of a Shiga toxin B Subunit.
  • Archetypal Shiga toxins naturally target the human cell-surface receptors globotriaosylceramide (Gb3, Gb3Cer, or CD77) and globotetraosylceramide (Gb4 or Gb4Cer) via the Shiga toxin B Subunit, which severely limits potential applications by restricting targeting cell-types and potentially unwanted targeting of vascular endothelial cells, certain renal epithelial cells, and/or respiratory epithelial cells (Tesh V et al., Infect Immun 61: 3392-402 (1993); Ling H et al., Biochemistry 37: 1777-88 (1998); Bast D et al., Mol Microbiol 32: 953-60 (1999); Rutjes N et al., Kidney Int 62: 832-45 (2002);
  • Gb3 and Gb4 are a common, neutral sphingolipid present on the extracellular leaflet of cell membranes of various, healthy cell-types, such as polymorphonuclear leukocvtes and human endothelial cells from various vascular beds.
  • the cell-targeting molecules of the present invention do not comprise any polypeptide comprising or consisting essentially of a functional binding domain of a native Shiga toxin B subunit. Rather, the Shiga toxin effector polypeptides of the present invention may be functionally associated with heterologous binding regions to effectuate cell targeting.
  • a Shiga toxin effector polypeptide of the present invention may comprise or consist essentially of a full-length Shiga toxin A Subunit (e.g. any one of SEQ ID NOs: 1-18), noting that naturally occurring Shiga toxin A Subunits may comprise precursor forms containing signal sequences of about 22 amino acids at their amino-terminals which are removed to produce mature Shiga toxin A Subunits and are recognizable to the skilled worker.
  • the Shiga toxin effector polypeptide of the invention comprises or consists essentially of a truncated Shiga toxin A Subunit which is shorter than a full-length Shiga toxin A Subunit, such as, e.g., a truncation known in the art (see e.g., WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO 2015/191764).
  • the Shiga toxin effector polypeptide of the present invention comprises a disruption of an endogenous epitope or epitope region, such as, e.g., a B-cell and/or CD4+ T-cell epitope.
  • the Shiga toxin effector polypeptide of the present invention comprises a disruption of at least one, endogenous, epitope region described herein, wherein the disruption reduces the antigenic and/or immunogenic potential of the Shiga toxin effector polypeptide after administration of the polypeptide to a chordate, and wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin A Subunit functions, such as, e.g., a significant level of Shiga toxin cytotoxicity.
  • disrupted or “disruption” as used herein with regard to an epitope region refers to the deletion of at least one amino acid residue in an epitope region, inversion of two or more amino acid residues where at least one of the inverted amino acid residues is in an epitope region, insertion of at least one amino acid into an epitope region, and a substitution of at least one amino acid residue in an epitope region.
  • An epitope region disruption by mutation includes amino acid substitutions with non-standard amino acids and/or non-natural amino acids.
  • Epitope regions may alternatively be disrupted by mutations comprising the modification of an amino acid by the addition of a covalently-linked chemical structure which masks at least one amino acid in an epitope region, see, e.g. PEGylation (see Zhang C et al., BioDrugs 26: 209-15 (2012), small molecule adjuvants (Flower D, Expert Opin Drug Discov 7: 807-17 (2012), and site-specific albumination (Lim S et al., J Control Release 207-93 (2015)).
  • Certain epitope regions and disruptions are indicated herein by reference to specific amino acid positions of native Shiga toxin A Subunits provided in the Sequence Listing, noting that naturally occurring Shiga toxin A Subunits may comprise precursor forms containing signal sequences of about 22 amino acids at their amino-terminals which are removed to produce mature Shiga toxin A Subunits and are recognizable to the skilled worker. Further, certain epitope region disruptions are indicated herein by reference to specific amino acids (e.g. S for a serine residue) natively present at specific positions within native Shiga toxin A Subunits (e.g.
  • S33 for the serine residue at position 33 from the amino-terminus followed by the amino acid with which that residue has been substituted in the particular mutation under discussion (e.g. S331 represents the amino acid substitution of isoleucine for serine at amino acid residue 33 from the amino-terminus).
  • the de-immunized, Shiga toxin effector polypeptide of the present invention comprises a disruption of at least one epitope region described in WO 2015/113005, WO 2015/113007 and/or WO 2016/196344.
  • the de-immunized, Shiga toxin effector polypeptide of the present invention comprises or consists essentially of a full-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO: 1), StxA (SEQ ID NO:2), or SLT-2A (SEQ ID NO:3), or variants thereof (e.g.
  • SEQ ID NOs: 4-18) comprising at least one disruption of the amino acid sequence selected from the group of natively positioned amino acids consisting of: 1-15 of any one of SEQ ID NOs: 1-2 and 4-6; 3-14 of any one of SEQ ID NOs: 3 and 7-18; 26-37 of any one of SEQ ID NOs: 3 and 7-18: 27-37 of any one of SEQ ID NOs: 1-2 and 4-6; 39-48 of any one of SEQ ID NOs: 1-2 and 4-6: 42-48 of any one of SEQ ID NOs: 3 and 7-18; and 53-66 of any one of SEQ ID NOs: 1-18: 94-115 of any one of SEQ ID NOs: 1-18; 141-153 of any one of SEQ ID NOs: 1-2 and 4-6: 140-156 of any one of SEQ ID NOs: 3 and 7-18; 179-190 of any one of SEQ ID NOs: 1-2 and 4-6; 179-191 of any one of SEQ ID NOs: 3 and 7-18
  • the Shiga toxin effector polypeptide of the present invention comprises or consists essentially of a truncated Shiga toxin A Subunit. Truncations of Shiga toxin A Subunits might result in the deletion of an entire epitope region(s) without affecting Shiga toxin effector function(s). The smallest, Shiga toxin A Subunit fragment shown to exhibit significant enzymatic activity was a polypeptide composed of residues 75-247 of StxA (A1-Jaufy A et al., Infect Immun 62: 956-60 (1994)).
  • Truncating the carboxy-terminus of SLT-1A, StxA, or SLT-2A to amino acids 1-251 removes two predicted B-cell epitope regions, two predicted CD4 positive (CD4+) T-cell epitopes, and a predicted, discontinuous, B-cell epitope.
  • Truncating the amino-terminus of SLT-1A, StxA, or SLT-2A to 75-293 removes at least three, predicted, B-cell epitope regions and three predicted CD4+ T-cell epitopes.
  • Truncating both amino- and carboxy-terminals of SLT-1A, StxA, or SLT-2A to 75-251 deletes at least five, predicted, B-cell epitope regions; four, putative, CD4+ T-cell epitopes; and one, predicted, discontinuous, B-cell epitope.
  • a Shiga toxin effector polypeptide of the invention may comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with at least one mutation, e.g. deletion, insertion, inversion, or substitution, in a provided epitope region.
  • the polypeptides comprise a disruption which comprises a deletion of at least one amino acid within the epitope region.
  • the polypeptides comprise a disruption which comprises an insertion of at least one amino acid within the epitope region.
  • the polypeptides comprise a disruption which comprises an inversion of amino acids, wherein at least one inverted amino acid is within the epitope region.
  • the polypeptides comprise a disruption which comprises a mutation, such as an amino acid substitution to a non-standard amino acid or an amino acid with a chemically modified side chain. Numerous examples of single amino acid substitutions are provided in the Examples below.
  • the Shiga toxin effector polypeptides of the invention may comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with one or more mutations as compared to the native sequence which comprises at least one amino acid substitution selected from the group consisting of: A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and K.
  • the polypeptide may comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with a single mutation as compared to the native sequence wherein the substitution is selected from the group consisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, G to A, H to A H to G, H to V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R to R to
  • the Shiga toxin effector polypeptides of the invention comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with one or more mutations as compared to the native amino acid residue sequence which comprises at least one amino acid substitution of an immunogenic residue and/or within an epitope region, wherein at least one substitution occurs at the natively positioned group of amino acids selected from the group consisting of: 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO: 1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1
  • the Shiga toxin effector polypeptides of the invention comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with at least one substitution of an immunogenic residue and/or within an epitope region, wherein at least one amino acid substitution is to a non-conservative amino acid (see, e.g., Table B, infra) relative to a natively occurring amino acid positioned at one of the following native positions; 1 of SEQ ID NO: 1 or SEQ ID NO:2; 4 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:1 or S
  • the Shiga toxin effector polypeptides of the invention comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with at least one amino acid substitution selected from the group consisting of K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S, and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, 1, L, F, M, and S; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11 to A, G, V, L, I, F, M and H; T12 to A, G, V, 1, L, F, M, and S; S33 to A, G, V, L, 1, F, and M; S43 to A
  • the Shiga toxin effector polypeptides of the invention comprise or consist essentially of a full-length or truncated Shiga toxin A Subunit with at least one of the following amino acid substitutions K1A, KIM, T41, D6R, S8I, T8V, T91, S91, K11A, K11H, T12K, S331, S33C, S43N, G44L, S45V, S451, T45V, T451, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V541, R55A, R55V, R55L, G56P, 157F, 157M, D58A, D58V, D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V
  • epitope-disrupting substitutions may be combined to form a de-immunized, Shiga toxin effector polypeptide with multiple substitutions per epitope region and/or multiple epitope regions disrupted while still retaining Shiga toxin effector function.
  • any of the de-immunized, Shiga toxin effector polypeptide sub-regions and/or epitope disrupting mutations described herein may be used alone or in combination with each individual embodiment of the present invention, including methods of the present invention.
  • the Shiga toxin effector polypeptide of the present invention comprises (1) a Shiga toxin A1 fragment derived region having a carboxy-terminus and (2) a disrupted furin-cleavage motif at the carboxy-terminus of the Shiga toxin A1 fragment region.
  • Shiga toxin A Subunits of members of the Shiga toxin family comprise a conserved, furin-cleavage site at the carboxy-terminal of their A1 fragment regions important for Shiga toxin function.
  • Furin-cleavage site motifs and furin-cleavage sites can be identified by the skilled worker using standard techniques and/or by using the information herein.
  • the model of Shiga toxin cytotoxicity is that intracellular proteolytic processing of Shiga toxin A Subunits by furin in intoxicated cells is essential for 1) liberation of the A1 fragment from the rest of the Shiga holotoxin, 2) escape of the A1 fragment from the endoplasmic reticulum by exposing a hydrophobic domain in the carboxy-terminus of the A1 fragment, and 3) enzymatic activation of the A1 fragment (see Johannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)).
  • the efficient liberation of the Shiga toxin A1 fragment from the A2 fragment and the rest of the components of the Shiga holotoxin in the endoplasmic reticulum of intoxicated cells is essential for efficient intracellular routing to the cytosol, maximal enzymatic activity, efficient ribosome inactivation, and achieving optimal cytotoxicity, i.e. comparable to a wild-type Shiga toxin (see e.g. WO 2015/191764 and references therein).
  • the A Subunit is proteolytically cleaved by furin at the carboxy bond of a conserved arginine residue (e.g. the arginine residue at position 251 in StxA and SLT-A variants and the arginine residue at position 250 in Stx2A and SLT-2A variants).
  • Furin cleavage of Shiga toxin A Subunits occurs in endosomal and/or Golgi compartments.
  • Furin is a specialized serine endoprotease which is expressed by a wide variety of cell types, in all human tissues examined, and by most animal cells.
  • Furin cleaves polypeptides comprising accessible motifs often centered on the minimal, dibasic, consensus motif R-x-(R/K/x)-R.
  • the A Subunits of members of the Shiga toxin family comprise a conserved, surface-exposed, extended loop structure (e.g. 242-261 in StxA and SLT-1A, and 241-260 in SLT-2) with a conserved S-R/Y-x-x-R motif which is cleaved by furin.
  • the surface exposed, extended loop structure positioned at amino acid residues 242-261 in StxA is required for furin-induced cleavage of StxA, including features flanking the minimal, furin-cleavage motif R-x-x-R.
