WO2022125632A1 - Cellules exprimant un récepteur antigénique chimérique pour cibler des motifs moléculaires associés aux microbes - Google Patents

Cellules exprimant un récepteur antigénique chimérique pour cibler des motifs moléculaires associés aux microbes Download PDF

Info

Publication number
WO2022125632A1
WO2022125632A1 PCT/US2021/062362 US2021062362W WO2022125632A1 WO 2022125632 A1 WO2022125632 A1 WO 2022125632A1 US 2021062362 W US2021062362 W US 2021062362W WO 2022125632 A1 WO2022125632 A1 WO 2022125632A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
mtm
microbe
based car
binding
Prior art date
Application number
PCT/US2021/062362
Other languages
English (en)
Inventor
Keith Crawford
Original Assignee
Miraki Innovation Think Tank Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Miraki Innovation Think Tank Llc filed Critical Miraki Innovation Think Tank Llc
Publication of WO2022125632A1 publication Critical patent/WO2022125632A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4726—Lectins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • ASCII text file A sequence listing in electronic (ASCII text file) format is filed with this application and incorporated herein by reference.
  • the name of the ASCII text file is
  • the technology described herein generally relates to immune cell-based tools for use in detecting, identifying, removing and/or killing microbes, microbe components and microbe- infected cells that are present in a sample or a target area, including bodily fluids (e.g. blood and tissues), food, water, and environmental surfaces.
  • bodily fluids e.g. blood and tissues
  • T cells capable of recognizing selected antigens on the surface of cells has important clinical applications in T cell-based cancer therapies.
  • One current strategy incorporates the use of genetic engineering to express a chimeric antigen receptor (CAR) on T cells.
  • CAR chimeric antigen receptor
  • the extracellular domain of a typical CAR consists of the VH and VL domains - singlechain fragment variable (scFv) - from the antigen binding sites of a monoclonal antibody.
  • the scFv is linked to a flexible transmembrane domain followed by a tyrosine-based activation motif such as that from CD3 ⁇ (Sadelain et al. Curr. Opin. Immunol. 21, 215-223 (2009); Gross et al. Proc. Natl.
  • CAR T cells offer the opportunity to seek out and destroy cancer cells by recognizing tumor-associated antigens (TAA) expressed on their surface (Sadelain et al. Curr. Opin. Immunol. 21, 215-223 (2009)).
  • TAA tumor-associated antigens
  • CAR-based T cells recognize and bind to cell surface markers associated with cancer
  • such cells might be engineered to recognize and bind microbial pathogens, whether free floating or associated with a cell.
  • Important microbes and their components include, for example, environmental and pathogenic agents, including bacteria, viruses, toxins, and antigens.
  • a variety of cellbased biosensor products have been commercially developed and released.
  • a specific example of a cell-based biosensor platform currently in use is the CANARY® biosensor technology of PathSensors, Inc. This platform, based on the work of Rider et al. (Science 301 :213-215 (2003)), enables reliable identification of specific airborne and liquid-based pathogens.
  • the biological backbone of the CANARY® biosensor is comprised of a genetically-engineered B cell expressing an extracellularly bound, antigen-specific antibody that can bind its cognate antigen or pathogenic agent.
  • an antigen-containing sample interacts with the antibody on the extracellular surface of the biosensor, an intracellular signaling cascade is activated resulting in the release of Ca 2+ within the B cells.
  • the B cells express aequorin, a Ca 2+ -sensitive photoprotein, which results in cell luminescence in the presence of elevated intracellular Ca 2+ levels.
  • the luminescence can be used to indicate antigen binding.
  • the CANARY® system can be used to efficiently identify a number of specific antigens, including those from bacteria, viruses, and toxins.
  • expansion of the antigen test repertoire is complex and costly.
  • Different antigen- or pathogen-specific biosensors must be constructed to recognized each and every selected antigen, which requires multiple steps including production of hybridoma cell lines, cloning of nucleic acid sequences encoding the antibodies, and expressing cloned antibodies as transmembrane proteins on the surface of a B cell line genetically engineered to luminesce upon binding of the cognate antigen (e.g., a pathogen) by the antibody.
  • the present invention provides chimeric antigen receptors (CARs) and CAR- expressing cells that recognize and bind to one or more targets, where at least one of the targets is a microbe-associated molecular pattern (MAMP).
  • the CARs are integral membrane proteins that comprise (i) one or more primary target-binding domains, (ii) optionally one or more secondary target-binding domains, (iii) a transmembrane domain, and (iv) an intracellular activation domain.