  • Furin-cleavage motifs and furin-cleavage sites in Shiga toxin A Subunits and Shiga toxin effector polypeptides can be identified by the skilled worker using standard methods and/or by using the information herein.
  • Furin cleaves the minimal, consensus motif R-x-x-R (Schalken J et al., J Clin Invest 80: 1545-9 (1987); Bresnahan P et al., J Cell Biol 111: 2851-9 (1990); Hatsuzawa K et al., J Biol Chem 265: 22075-8 (1990); Wise R et al., Proc Natl Acad Sci USA 87: 9378-82 (1990); Molloy S et al., J Biol Chem 267: 16396-402 (1992)).
  • furin inhibitors comprise peptides comprising the motif R-x-x-R.
  • An example of a synthetic inhibitor of furin is a molecule comprising the peptide R—V-K-R (Henrich S et al., Nat Struct Biol 10: 520-6 (2003)).
  • a peptide or protein comprising a surface accessible, dibasic amino acid motif with two positively charged, amino acids separated by two amino acid residues may be predicted to be sensitive to furin-cleavage with cleavage occurring at the carboxy bond of the last basic amino acid in the motif.
  • furin-cleavage site motif comprises a region of twenty, continuous, amino acid residues, which can be labeled P14 through P6′ (Tian S et al., Int J Mol Sci 12: 1060-5 (2011)) using the nomenclature described in Schechter I, Berger, A, Biochem Biophys Res Commun 32: 898-902 (1968). According to this nomenclature, the furin-cleavage site is at the carboxy bond of the amino acid residue designated P1, and the amino acid residues of the furin-cleavage motif are numbered P2.
  • P3, P4, etc. in the direction going toward the amino-terminus from this reference P1 residue.
  • the amino acid residues of the motif going toward the carboxy-terminus from the P1 reference residue are numbered with the prime notation P2′, P3′, P4′, etc.
  • the P6 to P2′ region delineates the core substrate of the furin cleavage motif which is bound by the enzymatic domain of furin.
  • the two flanking regions P14 to P7 and P3′ to P6′ are often rich in polar, amino acid residues to increase the accessibility to the core furin cleavage site located between them.
  • furin-cleavage site is often described by the consensus motif R-x-x-R which corresponds to P4-P3-P2-P1; where “R” represents an arginine residue (see Table A, supra), a dash “-” represents a peptide bond, and a lowercase “x” represents any amino acid residue.
  • R represents an arginine residue (see Table A, supra)
  • a dash “-” represents a peptide bond
  • a lowercase “x” represents any amino acid residue.
  • other residues and positions may help to further define furin-cleavage motifs.
  • consensus motif is often reported as the consensus motif R-x-[K/R]-R (where a forward slash “/” means “or” and divides alternative amino acid residues at the same position), which corresponds to P4-P3-P2-P1, because it was observed that furin has a strong preference for cleaving substrates containing this motif.
  • furin-cleavage site R-x-x-R a larger, furin-cleavage motif has been described with certain amino acid residue preferences at certain positions.
  • certain physicochemical properties have been characterized for the amino acid residues in a amino acid residue long, furin-cleavage site motif.
  • the P6 to P2′ region of the furin-cleavage motif delineates the core furin-cleavage site which physically interacts with the enzymatic domain of furin.
  • the two flanking regions P14 to P7 and P3′ to P6′ are often hydrophilic being rich in polar, amino acid residues to increase the surface accessibility of the core furin-cleavage site located between them.
  • the furin-cleavage motif region from position P5 to P1 tends to comprise amino acid residues with a positive charge and/or high isoelectric points.
  • the P1 position which marks the position of furin proteolysis, is generally occupied by an arginine but other positively charged, amino acid residues may occur in this position.
  • Positions P2 and P3 tend to be occupied by flexible, amino acid residues, and in particular P2 tends to be occupied by arginine, lysine, or sometimes by very small and flexible amino acid residues like glycine.
  • the P4 position tends to be occupied by positively charged, amino acid residues in furin substrates.
  • Positions P1′ and P2′ are commonly occupied by aliphatic and/or hydrophobic amino acid residues, with the P1 position most commonly being occupied by a serine.
  • flanking regions tend to be occupied by amino acid residues which are polar, hydrophilic, and have smaller amino acid functional groups, however, in certain verified furin substrates, the flanking regions do not contain any hydrophilic, amino acid residues (see Tian S, Biochem Insights 2: 9-20 (2009)).
  • the twenty amino acid residue, furin-cleavage motif and furin-cleavage site found in native, Shiga toxin A Subunits at the junction between the Shiga toxin A1 fragment and A2 fragment is well characterized in certain Shiga toxins.
  • StxA SEQ ID NO:2
  • SLT-1A SEQ ID NO: 1
  • this furin-cleavage motif is natively positioned from L238 to F257
  • SLT-2A SEQ ID NO:3
  • furin-cleavage motifs in other native, Shiga toxin A Subunits or Shiga toxin effector polypeptides, where the motifs are actual furin-cleavage motifs or are predicted to result in the production of A1 and A2 fragments after furin cleavage of those molecules within a eukaryotic cell.
  • the Shiga toxin effector polypeptide of the present invention comprises (1) a Shiga toxin A1 fragment derived polypeptide having a carboxy-terminus and (2) a disrupted furin-cleavage motif at the carboxy-terminus of the Shiga toxin A1 fragment derived polypeptide.
  • the carboxy-terminus of a Shiga toxin A1 fragment derived polypeptide may be identified by the skilled worker by using techniques known in the art, such as, e.g., by using protein sequence alignment software to identify (i) a furin-cleavage motif conserved with a naturally occurring Shiga toxin, (ii) a surface exposed, extended loop conserved with a naturally occurring Shiga toxin, and/or (iii) a stretch of amino acid residues which are predominantly hydrophobic (i.e. a hydrophobic “patch”) that may be recognized by the ERAD system
  • a protease-cleavage resistant, Shiga toxin effector polypeptide of the present invention (1) may be completely lacking any furin-cleavage motif at a carboxy-terminus of its Shiga toxin A1 fragment region and/or (2) comprise a disrupted furin-cleavage motif at the carboxy-terminus of its Shiga toxin A1 fragment region and/or region derived from the carboxy-terminus of a Shiga toxin A1 fragment.
  • a disruption of a furin-cleavage motif includes various alterations to an amino acid residue in the furin-cleavage motif, such as, e.g., a post-translation modification(s), an alteration of one or more atoms in an amino acid functional group, the addition of one or more atoms to an amino acid functional group, the association to a non-proteinaceous moiety(ies), and/or the linkage to an amino acid residue, peptide, polypeptide such as resulting in a branched proteinaceous structure.
  • a post-translation modification(s) an alteration of one or more atoms in an amino acid functional group
  • the addition of one or more atoms to an amino acid functional group the association to a non-proteinaceous moiety(ies)
  • linkage to an amino acid residue, peptide, polypeptide such as resulting in a branched proteinaceous structure.
  • Shiga toxin effector polypeptides may be created from a Shiga toxin effector polypeptide and/or Shiga toxin A Subunit polypeptide, whether naturally occurring or not, using a method described herein, described in WO 2015/191764 and WO 2016/196344, and/or known to the skilled worker, wherein the resulting molecule still retains one or more Shiga toxin A Subunit functions.
  • the term “disruption” or “disrupted” refers to an alteration from the naturally occurring furin-cleavage site and/or furin-cleavage motif, such as, e.g., a mutation, that results in a reduction in furin-cleavage proximal to the carboxy-terminus of a Shiga toxin A1 fragment region, or identifiable region derived thereof, as compared to the furin-cleavage of a wild-type Shiga toxin A Subunit or a polypeptide derived from a wild-type Shiga toxin A Subunit comprising only wild-type polypeptide sequences.
  • An alteration to an amino acid residue in the furin-cleavage motif includes a mutation in the furin-cleavage motif, such as, e.g., a deletion, insertion, inversion, substitution, and/or carboxy-terminal truncation of the furin-cleavage motif, as well as a post-translation modification, such as, e.g., as a result of glycosylation, albumination, and the like which involve conjugating or linking a molecule to the functional group of an amino acid residue.
  • a mutation in the furin-cleavage motif such as, e.g., a deletion, insertion, inversion, substitution, and/or carboxy-terminal truncation of the furin-cleavage motif
  • a post-translation modification such as, e.g., as a result of glycosylation, albumination, and the like which involve conjugating or linking a molecule to the functional group of an amino acid residue.
  • furin-cleavage motif is comprised of about twenty, amino acid residues, in theory, alterations, modifications, mutations, deletions, insertions, and/or truncations involving one or more amino acid residues of any one of these twenty positions might result in a reduction of furin-cleavage sensitivity (Tian S et al., Sci Rep 2: 261 (2012)).
  • the disruption of a furin-cleavage site and/or furin-cleavage motif may or may not increase resistance to cleavage by other proteases, such as, e.g., trypsin and extracellular proteases common in the vascular system of mammals.
  • proteases such as, e.g., trypsin and extracellular proteases common in the vascular system of mammals.
  • the effects of a given disruption to cleavage sensitivity of a given protease may be tested by the skilled worker using techniques known in the art.
  • a “disrupted furin-cleavage motif” is furin-cleavage motif comprising an alteration to one or more amino acid residues derived from the 20 amino acid residue region representing a conserved, furin-cleavage motif found in native, Shiga toxin A Subunits at the junction between the Shiga toxin A1 fragment and A2 fragment regions and positioned such that furin cleavage of a Shiga toxin A Subunit results in the production of the A1 and A2 fragments; wherein the disrupted furin-cleavage motif exhibits reduced furin cleavage in an experimentally reproducible way as compared to a reference molecule comprising a wild-type, Shiga toxin A1 fragment region fused to a carboxy-terminal polypeptide of a size large enough to monitor furin cleavage using the appropriate assay known to the skilled worker and/or described herein.
  • furin-cleavage sites and furin-cleavage motifs can be disrupted by mutations comprising the modification of an amino acid by the addition of a covalently-linked structure which masks at least one amino acid in the site or motif, such as, e.g., as a result of PEGylation, the coupling of small molecule adjuvants, and/or site-specific albumination.
  • a disrupted furin-cleavage motif may comprise less than the twenty, amino acid residues of the furin-cleavage motif due to a carboxy-terminal truncation as compared to a Shiga toxin A Subunit and/or Shiga toxin A1 fragment.
  • the Shiga toxin effector polypeptide of the present invention comprises (1) a Shiga toxin A1 fragment derived polypeptide having a carboxy-terminus and (2) a disrupted furin-cleavage motif at the carboxy-terminus of the Shiga toxin A1 fragment polypeptide region; wherein the Shiga toxin effector polypeptide (and any cell-targeting molecule comprising it) is more furin-cleavage resistant as compared to a reference molecule, such as, e.g., a wild-type Shiga toxin polypeptide comprising the carboxy-terminus of an A1 fragment and/or the conserved, furin-cleavage motif between A1 and A2 fragments.
  • a reference molecule such as, e.g., a wild-type Shiga toxin polypeptide comprising the carboxy-terminus of an A1 fragment and/or the conserved, furin-cleavage motif between A1 and A2 fragments.
  • a reduction in furin cleavage of one molecule compared to a reference molecule may be determined using an in vitro, furin-cleavage assay described in the Examples below, conducted using the same conditions, and then performing a quantitation of the band density of any fragments resulting from cleavage to quantitatively measure in change in furin cleavage.
  • the Shiga toxin effector polypeptide is more resistant to furin-cleavage in vitro and/or in vivo as compared to a wild-type, Shiga toxin A Subunit.
  • the protease-cleavage sensitivity of a cell-targeting molecule of the present invention is tested by comparing it to the same molecule having its furin-cleavage resistant, Shiga toxin effector polypeptide replaced with a wild-type, Shiga toxin effector polypeptide comprising a Shiga toxin A1 fragment.
  • the molecules of the present invention comprising a disrupted furin-cleavage motif exhibits a reduction in in vitro furin cleavage of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or greater compared to a reference molecule comprising a wild-type, Shiga toxin A1 fragment fused at its carboxy-terminus to a peptide or polypeptide, such as, e.g., the reference molecule SLT-1A-WT::scFv1::C2 (SEQ ID NO:278) described in the Examples below.
  • furin-cleavage motif disruptions have been described. For example, mutating the two conserved arginines to alanines in the minimal R-x-x-R motif completely blocked processing by furin and/or furin-like proteases (see e.g. Duda A et al., J Virology 78: 13865-70 (2004)). Because the furin-cleavage site motif is comprised of about twenty amino acid residues, in theory, certain mutations involving one or more of any one of these twenty, amino acid residue positions might abolish furin cleavage or reduce furin cleavage efficiency (see e.g. Tian S et al., Sci Rep 2: 261 (2012)).
  • the molecules of the present invention comprise a Shiga toxin effector polypeptide derived from at least one A Subunit of a member of the Shiga toxin family wherein the Shiga toxin effector polypeptide comprises a disruption in one or more amino acids derived from the conserved, highly accessible, protease-cleavage sensitive loop of Shiga toxin A Subunits.
  • the conserved, highly accessible, protease-cleavage sensitive loop of Shiga toxin A Subunits For example, in StxA and SLT-1A, this highly accessible, protease-sensitive loop is natively positioned from amino acid residues 242 to 261, and in SLT-2A, this conserved loop is natively positioned from amino acid residues 241 to 260.
  • a molecule of the present invention comprises a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif comprising a mutation in the surface-exposed, protease sensitive loop conserved among Shiga toxin A Subunits.
  • a molecule of the present invention comprises a Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif comprising a mutation in this protease-sensitive loop of Shiga toxin A Subunits, the mutation which reduce the surface accessibility of certain amino acid residues within the loop such that furin-cleavage sensitivity is reduced.
  • the disrupted furin-cleavage motif of a Shiga toxin effector polypeptide of the present invention comprises a disruption in terms of existence, position, or functional group of one or both of the consensus amino acid residues P1 and P4, such as, e.g., the amino acid residues in positions I and 4 of the minimal furin-cleavage motif R/Y-x-x-R.
  • mutating one or both of the two arginine residues in the minimal, furin consensus site R-x-x-R to alanine will disrupt a furin-cleavage motif and prevent furin-cleavage at that site.
  • amino acid residue substitutions of one or both of the arginine residues in the minimal furin-cleavage motif R-x-x-R to any non-conservative amino acid residue known to the skilled worker will reduced the furin-cleavage sensitivity of the motif.
  • amino acid residue substitutions of arginine to any non-basic amino acid residue which lacks a positive charge such as, e.g., A, G, P, S, T, D, E, Q, N, C, I, L, M, V, F, W, and Y, will result in a disrupted furin-cleavage motif.
  • the disrupted furin-cleavage motif of a Shiga toxin effector polypeptide of the present invention comprises a disruption in the spacing between the consensus amino acid residues P4 and P1 in terms of the number of intervening amino acid residues being other than two, and, thus, changing either P4 and/or P1 into a different position and eliminating the P4 and/or PI designations.
  • deletions within the furin-cleavage motif of the minimal furin-cleavage site or the core, furin-cleavage motif will reduce the furin-cleavage sensitivity of the furin-cleavage motif.
  • the disrupted furin-cleavage motif comprises one or more amino acid residue substitutions, as compared to a wild-type, Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif comprises one or more amino acid residue substitutions within the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, the natively positioned amino acid residue R248 substituted with any non-positively charged, amino acid residue and/or R251 substituted with any non-positively charged, amino acid residue; and for SLT-2A derived Shiga toxin effector polypeptides, the natively positioned amino acid residue Y247 substituted with any non-positively charged, amino acid residue and/or R250 substituted with any non-positively charged, amino acid residue.
  • the disrupted furin-cleavage motif comprises an un-disrupted, minimal furin-cleavage site R/Y-x-x-R but instead comprises a disrupted flanking region, such as, e.g., amino acid residue substitutions in one or more amino acid residues in the furin-cleavage motif flanking regions natively position at, e.g., 241-247 and/or 252-259.
  • the disrupted furin cleavage motif comprises a substitution of one or more of the amino acid residues located in the P1-P6 region of the furin-cleavage motif; mutating P1′ to a bulky amino acid, such as, e.g., R, W, Y, F, and H; and mutating P2′ to a polar and hydrophilic amino acid residue; and substituting one or more of the amino acid residues located in the P1′-P6′ region of the furin-cleavage motif with one or more bulky and hydrophobic amino acid residues
  • the disruption of the furin-cleavage motif comprises a deletion, insertion, inversion, and/or mutation of at least one amino acid residue within the furin-cleavage motif.
  • a protease-cleavage resistant, Shiga toxin effector polypeptide of the present invention may comprise a disruption of the amino acid sequence natively positioned at 248-251 of the A Subunit of Shiga toxin (SEQ ID NOs: 1-2 and 4-6), at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NOs: 3 and 7-18), or at the equivalent position in a conserved Shiga toxin effector polypeptide and/or non-native Shiga toxin effector polypeptide sequence.
  • protease-cleavage resistant, Shiga toxin effector polypeptides comprise a disruption which comprises a deletion of at least one amino acid within the furin-cleavage motif. In certain further embodiments, protease-cleavage resistant, Shiga toxin effector polypeptides comprise a disruption which comprises an insertion of at least one amino acid within the protease-cleavage motif region. In certain further embodiments, the protease-cleavage resistant, Shiga toxin effector polypeptides comprise a disruption which comprises an inversion of amino acids, wherein at least one inverted amino acid is within the protease motif region.
  • the protease-cleavage resistant, Shiga toxin effector polypeptides comprise a disruption which comprises a mutation, such as an amino acid substitution to a non-standard amino acid or an amino acid with a chemically modified side chain. Examples of single amino acid substitutions are provided in the Examples below.
  • the disrupted furin-cleavage motif comprises the deletion of nine, ten, eleven, or more of the carboxy-terminal amino acid residues within the furin-cleavage motif.
  • the disrupted furin-cleavage motif will not comprise a furin-cleavage site or a minimal furin-cleavage motif.
  • certain embodiments lack a furin-cleavage site at the carboxy-terminus of the A1 fragment region.
  • the disrupted furin-cleavage motif comprises both an amino acid residue deletion and an amino acid residue substitution as compared to a wild-type, Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif comprises one or more amino acid residue deletions and substitutions within the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, the natively positioned amino acid residue R248 substituted with any non-positively charged, amino acid residue and/or R251 substituted with any non-positively charged, amino acid residue; and for SLT-2A derived Shiga toxin effector polypeptides, the natively positioned amino acid residue Y247 substituted with any non-positively charged, amino acid residue and/or R250 substituted with any non-positively charged, amino acid residue.
  • the disrupted furin-cleavage motif comprises an amino acid residue deletion and an amino acid residue substitution as well as a carboxy-terminal truncation as compared to a wild-type, Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif comprises one or more amino acid residue deletions and substitutions within the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, the natively positioned amino acid residue R248 substituted with any non-positively charged, amino acid residue and/or R251 substituted with any non-positively charged, amino acid residue, and for SLT-2A derived Shiga toxin effector polypeptides, the natively positioned amino acid residue Y247 substituted with any non-positively charged, amino acid residue and/or R250 substituted with any non-positively charged, amino acid residue.
  • the disrupted furin-cleavage motif comprises both an amino acid substitution within the minimal furin-cleavage site R/Y-x-x-R and a carboxy-terminal truncation as compared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, truncations ending at the natively amino acid position 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, or greater and comprising the natively positioned amino acid residue R248 and/or R251 substituted with any non-positively charged
  • the disrupted furin-cleavage motif comprises an insertion of one or more amino acid residues as compared to a wild-type, Shiga toxin A Subunit as long as the inserted amino residue(s) does not create a de novo furin-cleavage site.
  • the insertion of one or more amino acid residues disrupts the natural spacing between the arginine residues in the minimal, furin-cleavage site R/Y-x-x-R, such as, e.g., StxA and SLT-1A derived polypeptides comprising an insertion of one or more amino acid residues at 249 or 250 and thus between R248 and R251; or SLT-2A derived polypeptides comprising an insertion of one or more amino acid residues at 248 or 249 and thus between Y247 and R250.
  • StxA and SLT-1A derived polypeptides comprising an insertion of one or more amino acid residues at 249 or 250 and thus between R248 and R251
  • SLT-2A derived polypeptides comprising an insertion of one or more amino acid residues at 248 or 249 and thus between Y247 and R250.
  • the disrupted furin-cleavage motif comprises both an amino acid residue insertion and a carboxy-terminal truncation as compared to a wild-type, Shiga toxin A Subunit. In certain embodiments, the disrupted furin-cleavage motif comprises both an amino acid residue insertion and an amino acid residue substitution as compared to a wild-type, Shiga toxin A Subunit. In certain embodiments, the disrupted furin-cleavage motif comprises both an amino acid residue insertion and an amino acid residue deletion as compared to a wild-type, Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif comprises an amino acid residue deletion, an amino acid residue insertion, and an amino acid residue substitution as compared to a wild-type, Shiga toxin A Subunit.
  • the disrupted furin-cleavage motif comprises an amino acid residue deletion, insertion, substitution, and carboxy-terminal truncation as compared to a wild-type, Shiga toxin A Subunit.
  • the Shiga toxin effector polypeptide comprising a disrupted furin-cleavage motif is directly fused by a peptide bond to a molecular moiety comprising an amino acid, peptide, and/or polypeptide wherein the fused structure involves a single, continuous polypeptide.
  • the amino acid sequence following the disrupted furin-cleavage motif should not create a de novo, furin-cleavage site at the fusion junction.
  • any of the above protease-cleavage resistant, Shiga toxin effector polypeptide disrupted furin-cleavage motifs may be used alone or in combination with each individual embodiment of the present invention, including methods of the present invention.
  • the Shiga toxin effector polypeptide of the present invention comprises an embedded or inserted epitope-peptide.
  • the epitope-peptide is a heterologous, T-cell epitope-peptide, such as, e.g., an epitope considered heterologous to Shiga toxin A Subunits.
  • the epitope-peptide is a CD8+ T-cell epitope.
  • the CD8+ T-cell epitope-peptide has a binding affinity to a MHC class I molecule characterized by a dissociation constant (K D ) of 10 ⁇ 4 molar or less and/or the resulting MHC class I-epitope-peptide complex has a binding affinity to a T-cell receptor (TCR) characterized by a dissociation constant (K D ) of 10 ⁇ 4 molar or less.
  • TCR T-cell receptor
  • the Shiga toxin effector polypeptide of the present invention comprises an embedded or inserted, heterologous, T-cell epitope, such as, e.g., a human CD8+ T-cell epitope.
  • the heterologous, T-cell epitope is embedded or inserted so as to disrupt an endogenous epitope or epitope region (e.g. a B-cell epitope and/or CD4+ T-cell epitope) identifiable in a naturally occurring Shiga toxin polypeptide or parental Shiga toxin effector polypeptide from which the Shiga toxin effector polypeptide of the present invention is derived.
  • the Shiga toxin effector polypeptide (and any cell-targeting molecule comprising it) is CD8+ T-cell hyper-immunized, such as, e.g., as compared to a wild-type Shiga toxin polypeptide.
  • the CD8+ T-cell hyper-immunized, Shiga toxin effector polypeptides of the present invention each comprise a T-cell epitope-peptide.