  • the CARs may optionally further comprise a hinge domain.
  • the primary target-binding domains are microbe-binding domains, which comprise at least a portion of a microbe-targeting molecule (MTM) or engineered MTM that has the shared characteristic of binding to one or more MAMPs.
  • MTM microbe-targeting molecule
  • the optional secondary target-binding domains do not comprise an MTM or engineered MTM.
  • the optional secondary target-binding domains help to stabilize interactions between the CAR and a microbe bound by the protein.
  • MTM-based CARs of the invention can be used to contact, and optionally isolate, microbes and microbial components from a sample based on the identity of the MAMP produced by the microbe, rather than the identity of microbe itself. While some MAMPs are produced by only a single species of microbe, other MAMPs are shared across species. Thus, while some MTM-based CARs of the invention bind to only MAMPs of a particular species of microbe, other MTM-based CARs of the invention can bind to MAMPs produced by all members of a particular class, order, family, genus or sub-genus of microbe.
  • MTM-based CARs and “engineered MTM-based CARs” refers to any of the molecules described herein (or described in patents or patent application incorporated by reference) that can bind to a microbe or microbe component and that comprises at least one microbe-binding domain.
  • MTM-based CARs should be understood to include both CARs comprising a naturally-occurring MTM and CARs comprising an engineered MTM.
  • MTM-based CARs of the invention are defined based on their binding activity, it will be apparent that both naturally-occurring and engineered MTMs used in the CARs will comprise at least one microbe-binding domain, i.e. a domain that recognizes and binds to one or more MAMPs (including at least two, at least three, at least four, at least five, or more) as described herein.
  • a microbe-binding domain can be a naturally-occurring or a synthetic molecule.
  • a microbe-binding domain can be a recombinant molecule.
  • the MTMs of the invention may have one or more additional domains that may include, but are not limited to, an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain, and an immunoglobulin-like domain.
  • Non-limiting examples of the MTMs that may be used as the microbe-binding domain of the CARs of the invention include three broad categories of suitable MTMs, namely: (i) collectin-based MTMs, (ii) ficolin-based MTMs, and (iii) toll-like receptor-based MTMs.
  • the CARs of the invention include one or more primary targetbinding domains (i.e. microbe-binding domains), and optionally one or more secondary targetbinding domains.
  • Suitable secondary target-binding domains include, but are not limited to, universal capture sites, such a binding domain common to all CARs that allows the protein to be isolated from a sample after it has bound a MAMP.
  • the secondary domains may bind a target that stabilizes the binding between the microbe binding domain (i.e. the primary target-binding domain) and a MAMP. Such stabilization could serve to maintain the binding over longer periods of time and/or increase the affinity of the CAR for the MAMP.
  • the secondary domain may bind a non-mutable target of a pathogen, thus preventing the pathogen from escaping immune surveillance.
  • the target of the secondary domain may be, but is not limited to, a peptide, a polypeptide, a sugar, DNA, RNA, or other nucleic acid molecule.
  • the secondary binding domain may be a portion of an antibody, such as a single-chain variable fragment (scFv).
  • the target-binding domains (primary and secondary) present in the CARs of the invention are linked by short peptides of 1-50 amino acids in length.
  • the peptide linkers will vary widely based on the identity of the primary and secondary target-binding domains.
  • Suitable linkers include Gly x , wherein x is between 2 and 49 Gly, and where one or more of the Gly may be substituted by Ser.
  • Specific linkers include Alas, Gly x Ser (where x is 1-5 Gly), Gly x SerGly x Ser (where x is 1-5 Gly), Gly4Ser, and Gly4Ser repeats.
  • transmembrane domain of the CARs of the invention include the transmembrane regions of the human CD8a chain.
  • Non-limiting examples of the activation domain of the CARs of the invention include one or more of the cytoplasmic region of CD27, CD28, CD137 (41BB), 0X40, HVEM, CD3( ⁇ , and FcRs.
  • Non-limiting examples of the cells expressing the CARs of the invention include T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells and neutrophils.
  • the present invention is directed to CARs comprising at least one collectin-based MTM as a primary target-binding domain.
  • the collectinbased MTMs are either naturally-occurring collectin proteins or engineered MTM fusion proteins that comprise at least one collectin microbe-binding domain and at least one additional domain.