  • Shiga toxin effector polypeptides can be created from Shiga toxin effector polypeptides and/or Shiga toxin A Subunit polypeptides, whether naturally occurring or not, using a method described herein, described in WO 2015/113007, and/or known to the skilled worker, wherein the resulting molecule still retains one or more Shiga toxin A Subunit functions.
  • a T-cell epitope is a molecular structure which is comprised by an antigenic peptide and can be represented by a linear, amino acid sequence.
  • T-cell epitopes are peptides of sizes of eight to eleven amino acid residues (Townsend A, Bodmer H, Annu Rev Immunol 7: 601-24 (1989)); however, certain T-cell epitope-peptides have lengths that are smaller than eight or larger than eleven amino acids long (see e.g. Livingstone A, Fathman C, Annu Rev Immunol 5: 477-501 (1987); Green K et al., Eur J Immunol 34: 2510-9 (2004)).
  • the embedded or inserted epitope is at least seven amino acid residues in length.
  • the embedded or inserted epitope is bound by a TCR with a binding affinity characterized by a K D less than 10 mM (e.g. 1-100 pM) as calculated using the formula in Stone J et al., Immunology 126: 165-76 (2009).
  • a K D less than 10 mM (e.g. 1-100 pM) as calculated using the formula in Stone J et al., Immunology 126: 165-76 (2009).
  • the binding affinity within a given range between the MHC-epitope and TCR may not correlate with antigenicity and/or immunogenicity (see e.g.
  • a heterologous, T-cell epitope is an epitope not already present in a wild-type Shiga toxin A Subunit; a naturally occurring Shiga toxin A Subunit; and/or a parental, Shiga toxin effector polypeptide used as a source polypeptide for modification by a method described herein, described in WO 2015/113007 and/or WO 2016/196344, and/or known to the skilled worker.
  • a heterologous, T-cell epitope-peptide may be incorporated into a source polypeptide via numerous methods known to the skilled worker, including, e.g., the processes of creating one or more amino acid substitutions within the source polypeptide, fusing one or more amino acids to the source polypeptide, inserting one or more amino acids into the source polypeptide, linking a peptide to the source polypeptide, and/or a combination of the aforementioned processes.
  • the result of such a method is the creation of a modified variant of the source polypeptide which comprises one or more embedded or inserted, heterologous, T-cell epitope-peptides.
  • T-cell epitopes may be chosen or derived from a number of source molecules for use in the present invention.
  • T-cell epitopes may be created or derived from various naturally occurring proteins.
  • T-cell epitopes may be created or derived from various naturally occurring proteins foreign to mammals, such as, e.g., proteins of microorganisms.
  • T-cell epitopes may be created or derived from mutated human proteins and/or human proteins aberrantly expressed by malignant human cells.
  • T-cell epitopes may be synthetically created or derived from synthetic molecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); Del Val M et al., 0.1 Virol 65: 3641-6 (1991); Appella E et al., Biomed Pept Proteins Nucleic Acids 1: 177-84 (1995); Perez S et al., Cancer 116: 2071-80 (2010)).
  • T-cell epitope-peptide is contemplated as being used as a heterologous, T-cell epitope of the present invention
  • certain epitopes may be selected based on desirable properties.
  • One objective of the present invention is to create CD8+ T-cell hyper-immunized, Shiga toxin effector polypeptides for administration to vertebrates, meaning that the heterologous, T-cell epitope is highly immunogenic and can elicit robust immune responses in vivo when displayed complexed with a MHC class I molecule on the surface of a cell.
  • the Shiga toxin effector polypeptide of the present invention comprises one or more, embedded or inserted, heterologous, T-cell epitopes which are CD8+ T-cell epitopes.
  • a Shiga toxin effector polypeptide of the present invention that comprises a heterologous, CD8+ T-cell epitope is considered a CD8+ T-cell hyper-immunized, Shiga toxin effector polypeptide.
  • T-cell epitope components of the present invention may be chosen or derived from a number of source molecules already known to be capable of eliciting a vertebrate immune response.
  • T-cell epitopes may be derived from various naturally occurring proteins foreign to vertebrates, such as, e.g., proteins of pathogenic microorganisms and non-self, cancer antigens.
  • infectious microorganisms may contain numerous proteins with known antigenic and/or immunogenic properties.
  • infectious microorganisms may contain numerous proteins with known antigenic and/or immunogenic sub-regions or epitopes.
  • T-cell epitopes the proteins of intracellular pathogens with mammalian hosts are sources for T-cell epitopes.
  • intracellular pathogens such as viruses, bacteria, fungi, and single-cell eukaryotes, with well-studied antigenic proteins or peptides.
  • T-cell epitopes can be selected or identified from human viruses or other intracellular pathogens, such as, e.g., bacteria like mycobacterium, fungi like toxoplasmae, and protists like trypanosomes.
  • HCMV human cytomegaloviruses
  • proteins from human cytomegaloviruses such as peptides in the proteins pp65 (UL83), UL128-131, immediate-early 1 (IE-1; UL123), glycoprotein B, tegument proteins, and many of these peptides have been shown to elicit human immune responses, such as by using ex vivo assays.
  • CD8+ T-cell epitopes of cancer and/or tumor cell antigens can be identified by the skilled worker using techniques known in the art, such as, e.g., differential genomics, differential proteomics, immunoproteomics, prediction then validation, and genetic approaches like reverse-genetic transfection (see e.g., Admon A et al., Mol Cell Proteomics 2: 388-98 (2003); Purcell A, Gorman J, Mol Cell Proteomics 3: 193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)).
  • T-cell epitopes There are many antigenic and/or immunogenic T-cell epitopes already identified or predicted to occur in human cancer and/or tumor cells. For example, T-cell epitopes have been predicted in human proteins commonly mutated or overexpressed in neoplastic cells, such as, e.g., ALK.
  • CEA N-acetylglucosaminyl-transferase V (GnT-V), HCA587, HER-2/neu, MAGE, Melan-A/MART-1, MUC-1, p53, and TRAG-3
  • GnT-V N-acetylglucosaminyl-transferase V
  • HCA587 HCA587
  • HER-2/neu HCA587
  • MAGE Melan-A/MART-1
  • MUC-1 MUC-1
  • p53 p53
  • TRAG-3 see e.g., van der Bruggen P et al., Science 254: 1643-7 (1991); Kawakami Y et al., J Exp Med 180: 347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); Guilloux Y et al., J Exp Med 183: 1173 (1996); Skipper J et al.
  • T-cell epitopes from human cancer cells have been created (see e.g., Lazoura E, strigopoulos V, Curr Med Chem 12: 629-39 (2005); Douat-Casassus C et al., J Med Chem 50: 1598-609 (2007)).
  • T-cell epitopes may be used in the polypeptides and molecules of the present invention, certain T-cell epitopes may be preferred based on their known and/or empirically determined characteristics. For example, in many species, the MHC alleles in its genome encode multiple MHC-I molecular variants. Because MHC class I protein polymorphisms can affect antigen-MHC class I complex recognition by CD8+ T-cells, T-cell epitopes may be chosen for use in the present invention based on knowledge about certain MHC class I polymorphisms and/or the ability of certain antigen-MHC class I complexes to be recognized by T-cells having different genotypes.
  • HLA-class I-restricted epitopes can be selected or identified by the skilled worker using standard techniques known in the art.
  • the ability of peptides to bind to human MHC class I molecules can be used to predict the immunogenic potential of putative T-cell epitopes.
  • the ability of peptides to bind to human MHC class I molecules can be scored using software tools.
  • T-cell epitopes may be chosen for use as a heterologous, T-cell epitope component of the present invention based on the peptide selectivity of the HLA variants encoded by the alleles more prevalent in certain human populations.
  • the human population is polymorphic for the alpha chain of MHC class I molecules due to the varied alleles of the HLA genes from individual to individual.
  • T-cell epitopes may be more efficiently presented by a specific HLA molecule, such as, e.g., the commonly occurring HLA variants encoded by the HLA-A allele groups HLA-A2 and HLA-A3.
  • T-cell epitopes When choosing T-cell epitopes for use as a heterologous, T-cell epitope component of the present invention, multiple factors may be considered that can influence epitope generation and transport to receptive MHC class I molecules, such as, e.g., the presence and epitope specificity of the following factors in the target cell: proteasome, ERAAP/ERAP1, tapasin, and TAPs.
  • factors may be considered that can influence epitope generation and transport to receptive MHC class I molecules, such as, e.g., the presence and epitope specificity of the following factors in the target cell: proteasome, ERAAP/ERAP1, tapasin, and TAPs.
  • epitope may be selected which best match the MHC class I molecules present in the cell-type or cell populations to be targeted. Different MHC class I molecules exhibit preferential binding to particular peptide sequences, and particular peptide-MHC class I variant complexes are specifically recognized by the T-cell receptors (TCRs) of effector T-cells.
  • TCRs T-cell receptors
  • the skilled worker can use knowledge about MHC class I molecule specificities and TCR specificities to optimize the selection of heterologous, T-cell epitopes used in the present invention.
  • multiple, immunogenic, T-cell epitopes for MHC class I presentation may be embedded in the same Shiga toxin effector polypeptide of the present invention, such as, e.g., for use in the targeted delivery of a plurality of T-cell epitopes simultaneously.
  • An example of a cell-targeting molecule of the present invention comprising multiple, CD8+ T-cell epitopes is SEQ ID NO:253.
  • the Shiga toxin effector polypeptide comprises a unique amino acid residue, such as, e.g., a cysteine, lysine, selenocysteine, or pyrroline-carboxy-lysine residue, which may optionally be linked, either directly or indirectly, to an agent or cargo for targeted delivery, including, e.g., a cell-targeting molecule altering agent which confers a desirable property to a cell-targeting molecule comprising the Shiga toxin effector polypeptide upon administration to a mammal (see e.g. WO 2018/106895).
  • a unique amino acid residue such as, e.g., a cysteine, lysine, selenocysteine, or pyrroline-carboxy-lysine residue
  • an agent or cargo for targeted delivery including, e.g., a cell-targeting molecule altering agent which confers a desirable property to a cell-targeting molecule comprising the Shig
  • Molecules comprising such Shiga toxin effector polypeptides may be equipped with a site-specific position, such as, e.g., a unique amino acid residue in the molecule, for linking other molecules while retaining Shiga toxin function(s), such as, e.g., stimulating cellular internalization, directing efficient intracellular routing, and/or potent cytotoxicity.
  • the Shiga toxin effector polypeptide is conjugated to another moiety, agent, and/or cargo, either directly or indirectly, via the unique amino acid residue, such as, e.g., via the functional group of the unique amino acid (see e.g. WO 2018/106895).
  • the cell-targeting molecules of the present invention each comprise one or more CD8+ T-cell epitope-peptides that are heterologous to their respective Shiga toxin effector polypeptide(s) and binding region(s) and which are not embedded or inserted within a Shiga toxin effector polypeptide component.
  • a CD8+ T-cell epitope (also known as a MHC class I epitope or MHC class I peptide) is a molecular structure which is comprised by an antigenic peptide and can be represented by a linear, amino acid sequence.
  • CD8+ T-cell epitopes are peptides of sizes of eight to eleven amino acid residues (Townsend A, Bodmer H, Annu Rev Immunol 7: 601-24 (1989)); however, certain CD8+ T-cell epitopes have lengths that are smaller than eight or larger than eleven amino acids long (see e.g. Livingstone A, Fathnman C, Annu Rev Immunol 5: 477-501 (1987); Green K et al., Eur J Immunol 34: 2510-9 (2004)).
  • a CD8+ T-cell epitope is a molecular structure recognizable by an immune system of at least one individual, i.e. an antigenic peptide.
  • the heterologous, CD8+ T-cell epitope-peptide cargo of the cell-targeting molecule of the present invention can be chosen from virtually any CD8+ T-cell epitope.