  • the naturally-occurring collectin protein may be any one of (i) mannose-binding lectin (MBL), (ii) surfactant protein A (SP-A), (iii) surfactant protein D (SP-D), (iv) collectin liver 1 (CL-L1), (v) collectin placenta 1 (CL-P1), (vi) conglutinin collectin of 43 kDa (CL-43), (vii) collectin of 46 kDa (CL-46), (viii) collectin kidney 1 (CL-K1), (ix) conglutinin, and (x) a sequence variant having at least 85% sequence identity to any one of (i)-(ix).
  • the collectin is (i) a naturally-occurring MBL, (ii) a truncated form of naturally-occurring MBL, (iii) an engineered form of MBL, or (iv) a sequence variant having at least 85% sequence identity to any one of (i), (ii) or (iii).
  • the collectin is a naturally-occurring MBL as set forth in SEQ ID NO: 1 or a sequence variant having at least 85% sequence identity with SEQ ID NO: 1 that retains the activity of the native protein.
  • the collectin is a truncated form of naturally-occurring MBL as set forth in any one of SEQ ID NOs:2-5 or a sequence variant having at least 85% sequence identity with any one of SEQ ID NOs:2-5 that retains the activity of the native protein.
  • the collectin microbe-binding domain of the collectin-based engineered MTMs comprises a carbohydrate recognition domain (CRD) of a collectin.
  • the collectin may be any one of (i) mannose-binding lectin (MBL), (ii) surfactant protein A (SP-A), (iii) surfactant protein D (SP-D), (iv) collectin liver 1 (CL-L1), (v) collectin placenta 1 (CL-P1), (vi) conglutinin collectin of 43 kDa (CL-43), (vii) collectin of 46 kDa (CL-46), (viii) collectin kidney 1 (CL- Kl), (ix) conglutinin, and (x) a sequence variant having at least 85% sequence identity to any one of (i)-(ix).
  • the at least one additional domain of the collectin-based engineered MTMs may be one or more of (xi) a collectin cysteine-rich domain, (xii) a collectin collagen-like domain, (xiii) a collectin coiled-coil neck domain, (xiv) a ficolin short N-terminal domain, (xv) a ficolin collagen-like domain, (xvi) a Toll-like receptor (TLR) transmembrane helix, (xvii) a TLR C- terminal cytoplasmic signaling domain, (xviii) an oligomerization domain, (xix) a signal domain, (xx) an anchor domain, (xxi) a collagen-like domain, (xxii) a fibrinogen-like domain, (xxiii) an immunoglobulin domain, (xxiv) an immunoglobulin-like domain, and (xxv) a sequence variant having at least 85% sequence identity to any one of (xi)-
  • the at least one additional domain is an immunoglobulin domain.
  • the immunoglobulin domain may comprise the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the collectin microbe-binding domain comprises the CRD of MBL or a sequence variant thereof having at least 85% sequence identity to the CRD of MBL.
  • the CRD of MBL may comprise the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, and 5 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, and 5.
  • the collectin-based MTM comprises the CRD of MBL or a sequence variant thereof having at least 85% sequence identity to the CRD of MBL and an immunoglobulin domain.
  • the CRD of MBL may comprise the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, and 5 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, and 5, and an immunoglobulin domain comprising the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the CARs comprise at least one collectin-based engineered MTM, wherein the collectin-based engineered MTM is an FcMBL of SEQ ID NO:6, 7, 8 or 9, or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO:6, 7, 8 or 9.
  • FcMBL MTMs comprise a mannose-binding ligand (MBL) linked to the Fc domain of human IgG (Fc).
  • the CARs comprise at least two collectinbased engineered MTMs, wherein the two MTMs are selected from SEQ ID NO: 6, 7, 8 or 9, or sequence variants thereof having at least 85% sequence identity to SEQ ID NO: 6, 7, 8 or 9.
  • the CARs that comprise a collectin-based MTM as a primary target-binding domain further comprise (i) optionally, at least one secondary target-binding domain, (ii) a transmembrane domain and (iii) an activation domain.
  • suitable transmembrane domains include, but are not limited to, the hinge and transmembrane regions of the human CD8a chain.
  • suitable activation domains include, but are not limited to, the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), 0X40, HVEM, CD3 ⁇ ; and FcRs.
  • the invention is directed to CARs comprising at least one ficolin-based MTM as a primary target-binding domain.
  • the ficolin-based MTMs are either naturally-occurring ficolin proteins or engineered MTM fusion proteins that comprise at least one ficolin microbe-binding domain and at least one additional domain.