  • the heterologous, CD8+ T-cell epitope-peptide cargo has a binding affinity to a MHC class I molecule characterized by a dissociation constant (K D ) of 10′ molar or less and/or the resulting MHC class 1-epitope-peptide complex has a binding affinity to a T-cell receptor (TCR) characterized by a dissociation constant (K D ) of 10; molar or less.
  • TCR T-cell receptor
  • T-cell epitopes can be empirical characterized by a binding affinity, as described above.
  • the critical structural elements of a T-cell epitope can be identified and further analyzed using assays known to the skilled worker, such as, e.g., a combination of alanine scanning mutagenesis and binding affinity experiments, which may also take crystallographic or other empirical structural data into account.
  • the energetic contributions of some or all of the amino acid residues in the epitope-peptide to the epitope-MHC complex and/or epitope-MHC-TCR complex may be estimated or calculated. Certain residues/positions may be considered or categorized as either determinant for binding affinity or neutral.
  • determinant residues/positions/structures can be further categorized as contact-determining, specificity-determining, and/or affinity determining (see e.g. Langman R, Mol Immunol 37: 555-61 (2000): Greenspan N, Adv Cancer Res 80: 147-87 (2001); Cohn M, Mol Immunol 42: 651-5 (2005)).
  • Certain CD8+ T-cell epitopes may be represented by a consensus amino acid sequence, which allows for some variation in amino acid identity and/or positioning.
  • the heterologous, CD8+ T-cell epitope-peptide is at least seven amino acid residues in length.
  • the CD8+ T-cell epitope-peptide is bound by a TCR with a binding affinity characterized by a K D less than 10 millimolar (mM) (e.g. 1-100 ⁇ M) as calculated using the formula in Stone J et al., Immunology 126: 165-76 (2009).
  • mM millimolar
  • the binding affinity within a given range between the MHC-epitope and TCR may not correlate with antigenicity and/or immunogenicity (see e.g.
  • T-cell epitopes may be chosen or derived from a number of source molecules for use in the present invention.
  • T-cell epitopes may be created or derived from various naturally occurring proteins.
  • T-cell epitopes may be created or derived from various naturally occurring proteins foreign to mammals, such as, e.g., proteins of microorganisms.
  • T-cell epitopes may be created or derived from mutated human proteins and/or human proteins aberrantly expressed by malignant human cells.
  • T-cell epitopes may be synthetically created or derived from synthetic molecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); Del Val M et al., J Virol 65: 3641-6 (1991); Appella E et al., Biomed Pept Proteins Nucleic Acids 1: 177-84 (1995); Perez S et al., Cancer 116: 2071-80 (2010)).
  • the CD8+ T-cell epitope-peptide of the cell-targeting molecule of the present invention can be chosen from various known antigens, such as, e.g., well-characterized immunogenic epitopes from human pathogens, typically the most common pathogenic viruses and bacteria.
  • CD8+ T-cell epitopes can be identified by reverse immunology methods known to the skilled worker, such as, e.g., genetic approaches, library screening, and eluting peptides off of cells displaying MHC class I molecules and sequencing them by mass-spectrometry, (see e.g. Van Der Bruggen P et al., Immunol Rev 188: 51-64 (2002)).
  • MHC I-peptide binding assays based on a measure of the ability of a peptide to stabilize the ternary MHC-peptide complex for a given MHC class I allele, as a comparison to known controls, have been developed (e.g., MHC I-peptide binding assay from ProImmune, Inc., Sarasota, Fla., U.S.). Such approaches can help predict the effectiveness of a putative CD8+ T-cell epitope-peptide or to corroborate empirical evidence regarding a known CD8+ T-cell epitope.
  • CD8+ T-cell epitope is contemplated as being used as a heterologous, CD8+ T-cell epitope of the present invention
  • certain CD8+ T-cell epitopes may be selected based on desirable properties.
  • One objective is to create CD8+ T-cell hyper-immunized cell-targeting molecules, meaning that the heterologous, CD8+ T-cell epitope-peptide is highly immunogenic because it can elicit robust immune responses in vivo when displayed complexed with a MHC class I molecule on the surface of a cell.
  • CD8+ T-cell epitopes may be derived from a number of source molecules already known to be capable of eliciting a vertebrate immune response
  • CD8+ T-cell epitopes may be derived from various naturally occurring proteins foreign to vertebrates, such as, e.g., proteins of pathogenic microorganisms and non-self, cancer antigens.
  • infectious microorganisms may contain numerous proteins with known antigenic and/or immunogenic properties.
  • infectious microorganisms may contain numerous proteins with known antigenic and/or immunogenic sub-regions or epitopes
  • CD8+ T-cell epitopes may be derived from mutated human proteins and/or human proteins aberrantly expressed by malignant human cells, such as, e.g., mutated proteins expressed by cancer cells (see e.g.
  • CD8+ T-cell epitopes may be chosen or derived from a number of source molecules already known to be capable of eliciting a mammalian immune response, including peptides, peptide components of proteins, and peptides derived from proteins.
  • the proteins of intracellular pathogens with mammalian hosts are sources for CD8+ T-cell epitopes.
  • CD8+ T-cell epitopes can be selected or identified from human viruses or other intracellular pathogens, such as, e.g., bacteria like mycobacterium, fungi like toxoplasmae, and protists like trypanosomes.
  • peptides there are many known immunogenic viral peptide components of viral proteins from viruses that infect humans.
  • Numerous human CD8+ T-cell epitopes have been mapped to peptides within proteins from influenza A viruses, such as peptides in the proteins HA glycoproteins FE 17, S139/1, CH65, C05, hemagglutinin 1 (HA1), hemagglutinin 2 (HA2), nonstructural protein I and 2 (NS1 and NS 2), matrix protein 1 and 2 (M1 and M2), nucleoprotein (NP), neuraminidase (NA)), and many of these peptides have been shown to elicit human immune responses, such as by using ex vivo assay (see e.g.
  • HCMV human cytomegaloviruses
  • proteins from human cytomegaloviruses such as peptides in the proteins pp65 (UL83), UL128-131, immediate-early 1 (IE-1; UL123), glycoprotein B, tegument proteins, and many of these peptides have been shown to elicit human immune responses, such as by using ex vivo assays (Schoppel K et al., J Infect Dis 175: 533-44 (1997); Elkington R et al, J Virol 77: 5226-4) (2003); Gibson L et al., J Immunol 172: 2256-64 (2004); Ryckman B et al., J Virol 82: 60-70 (2008); Sacre K et al., J Virol 82: 10143-52 (2008)).
  • CD8+ T-cell epitopes of cancer and/or tumor cell antigens can be identified by the skilled worker using techniques known in the art, such as, e.g., differential genomics, differential proteomics, immunoproteomics, prediction then validation, and genetic approaches like reverse-genetic transfection (see e.g., Admon A et al., Mol Cell Proteomics 2: 388-98 (2003); Purcell A, Gorman J, Mol Cell Proteomics 3: 193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)).
  • T-cell epitopes There are many antigenic and/or immunogenic T-cell epitopes already identified or predicted to occur in human cancer and/or tumor cells.
  • T-cell epitopes have been predicted in human proteins commonly mutated or overexpressed in neoplastic cells, such as, e.g., ALK, CEA.
  • N-acetylglucosaminyl-transferase V (GnT-V), HCA587, HER-2/neu, MAGE, Melan-A/MART-1, MUC-I, p53, and TRAG-3 (see e.g., van der Bruggen P et al., Science 254: 1643-7 (1991); Kawakami Y et al., J Exp Med 180: 347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); Guilloux Y et al., J Exp Med 183: 1173 (1996); Skipper J et al., J Exp Med 183: 527 (1996); Brossart P et al., 93: 4309-17 (1999); Kawashima I et al., Cancer Res 59: 431-5 (1999); Papadopoulos K et al., Clin Cancer Res 5: 2089-93 (1999); Zh
  • T-cell epitopes from human cancer cells have been created (see e.g., Lazoura E, strigopoulos V, Curr Med Chem 12: 629-39 (2005); Douat-Casassus C et al., J Med Chem 50: 1598-609 (2007)).
  • heterologous, CD8+ T-cell epitope is used herein to signify that the heterologous, CD8+ T-cell epitope is not embedded or inserted within a Shiga toxin effector polypeptide's Shiga toxin A1 fragment derived region or is not embedded or inserted within a Shiga toxin effector polypeptide component (see e.g. WO 2015/113005).
  • a cell-targeting molecule of the present invention may comprise multiple, heterologous, CD8+ T-cell epitopes but only one which is a cargo because the other heterologous, CD8+ T-cell epitopes are embedded or inserted into a Shiga toxin A1 fragment region of a Shiga toxin effector polypeptide component of the cell-targeting molecule.
  • CD8+ T-cell epitopes While any heterologous, CD8+ T-cell epitope may be used in the compositions and methods of the present invention, certain CD8+ T-cell epitopes may be preferred based on their known and/or empirically determined characteristics. Immunogenic peptide-epitopes that elicit a human, CD8+ T-cell responses have been described and/or can be identified using techniques known to the skilled worker (see e.g.
  • the MHC gene encodes multiple MHC-I molecular variants. Because MHC class I protein polymorphisms can affect antigen-MHC class I complex recognition by CD8+ T-cells, heterologous T-cell epitopes may be chosen based on knowledge about certain MHC class I polymorphisms and/or the ability of certain antigen-MHC class I complexes to be recognized by T-cells of different genotypes.
  • HLA-Class I-restricted epitopes can be selected or identified by the skilled worker using standard techniques known in the art. The ability of peptides to bind to human MHC Class I molecules can be used to predict the immunogenic potential of putative, CD8+ T-cell epitopes.
  • CD8+ T-cell epitopes may be chosen for use as a CD8+ heterologous, T-cell epitope component of the present invention based on the peptide selectivity of the HLA variants encoded by the alleles more prevalent in certain human populations.
  • the human population is polymorphic for the alpha chain of MHC class I molecules, and the variable alleles are encoded by the HLA genes.
  • Certain T-cell epitopes may be more efficiently presented by a specific HLA molecule, such as, e.g., the commonly occurring HLA variants encoded by the HLA-A allele groups HLA-A2 and HLA-A3.
  • CD8+ T-cell epitopes When choosing CD8+ T-cell epitopes for use as a heterologous, CD8+ T-cell epitope-peptide component of the cell-targeting molecule of the present invention, CD8+ epitopes may be selected which best match the MHC Class I molecules present in the cell-type or cell populations to be targeted. Different MHC class I molecules exhibit preferential binding to particular peptide sequences, and particular peptide-MHC class I variant complexes are specifically recognized by the TCRs of effector T-cells. The skilled worker can use knowledge about MHC class I molecule specificities and TCR specificities to optimize the selection of heterologous T-cell epitopes used in the present invention.
  • the heterologous, CD8+ T-cell epitope-peptide is comprised within a heterologous polypeptide, such as, e.g., an antigen or antigenic protein.
  • a heterologous polypeptide such as, e.g., an antigen or antigenic protein.
  • the heterologous polypeptide is no larger than 27 kDa, 28 kDa, 29 kDa, or 30 kDa.
  • the cell-targeting molecule of the present invention comprises two or more heterologous, CD8+ T-cell epitope-peptides.
  • the combined size of all the heterologous, CD8+ T-cell epitope-peptides is no larger than 27 kDa, 28 kDa, 29 kDa, or 30 kDa.
  • the heterologous, CD8+ T-cell epitope-peptide is processed better in cells with more immunoproteasomes, intermediate proteasomes, and/or thymoproteasomes as compared to standard proteasomes; however, in other embodiments the opposite is true.
  • CD8+ T-cell epitope-peptides for use as a heterologous, CD8+ T-cell epitope-peptide component of a cell-targeting molecule of the present invention
  • multiple factors in the MHC class I presentation system may be considered that can influence CD8+ T-cell epitope generation and transport to receptive MHC class I molecules, such as, e.g., the epitope specificity of the following factors in the target cell: proteasome, ERAAP/ERAP1, tapasin, and TAPs can (see e.g. Akram A, Inman R. Clin Immunol 143: 99-115 (2012)).
  • the heterologous, CD8+ T-cell epitope-peptide is only proteolytically processed in an intact form by an intermediate proteasome (see e.g. Nicolas B et al., Proc Natl Acad Sci USA 107: 18599-604 (2010); criz B et al., J Immunol 189: 3538-47 (2012)).
  • the heterologous, CD8+ T-cell epitope-peptide is not destroyed by standard proteasomes, immunoproteasomes, intermediate proteasomes, and/or thymoproteasomes, which also may depend on the cell type, cytokine environment, tissue location, etc. (see e.g., Morel S et al., Immunity 12: 107-17 (2000); Chapiro J et al., J Immunol 176: 1053-61 (2006); criz B et al., Proc Natl Acad Sci U.S.A.
  • the heterologous, CD8+ T-cell epitope-peptide is considered a “weak” epitope, such as, e.g., “weak” in vivo at eliciting a CD8+ CTL response in a given subject or genotype group or cells derived from the aforementioned (see e.g. Cao W et al., J Immunol 157: 505-11 (1996)).
  • the heterologous, CD8+ T-cell epitope-peptide is a tumor cell epitope, such as, e.g., NY-ESO-1 157-165A (see e.g. Jager E et al. J Exp Med 187: 265-70 (1998)).
  • the heterologous, CD8+ T-cell epitope-peptide has been modified to have a bulky or a charged residue at its amino terminus in order to increase ubiquitination (see e.g., Grant E et al., J Immunol 155: 3750-8 (1995); Townsend A et al., J Exp Med 168: 1211-24 (1998); Kwon Y et al., Proc Natl Acad Sci U.S.A. 95: 7898-903 (1998)).
  • the heterologous, CD8+ T-cell epitope-peptide has been modified to have a hydrophobic amino acid residue at its carboxy terminus in order to increase proteolytic cleavage probability (see e.g., Driscoll J et al., Nature 365: 262-4 (1993); Gaczynska M et al., Nature 365: 264-7 (1993)).
  • the heterologous, CD8+ T-cell epitope-peptide is a Tregitope.
  • Tregitopes are functionally defined as epitope-peptides capable of inducing an immuno-suppressive result.
  • Tregitopes examples include sub-regions of human immunoglobulin G heavy chain constant regions (Fcs) and Fabs (see e.g., Sumida T et al., Arthritis Rheum 40: 2271-3 (1997); Bluestone J, Abbas A, Nat Rev Immunol 3: 253-7 (2003); Hahn B et al., J Immunol 175: 7728-37 (2005); Durinovic-Belló I et al., Proc Natl Acad Sci USA 103: 11683-8 (2006); Sharabi A et al., Proc Natl Acad Sci USA 103: 8810-5 (2006); De Groot A et al., Blood 112: 3303-11 (2008); Sharabi A et al., J Clin Immunol 30: 34-4 (2010); Mozes E, Sharabi A, Autoimmun Rev 10: 22-6 (2010)).
  • Fcs immunoglobulin G heavy chain constant regions
  • heterologous, CD8+ T-cell epitope-peptide of the cell-targeting molecule of the present invention is not generally restricted.
  • the heterologous, CD8+ T-cell epitope-peptide is linked to the cell-targeting molecule at a location carboxy-terminal to the Shiga toxin A1 fragment derived region.
  • the cell-targeting molecule comprises two or more heterologous, CD8+ T-cell epitope-peptides.
  • the multiple heterologous, CD8+ T-cell epitope-peptides are derived from different species, such as, e.g., different microorganism species.
  • the two or more, heterologous, CD8+ T-cell epitope peptides are from a bacterium and a virus or two or more different bacterial species (see e.g. Engelhorn M et al., Mol Ther 16: 773-81 (2008)).
  • the two or more, heterologous, CD8+ T-cell epitope peptides are from a microorganism and a human cancer cell.
  • the cell-targeting molecules of the present invention comprise a cell-targeting binding region capable of specifically binding an extracellular target biomolecule.
  • a binding region of a cell-targeting molecule of the present invention is a cell-targeting component, such as, e.g., a domain, molecular moiety, or agent, capable of binding specifically to an extracellular part of a target biomolecule (e.g. an extracellular target biomolecule) with high affinity.
  • a target biomolecule e.g. an extracellular target biomolecule
  • binding regions There are numerous types of binding regions known to skilled worker or which may be discovered by the skilled worker using techniques known in the art. For example, any cell-targeting component that exhibits the requisite binding characteristics described herein may be used as the binding region in certain embodiments of the cell-targeting molecules of the present invention.
  • An extracellular part of a target biomolecule refers to a portion of its structure exposed to the extracellular environment when the molecule is physically coupled to a cell, such as, e.g., when the target biomolecule is expressed at a cellular surface by the cell.
  • exposed to the extracellular environment means that part of the target biomolecule is accessible by, e.g., an antibody or at least a binding moiety smaller than an antibody such as a single-domain antibody domain, a nanobody, a heavy-chain antibody domain derived from camelids or cartilaginous fishes, a single-chain variable fragment, or any number of engineered alternative scaffolds to immunoglobulins (see below).
  • the exposure to the extracellular environment of or accessibility to a part of target biomolecule physically coupled to a cell may be empirically determined by the skilled worker using methods well known in the art.
  • a binding region of a cell-targeting molecule of the present invention may be, e.g., a ligand, peptide, immunoglobulin-type binding region, monoclonal antibody, engineered antibody derivative, or engineered alternative to antibodies.
  • the binding region of a cell-targeting molecule of the present invention is a proteinaceous moiety capable of binding specifically to an extracellular part of target biomolecule with high affinity.
  • a binding region of a cell-targeting molecule of the present invention may comprise one or more various peptidic or polypeptide moieties, such as randomly generated peptide sequences, naturally occurring ligands or derivatives thereof, immunoglobulin derived domains, synthetically engineered scaffolds as alternatives to immunoglobulin domains, and the like (see e.g., WO 2005/092917; WO 2007/033497; Cheung M et al., Mol Cancer 9: 28 (2010); US2013/196928; WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO 2015/191764).
  • a cell-targeting molecule of the present invention comprises a binding region comprising one or more polypeptides capable of selectively and specifically binding an extracellular target biomolecule.
  • binding regions known in the art that are useful for targeting molecules to specific cell-types via their binding characteristics, such as certain ligands, monoclonal antibodies, engineered antibody derivatives, and engineered alternatives to antibodies.
  • the binding region of a cell-targeting molecule of the present invention comprises a naturally occurring ligand or derivative thereof that retains binding functionality to an extracellular target biomolecule, commonly a cell surface receptor.
  • a naturally occurring ligand or derivative thereof that retains binding functionality to an extracellular target biomolecule, commonly a cell surface receptor.
  • various cytokines, growth factors, and hormones known in the art may be used to target the cell-targeting molecule of the present invention to the cell-surface of specific cell-types expressing a cognate cytokine receptor, growth factor receptor, or hormone receptor.
  • Certain non-limiting examples of ligands include (alternative names are indicated in parentheses) angiogenin.
  • CSFs colony stimulating factors
  • EGFs epidermal growth factors
  • FGFs fibroblast growth factors
  • VEGFs vascular endothelial growth factors
  • IGFs insulin-like growth factors
  • interferons interleukins (such as IL-2, IL-6, and IL-23), nerve growth factors (NGFs), platelet derived growth factors, transforming growth factors (TGFs), and tumor necrosis factors (TNFs).
  • NGFs nerve growth factors
  • TGFs tumor necrosis factors
  • the binding region comprises a synthetic ligand capable of binding an extracellular target biomolecule (see e.g. Liang S et al., J Mol Med 84: 764-73 (2006); Ahmed S et al., Anal Chem 82: 7533-41 (2010); Kaur K et al., Methods Mol Biol 1248: 239-47 (2015)).
  • the binding region comprises a peptidomimetic, such as, e.g., an AApeptide, gamma-AApeptide, and/or sulfono- ⁇ -AApeptide (see e.g., Pilsl L, Reiser O, Amino Acids 41: 709-18 (2011); Akram O et al., Mol Cancer Res 12: 967-78 (2014); Wu H et al., Chemistry 21: 2501-7 (2015); Teng P et al., Chemistry 2016 Mar. 4)).
  • a peptidomimetic such as, e.g., an AApeptide, gamma-AApeptide, and/or sulfono- ⁇ -AApeptide (see e.g., Pilsl L, Reiser O, Amino Acids 41: 709-18 (2011); Akram O et al., Mol Cancer Res 12: 967-78 (2014); Wu H et al., Chemistry 21: 2501-7 (2015); Teng P et al
  • the binding region may comprise an immunoglobulin-type binding region.
  • immunoglobulin-type binding region refers to a polypeptide region capable of binding one or more target biomolecules, such as an antigen or epitope. Binding regions may be functionally defined by their ability to bind to target molecules. Immunoglobulin-type binding regions are commonly derived from antibody or antibody-like structures; however, alternative scaffolds from other sources are contemplated within the scope of the term.
  • Immunoglobulin (Ig) proteins have a structural domain known as an Ig domain.
  • Ig domains range in length from about 70-110 amino acid residues and possess a characteristic Ig-fold, in which typically 7 to 9 antiparallel beta strands arrange into two beta sheets which form a sandwich-like structure. The Ig fold is stabilized by hydrophobic amino acid interactions on inner surfaces of the sandwich and highly conserved disulfide bonds between cysteine residues in the strands.
  • Ig domains may be variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgI or I-set).
  • Ig domains may be associated with a complementarity determining region (CDR), also called a “complementary determining region,” which is important for the specificity of antibodies binding to their epitopes.
  • CDR complementarity determining region
  • Ig-like domains are also found in non-immunoglobulin proteins and are classified on that basis as members of the Ig superfamily of proteins.
  • the HUGO Gene Nomenclature Committee (HGNC) provides a list of members of the Ig-like domain containing family.
  • An immunoglobulin-type binding region may be a polypeptide sequence of an antibody or antigen-binding fragment thereof wherein the amino acid sequence has been varied from that of a native antibody or an Ig-like domain of a non-immunoglobulin protein, for example by molecular engineering or selection by library screening. Because of the relevance of recombinant DNA techniques and in vitro library screening in the generation of immunoglobulin-type binding regions, antibodies can be redesigned to obtain desired characteristics, such as smaller size, cell entry, or other improvements for in vivo and/or therapeutic applications. The possible variations are many and may range from the changing of just one amino acid to the complete redesign of; for example, a variable region. Typically, changes in the variable region will be made in order to improve the antigen-binding characteristics, improve variable region stability, or reduce the potential for immunogenic responses.
  • the immunoglobulin-type binding region is derived from an immunoglobulin binding region, such as an antibody paratope capable of binding an extracellular target biomolecule.
  • the immunoglobulin-type binding region comprises an engineered polypeptide not derived from any immunoglobulin domain but which functions like an immunoglobulin binding region by providing high-affinity binding to an extracellular target biomolecule.
  • This engineered polypeptide may optionally include polypeptide scaffolds comprising or consisting essentially of complementary determining regions from immunoglobulins as described herein.