  • the naturally-occurring ficolin protein may be any one of (i) ficolin 1, (ii) ficolin 2, (iii) ficolin 3, and (iv) a sequence variant having at least 85% sequence identity to any one of (i)- (iii).
  • the ficolin microbe-binding domain of the ficolin-based engineered MTMs may comprise the fibrinogen-like domain of a ficolin.
  • the ficolin may be any one of (i) ficolin 1, (ii) ficolin 2, (iii) ficolin 3, and (iv) a sequence variant having at least 85% sequence identity to any one of (i)-(iii).
  • the at least one additional domain of the ficolin-based engineered MTMs may be one or more of (v) a ficolin short N-terminal domain, (vi) a ficolin collagen-like domain, (vii) a collectin cysteine-rich domain, (viii) a collectin collagen-like domain, (ix) a collectin coiled-coil neck domain, (x) a TLR transmembrane helix, (xi) a TLR C-terminal cytoplasmic signaling domain, (xii) an oligomerization domain, (xiii) a signal domain, (xiv) an anchor domain, (xv) a collagen-like domain, (xvi) a fibrinogen-like domain, (xvii) an immunoglobulin domain, (xviii) an immunoglobulin-like domain, and (xix) a sequence variant having at least 85% sequence identity to any one of (v)-(xviii).
  • the at least one additional domain is an immunoglobulin domain.
  • the immunoglobulin domain may comprise the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the ficolin-based engineered MTMs comprise a ficolin microbe-binding domain comprising the fibrinogen-like domain of any one of SEQ ID NOs: 21, 22 and 23 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs:21, 22 and 23.
  • the ficolin-based engineered MTMs comprise a ficolin microbe-binding domain comprising the fibrinogen-like domain of any one of SEQ ID NOs: 21, 22 and 23 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs: 21, 22 and 23 and an immunoglobulin domain comprising the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the CARs that comprise a ficolin-based MTM as a primary target-binding domain further comprise (i) optionally at least one secondary target-binding domain, (ii) a transmembrane domain and (iii) an activation domain.
  • suitable transmembrane domains include, but are not limited to, the hinge and transmembrane regions of the human CD8a chain.
  • suitable activation domains include, but are not limited to, the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), 0X40, HVEM, CD3 ⁇ ; and FcRs.
  • the invention is directed to CARs comprising at least one tolllike receptor (TLR)-based MTM as a primary target-binding domain.
  • TLR-based MTMs are either naturally-occurring TLR proteins or engineered MTM fusion proteins that comprise at least one TLR microbe-binding domain and at least one additional domain.
  • the naturally-occurring TLR protein may be any one of (i) TLR1, (ii) TLR2, (iii) TLR3, (iv) TLR4, (v) TLR5, (vi) TLR6, (vii) TLR7, (viii) TLR8, (ix) TLR9, (x) TLR10, and (xi) a sequence variant having at least 85% sequence identity to any one of (i)-(x).
  • the TLR microbe-binding domain of the TLR-based engineered MTMs may comprise the N-terminal ligand-binding domain of a TLR.
  • the TLR may be any one of (i) TLR1, (ii) TLR2, (iii) TLR3, (iv) TLR4, (v) TLR5, (vi) TLR6, (vii) TLR7, (viii) TLR8, (ix) TLR9, (x) TLR10, and (xi) a sequence variant having at least 85% sequence identity to any one of (i)-(x).
  • the at least one additional domain of the TLR-based engineered MTMs may be one or more of (xii) a TLR transmembrane helix, (xiii) a TLR C-terminal cytoplasmic signaling domain, (xiv) a ficolin short N-terminal domain, (xv) a ficolin collagen-like domain, (xvi) a collectin cysteine-rich domain, (xvii) a collectin collagen-like domain, (xviii) a collectin coiled- coil neck domain, (xix) an oligomerization domain, (xx) a signal domain, (xxi) an anchor domain, (xxii) a collagen-like domain, (xxiii) a fibrinogen-like domain, (xxiv) an immunoglobulin domain, (xxv) an immunoglobulin-like domain, and (xxvi) a sequence variant having at least 85% sequence identity to any one of (xii)-(xxv).
  • the at least one additional domain is an immunoglobulin domain.
  • the immunoglobulin domain may comprise the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the TLR-based engineered MTMs comprise a TLR microbe-binding domain comprising the N-terminal ligand-binding domain of any one of SEQ ID NOs:24-33 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs:24-33.