  • the binding region comprises immunoglobulin domain selected from the group which includes autonomous V H domains, single-domain antibody domains (sdAbs), heavy-chain antibody domains derived from camelids (V H H fragments or V H domain fragments), heavy-chain antibody domains derived from camelid V H H fragments or V H domain fragments, heavy-chain antibody domains derived from cartilaginous fishes, immunoglobulin new antigen receptors (IgNARs), V NAR fragments, single-chain variable (scFv) fragments, nanobodies, Fd fragments consisting of the heavy chain and CHI domains, antibody variable domain (Fv) fragments, permutated Fvs (pFv), single chain Fv-C H 3 minibodies, dimeric C H 2 domain fragments (C H 2D), Fc antigen binding
  • sdAbs single-domain antibody domains derived from camelids
  • V H H fragments or V H domain fragments heavy-chain antibody domains derived from camelid V H H fragments or
  • the binding region of the cell-targeting molecule of the present invention is selected from the group which includes autonomous V H domains (such as, e.g., from camelids, murine, or human sources), single-domain antibody domains (sdAbs), heavy-chain antibody domains derived from camelids (V H H fragments or V H domain fragments), heavy-chain antibody domains derived from camelid V H H fragments or V H domain fragments, heavy-chain antibody domains derived from cartilaginous fishes, immunoglobulin new antigen receptors (IgNARs), V NAR fragments, single-chain variable (scFv) fragments, nanobodies, “camelized” or “camelised” scaffolds comprising a V H domain, Fd fragments consisting of the heavy chain and CHI domains, single chain Fv-C H 3 minibodies, dimeric C H 2 domain fragments (C H 2D), Fc antigen binding domains (Fcabs), isolated complementary determining region 3 (CDR3)
  • the binding region of a molecule of the present invention is multivalent and bispecific but the specificity is to a single target, i.e. the bispecificity is due to different binding regions binding different extracellular epitopes present on the same or a single extracellular target biomolecule (see e.g. Schanzer J et al., Antimicrob Agents Chemother 55: 2369-78 (2011)).
  • Immunoglobulin domains and/or fragments may be modified for use as a cell-targeting moiety in a cell-targeting molecule of the present invention by the addition or removal of a cysteine residue(s) and/or disulfide bond(s) and/or modification of other residues (see e.g. Young N et al., FEBS Lett 377: 135-9 (1995); Wirtz P.
  • the cell-targeting molecule of the present invention comprises an immunoglobulin-type binding region which comprises an immunoglobulin domain and/or Ig-fold structure having an intra-domain disulfide bond, such as, e.g., the disulfide bond found natively between the B and F ⁇ strands of certain immunoglobulins and/or a disulfide bond between their heavy and light chains of or derived from an immunoglobulin.
  • the molecules are very stable even though they do not comprise an intra-domain disulfide bond or any intra-domain disulfide bond within one or more immunoglobulin-type binding regions (see e.g.
  • cell-targeting molecule of the present invention comprises an immunoglobulin-type binding region derived from an immunoglobulin which has been engineered with certain camelid V H H “tetrad” mutations to improve solubility, to improve stability, and/or otherwise “camelize” the binding region (see e.g. Vincke C et al., J Biol Chem 284: 3273-84 (2009); Perchiacca J et al., Proteins 79: 2637-47 (2011); Gil D, Schrum A, Adv Biosci Biotechnol 4: 73-84 (2013)).
  • the cell-targeting molecule of the present invention is engineered to minimize the formation of unwanted, intermolecular associations, multimers, and/or aggregates, such as, e.g., by using disulfide-stabilized scFvs, Fv fragments, or Fabs (see e.g.
  • the cell-targeting molecule of the present invention comprises an immunoglobulin-type binding region which is an scFv engineered not to aggregate, such as, e.g., by using a shorter linker (typically less than twelve amino acid residues) and/or disulfide-stabilized linker that links the heavy and light chain regions of the scFv (see e.g., Brinkmann U et al., Proc Natl Acad Sci USA 90: 7538-42 (1993): Whitlow M et al., Protein Engineering 6: 989-95 (1993); Reiter Y et al., Biochemistry 33: 5451-9 (1994); Gong R et al., Molecular Pharmaceutics 10: 2642-52 (2013)).
  • a shorter linker typically less than twelve amino acid residues
  • disulfide-stabilized linker that links the heavy and light chain regions of the scFv
  • binding regions comprising polypeptides derived from the constant regions of immunoglobulins which may be used a binding region(s) of a cell-targeting molecule of the present invention, such as, e.g., engineered dimeric Fc domains, monomeric Fcs (mFcs), scFv-Fcs, V H H-Fcs, C H 2 domains, monomeric C H 3s domains (mC H 3s), synthetically reprogrammed immunoglobulin domains, and/or hybrid fusions of immunoglobulin domains with ligands (Hofer T et al., Proc Natl Acad Sci USA 105: 12451-6 (2008): Xiao J et al., J Am Chem Soc 131: 13616-8 (2009); Xiao X et al., Biochem Biophys Res Commun 387: 387-92 (2009); Wozniak-Knopp G et al.,
  • the binding region comprises an immunoglobulin domain(s) that is not from a traditional immunoglobulin but rather is from a cell-membrane bound receptor which functions as part of the immune system.
  • the cell-targeting binding region of the cell-targeting molecule of the present invention comprises or consists essentially of a single-chain T-cell receptor variable fragment (scTv), a single-chain TCR (scTCR), disulfide-stabilized T-cell receptor variable fragment (dsTv), and/or T-cell receptor variable fragment disulfide-stabilized Fv heterodimer (TCR dsFv heterodimer).
  • the cell-targeting binding region of the cell-targeting molecule of the present invention is a soluble, single-chain T-cell receptor variable fragment (soluble scTv) and/or TCR dsFv heterodimer (see e.g.
  • scTvs Unlike most immunoglobulins, naturally occurring scTvs typically bind with moderate affinity to an epitope-peptide-MHC protein complex. While scTvs in isolation bind to such cell-surface targets (i.e. extracellular target biomolecules physically coupled to cells in the form of pMHCs), scTvs also can retain their binding specificity for cell-targeting upon fusion to an effector molecule, such as a toxin protein (see e.g. Epel M et al., Cancer Immunol Immunother 51: 563-73 (2002)).
  • an effector molecule such as a toxin protein
  • the targets are typically MHC protein-non-self epitope-peptide complexes which are displayed on the surfaces of vertebrate cells.
  • TCRs which have been deleted during thymic selection often bind self-epitope-MHC complexes with high affinities.
  • the scTv may be mutated in its variable domain(s) to improve the affinity and/or stability of desired binding interactions (see e.g. Shusta E et al., Nat Bioteclmol 18: 754-9 (2000): Richman S et al., Mol Immunol 46: 902-16 (2009)).
  • solubility increasing mutations in a scTCR and/or a non-native disulfide bond in the TCR invariant region can greatly increase the stability and folding characteristics of a scTv (see e.g. Molloy P et al., Curr Opin Pharmacol 5: 438-43 (2005); WO2003020763).
  • scTv may improve stability and production of a scTv by orienting the domains of the scTCR in the amino-to-carboxy orientation of Va domain-linker-VD3 domain (Loset G et al., Protein Eng Des Sel 20: 461-72 (2007): Richman S et al., Mol Immunol 46: 902-16 (2009)).
  • the introduction of various mutations may also improve expression in a host cell system, e.g. in yeast cells (see e.g. Richman S et al., Mol Immunol 46: 902-16 (2009)).
  • the binding region comprises an engineered, alternative scaffold to immunoglobulin domains.
  • Engineered alternative scaffolds are known in the art which exhibit similar functional characteristics to immunoglobulin-derived structures, such as high-affinity and specific binding of target biomolecules, and may provide improved characteristics to certain immunoglobulin domains, such as, e.g., greater stability or reduced immunogenicity.
  • alternative scaffolds to immunoglobulins are less than kilodaltons (kDa), consist of a single polypeptide chain, lack cysteine residues, and exhibit relatively high thermodynamic stability.
  • the immunoglobulin-type binding region is selected from the group which includes engineered, Armadillo repeat polypeptides (ArmRPs); engineered, fibronectin-derived, 10 th fibronectin type III (10Fn3) domains (monobodies, AdNectinsTM, or AdNexinsm); engineered, tenascin-derived, tenascin type III domains (CentrynsTM); engineered, ankyrin repeat motif containing polypeptides (DARPinsTM); engineered, low-density-lipoprotein-receptor-derived, A domains (LDLR-A) (AvimersTM); lipocalins (anticalins); engineered, protease inhibitor-derived, Kunitz domains; engineered, Protein-A-derived, Z domains (AffibodiesTM): engineered, gamma-B crystallin-derived scaffold or engineered, ubiquit
  • antibody-like binding abilities may be conferred by non-proteinaceous compounds, such as, e.g., oligomers.
  • RNA molecules, DNA molecules, carbohydrates, and glycocalyxcalixarenes see e.g. Sansone F, Casnati A, Chem Soc Rev 42: 4623-39 (2013)
  • partially proteinaceous compounds such as, e.g., phenol-formaldehyde cyclic oligomers coupled with peptides and calixarene-peptide compositions (see e.g. U.S. Pat. No. 5,770,380).
  • protease-resistant immunoglobulin domains and/or synthetically stabilized scFv fragments such as to avoid instability during storage or after administration but before reaching a target cell (see e.g. Ewert S et al., Methods 34: 184-99 (2004); Honegger et al., Protein Eng Des Sel 22: 135-47 (2009); Miller et al., Protein Eng Des Sel 23: 549-57 (2010); Hussack G et al., Protein Eng Des Sel 27: 191-8 (2014)).
  • binding region structures may be used as a component of a cell-targeting molecule of the present invention as long as the binding region component has a dissociation constant of 10 ⁇ 5 to 10 ⁇ 12 moles per liter, preferably less than 200 nanomolar (nM), towards an extracellular target biomolecule.
  • the cell-targeting molecules of the present invention comprise a Shiga toxin effector polypeptide of the present invention linked and/or fused to a binding region capable of specifically binding an extracellular part of a target biomolecule or an extracellular target biomolecule.
  • Extracellular target biomolecules may be selected based on numerous criteria, such as a criterion described herein.
  • the binding region of a cell-targeting molecules of the present invention comprises a proteinaceous region capable of binding specifically to an extracellular part of a target biomolecule or an extracellular target biomolecule, preferably which is physically coupled to the surface of a cell-type of interest, such as, e.g., a cancer cell, tumor cell, plasma cell, infected cell, or host cell harboring an intracellular pathogen.
  • a cell-type of interest such as, e.g., a cancer cell, tumor cell, plasma cell, infected cell, or host cell harboring an intracellular pathogen.
  • the targeted cell-type will be expressing a MHC class I molecule(s).
  • Target biomolecules bound by the binding region of a cell-targeting molecule of the present invention may include biomarkers over-proportionately or exclusively present on cancer cells, immune cells, and/or cells infected with intracellular pathogens, such as, e.g., viruses, bacteria, fungi, prions, or protozoans.
  • pathogens such as, e.g., viruses, bacteria, fungi, prions, or protozoans.
  • target biomolecule refers to a biological molecule, commonly a proteinaceous molecule or a protein modified by post-translational modifications, such as glycosylation, that is bound by a binding region of a cell-targeting molecule of the present invention resulting in the targeting of the cell-targeting molecule to a specific cell, cell-type, and/or location within a multicellular organism.
  • extracellular with regard to a target biomolecule refers to a biomolecule that has at least a portion of its structure exposed to the extracellular environment.
  • the exposure to the extracellular environment of or accessibility to a part of target biomolecule coupled to a cell may be empirically determined by the skilled worker using methods well known in the art.
  • Non-limiting examples of extracellular target biomolecules include cell membrane components, transmembrane spanning proteins, cell membrane-anchored biomolecules, cell-surface-bound biomolecules, and secreted biomolecules.
  • the phrase “physically coupled” when used to describe a target biomolecule means covalent and/or non-covalent intermolecular interactions couple the target biomolecule, or a portion thereof, to the outside of a cell, such as a plurality of non-covalent interactions between the target biomolecule and the cell where the energy of each single interaction is on the order of at least about 1-5 kiloCalories (e.g., electrostatic bonds, hydrogen bonds, ionic bonds, Van der Walls interactions, hydrophobic forces, etc.). All integral membrane proteins can be found physically coupled to a cell membrane, as well as peripheral membrane proteins.
  • an extracellular target biomolecule might comprise a transmembrane spanning region, a lipid anchor, a glycolipid anchor, and/or be non-covalently associated (e.g. via non-specific hydrophobic interactions and/or lipid binding interactions) with a factor comprising any one of the foregoing.
  • Extracellular parts of target biomolecules may include various epitopes, including unmodified polypeptides, polypeptides modified by the addition of biochemical functional groups, and glycolipids (see e.g. U.S. Pat. No. 5,091,178: EP2431743).
  • the binding regions of the cell-targeting molecules of the present invention may be designed or selected based on numerous criteria, such as the cell-type specific expression of their target biomolecules, the physical localization of their target biomolecules with regard to specific cell-types, and/or the properties of their target biomolecules.
  • certain cell-targeting molecules of the present invention comprise binding regions capable of binding cell-surface target biomolecules that are expressed at a cellular surface exclusively by only one cell-type of a species or only one cell-type within a multicellular organism. It is desirable, but not necessary, that an extracellular target biomolecule be intrinsically internalized or be readily forced to internalize upon interacting with a cell-targeting molecule of the present invention.
  • any desired target biomolecule may be used to design or select a suitable binding region to be associated and/or coupled with a Shiga toxin effector polypeptide to produce a cell-targeting molecule of the present invention.
  • a cell-targeting molecule of the invention may further comprise a carboxy-terminal endoplasmic retention/retrieval signal motif, such as, e.g., the amino acids KDEL at the carboxy terminus of a proteinaceous component of the cell-targeting molecule (see e.g. PCT/US2015/19684).
  • Individual cell-targeting binding regions, Shiga toxin effector polypeptides, CD8+ T-cell epitope cargos, and/or other components of the cell-targeting molecules present invention may be suitably linked to each other via one or more linkers well known in the art and/or described herein (see e.g., WO 2014/164693; WO 2015/113005: WO 2015/113007; WO 2015/138452: WO 2015/191764).
  • Individual polypeptide subcomponents of the binding regions e.g. heavy chain variable regions (V H ), light chain variable regions (V L ), CDR, and/or ABR regions, may be suitably linked to each other via one or more linkers well known in the art and/or described herein.
  • Proteinaceous components of the invention may be suitably linked to each other or other polypeptide components of the invention via one or more linkers well known in the art.
  • Peptide components of the invention e.g., a heterologous, CD8+ T-cell epitope-peptide cargos, may be suitably linked to another component of the invention via one or more linkers, such as a proteinaceous linker, which is well known in the art.
  • Suitable linkers are generally those which allow each polypeptide component of the present invention to fold with a three-dimensional structure very similar to the polypeptide components produced individually without any linker or another component associated with it.
  • Suitable linkers include single amino acids, peptides, polypeptides, and linkers lacking any of the aforementioned, such as various non-proteinaceous carbon chains, whether branched or cyclic (see e.g. Alley S et al., Bioconjug Chem 19: 759-65 (2008); Ducry L, Stump B. Bioconjug Chem 21: 5-13 (2010)).
  • Suitable linkers may be proteinaceous and comprise one or more amino acids, peptides, and/or polypeptides. Proteinaceous linkers are suitable for both recombinant fusion proteins and chemically linked conjugates.
  • a proteinaceous linker typically has from about 2 to about 50 amino acid residues, such as, e.g., from about 5 to about 30 or from about 6 to about 25 amino acid residues. The length of the linker selected will depend upon a variety of factors, such as, e.g., the desired property or properties for which the linker is being selected.
  • the linker is proteinaceous and is linked near the terminus of a protein component of the present invention, typically within about 20 amino acids of the terminus.
  • suitable proteinaceous linkers comprise stretches of glycines and/or serines for flexibility combined with one or more charged residues, such as, e.g., a glutamate and/or lysine residue(s) for solubility (see e.g. Whitlow M et al., Protein Engineering 6: 989-95 (1993)).
  • Suitable linkers may be non-proteinaceous, such as, e.g. chemical linkers.
  • Suitable methods for linkage of the components of the cell-targeting molecules of the present invention may be by any method presently known in the art for accomplishing such, as long as the attachment does not substantially impede the binding capability of the cell-targeting binding region and/or when appropriate the desired Shiga toxin effector function(s) as measured by an appropriate assay, including assays described herein.
  • disulfide bonds and thioether bonds may be used to link two or more proteinaceous components of a cell-targeting molecule of the present invention.
  • the specific order or orientation is not fixed for the components unless stipulated.
  • the arrangement of the Shiga toxin effector polypeptide(s), heterologous, CD8+ T-cell epitope cargo(s), the binding region(s), and any optional linker(s), in relation to each other or the entire cell-targeting molecule is not fixed (see e.g. FIG. 1 ) unless specifically noted.
  • the components of the cell-targeting molecules of the present invention may be arranged in any order provided that the desired activity(ies) of the binding region, Shiga toxin effector polypeptide, and heterologous, CD8+ T-cell epitope are not eliminated.
  • the cell-targeting molecules of the present invention comprise a Shiga toxin A Subunit effector polypeptide, a cell-targeting binding region, and a heterologous, CD8+ T-cell epitope-peptide cargo which is not embedded or inserted in the Shiga toxin A1 fragment region and/or the Shiga toxin A Subunit effector polypeptide.
  • a cell-targeting molecule with the ability to deliver a CD8+ T-cell epitope cargo to the MHC class I presentation pathway of a target cell may be created, in principle, by linking any heterologous, CD8+ T-cell epitope-peptide to any combination of cell-targeting binding region and Shiga toxin A Subunit effector polypeptide as long as the resulting cell-targeting molecule has a cellular internalization capability (such as, e.g., via endocytosis) provided by at least the Shiga toxin effector, the cell-targeting moiety, or the structural combination of them together, and as long as the Shiga toxin effector polypeptide component or the cell-targeting molecule structure as a whole, provides, once inside a target cell, sufficient subcellular routing to a subcellular compartment competent for delivery of the T-cell epitope-peptide to the MHC class I presentation pathway of the target cell, such as, e.g., to the cytosol or the endoplasmic reti
  • the cell-targeting molecules of the present invention each comprise at least one Shiga toxin A Subunit effector polypeptide derived from at least one A Subunit of a member of the Shiga toxin family.
  • the Shiga toxin effector polypeptide of the cell-targeting molecule of the present invention comprises or consists essentially of a truncated Shiga toxin A Subunit.
  • Truncations of Shiga toxin A Subunits might result in the deletion of an entire epitope(s) and/or epitope region(s), B-cell epitopes, CD4+ T-cell epitopes, and/or furin-cleavage sites without affecting Shiga toxin effector functions, such as, e.g., catalytic activity and cytotoxicity.
  • the smallest Shiga toxin A Subunit fragment shown to exhibit full enzymatic activity was a polypeptide composed of residues 1-239 of Slt1A (LaPointe P et al., J Biol Chem 280: 23310-18 (2005)).
  • Shiga toxin A Subunit fragment shown to exhibit significant enzymatic activity was a polypeptide composed of residues 75-247 of StxA (A1-Jaufy A et al., Infect Immun 62; 956-60 (1994)).
  • Shiga toxin effector polypeptides of the present invention may commonly be smaller than the full-length Shiga toxin A Subunit, the Shiga toxin effector polypeptide of a cell-targeting molecule of the present invention may need to maintain the polypeptide region from amino acid position 77 to 239 (SLT-1A (SEQ ID NO: 1) or StxA (SEQ ID NO:2)) or the equivalent in other A Subunits of members of the Shiga toxin family (e.g. 77 to 238 of (SEQ ID NOs: 3 and 7-18)).
  • the Shiga toxin effector polypeptides of the present invention derived from a Shiga toxin may comprise or consist essentially of the polypeptide represented by the amino acid sequence selected from amino acids 75 to 251, 1 to 241, 1 to 251, and I to 261 of any one of SEQ ID NOs: 1-2 and 4-6.
  • Shiga toxin effector polypeptides derived from a Shiga toxin 2 may comprise or consist essentially of the polypeptide represented by the amino acid sequence selected from amino acids 75 to 250, 1 to 241, 1 to 250, and 1 to 260 of any one of SEQ ID NOs: 3 and 7-18.
  • the Shiga toxin effector polypeptide differs from a naturally occurring Shiga toxin A Subunit by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by no more than that which retains at least 85%, 90%, 95%, 99%, or more amino acid sequence identity).
  • the invention further provides variants of the cell-targeting molecules of the present invention, wherein the Shiga toxin effector polypeptide differs from a naturally occurring Shiga toxin A Subunit by only or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by no more than that which retains at least 85%, 90% o, 95%, 99% or more amino acid sequence identity).
  • a molecule of the present invention derived from an A Subunit of a member of the Shiga toxin family may comprise additions, deletions, truncations, or other alterations from the original sequence as long as at least 85%, 90%, 95%, 99% or more amino acid sequence identity is maintained to a naturally occurring Shiga toxin A Subunit, such as, e.g., wherein there is a disrupted, furin-cleavage motif at the carboxy terminus of a Shiga toxin A1 fragment derived region.
  • the Shiga toxin effector polypeptide of a molecule of the present invention comprises or consists essentially of amino acid sequences having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7% overall sequence identity to a naturally occurring Shiga toxin A Subunit (e.g., any one of SEQ ID NOs: 1-18), such as, e.g., wherein there is a disrupted, furin-cleavage motif at the carboxy terminus of a Shiga toxin A1 fragment derived region.
  • a naturally occurring Shiga toxin A Subunit e.g., any one of SEQ ID NOs: 1-18
  • a disrupted, furin-cleavage motif at the carboxy terminus of a Shiga toxin A1 fragment derived region.
  • Shiga toxin effector polypeptide of a cell-targeting molecule of the present of invention wherein the Shiga toxin derived polypeptide comprises one or more mutations (e.g. substitutions, deletions, insertions, or inversions) as compared to a naturally occurring Shiga toxin A Subunit.
  • mutations e.g. substitutions, deletions, insertions, or inversions
  • the Shiga toxin effector polypeptides have sufficient sequence identity to a wild-type Shiga toxin A Subunit to retain cytotoxicity after entry into a cell, either by well-known methods of host cell transformation, transfection, infection or induction, or by internalization mediated by a cell-targeting binding region linked with the Shiga toxin effector polypeptide.
  • the most critical region of a Shiga toxin A Subunit for enzymatic activity is the active site, which is positioned around amino acid residues 137/138 to 209/210, depending on the variant, such as any one of SEQ ID NOs: 1-18.
  • the Shiga toxin effector polypeptides may preferably but not necessarily maintain one or more conserved amino acids at positions, such as those found at positions 77, 167, 170, and 176 in StxA, SLT-1A, or the equivalent conserved position in other members of the Shiga toxin family which are typically required for potent cytotoxic activity.
  • the capacity of a cytotoxic cell-targeting molecule of the present invention to cause cell death, e.g. its cytotoxicity may be measured using any one or more of a number of assays well known in the art.
  • cell-targeting molecules of the invention that comprise Shiga toxin effector polypeptides with even considerable reductions in the Shiga toxin effector function(s) of subcellular routing as compared to wild-type Shiga toxin effector polypeptides may still be capable of delivering their heterologous, CD8+ T-cell epitope-peptide cargos to the MHC class I presentation pathway of a target cell, such as, e.g., in sufficient quantities to induce an immune response involving intercellular engagement of a CD8+ immune cell and/or to detect certain subcellular compartments of specific cell-types as even presentation of a single pMHC I complex is sufficient for intercellular engagement of a presenting cell by a CTL for cytolysis (Sykulev Y et al., Immunity 4: 565-71 (1996)).

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