  • the TLR-based engineered MTMs comprise a TLR microbe-binding domain comprising the N-terminal ligand-binding domain of any one of SEQ ID NOs:24-33 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ NOs:24-33 and an immunoglobulin domain comprising the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12
  • the CARs that comprise a TLR-based MTM as a primary target-binding domain further comprise (i) optionally at least one secondary target-binding domain, (ii) a transmembrane domain and (iii) an activation domain.
  • suitable transmembrane domains include, but are not limited to, the hinge and transmembrane regions of the human CD8a chain.
  • suitable activation domains include, but are not limited to, the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), 0X40, HVEM, CD3 ⁇ ; and FcRs.
  • the invention is directed to cells that express one or more of the MTM-based CARs as defined herein.
  • Suitable cells expressing the CARs of the invention include T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells and neutrophils.
  • the invention is directed to (i) cells expressing a CAR comprising at least one collectin-based engineered MTM as a primary target-binding domain, (ii) cells expressing a CAR comprising at least one fi colin-based engineered MTM as a primary target-binding domain, and (iii) cells expressing a CAR comprising at least one TLR-based engineered MTM as a primary target-binding domain.
  • the invention is directed to a composition comprising one or more of the MTM-based CARs of the invention.
  • the composition further comprises at least one naturally-occurring MTM.
  • the composition further comprises one or more antimicrobial agents.
  • the invention is directed to a composition comprising one or more of the MTM-based CAR-expressing cells of the invention.
  • the invention is directed to a method of treating a microbial infection in a subject comprising administering to a subject in need thereof a therapeutically- effective amount of one or more of the MTM-based -expressing cells of the invention, thereby treating a microbial infection in a subject.
  • Figure 1 Diagram showing the structure of exemplary collectins and ficolins.
  • Figure 2 Diagram showing the structure of an exemplary CAR comprising a collectin-based engineered MTM.
  • Figure 3 Diagram showing a portion of a CAR having four linked target-binding domains.
  • Figure 4 Diagram showing a portion of a CAR having three linked target-binding domains, wherein the domains bind three different targets.
  • Figure 5. Diagram showing a portion of a CAR having binding specificity for two different targets, namely SARS-CoV-2 spike protein (bound by a secondary target-binding domain) and a lectin (bound by a primary target-binding domain).
  • “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term “about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
  • the present invention is generally directed to chimeric antigen receptors (CARs) and CAR-expressing cells that recognize and bind to at least one target, where at least one of the targets is a microbe-associated molecular pattern (MAMP).
  • the CARs are integral membrane proteins that comprise (i) one or more primary target-binding domains, (ii) optionally one or more secondary target binding domains, (iii) a transmembrane domain, and (iv) an intracellular activation domain.
  • the CARs may optionally further comprise a hinge domain (see Figure 2).
  • the primary target-binding domains are microbe-binding domains, which comprise at least a portion of a microbe-targeting molecule (MTM) or engineered MTM that has the shared characteristic of binding to one or more MAMPs.
  • MTM microbe-targeting molecule
  • the optional secondary target-binding domains do not comprise an MTM or engineered MTM.
  • the secondary target-binding domains help to stabilize interactions between the CAR and a microbe bound by the protein.
  • the MTM-based CARs and MTM-based CAR-expressing cells of the invention can be used to contact, and optionally isolate, microbes and microbial components from a sample based on the identity of the MAMP produced by the microbe, rather than the identity of microbe itself, and they can be used in vitro, in vivo and ex vivo. While some MAMPs are produced by only a single species of microbe, other MAMPs are shared across species. Thus, while some MTM-based CARs of the invention bind to only MAMPs of a particular species of microbe, other MTM-based CARs of the invention can bind to MAMPs produced by all members of a particular class, order, family, genus or sub-genus of microbe.
  • MTM-based CAR and “engineered MTM-based CAR” refers to any of the molecules described herein (or described in patents or patent application incorporated by reference) that can bind to a microbe or microbe component and that comprises at least one microbe-binding domain (as a primary target-binding domain).
  • MTM-based CARs should be understood to include both CARs comprising a naturally-occurring MTM and CARs comprising an engineered MTM.
  • MTM-based CARs and “CARs” are used interchangeably, unless indicated otherwise by context.
  • the CARs of the invention may bind to a target that is something other than a microbe or microbe component through the secondary target-binding domains of the proteins.
  • the secondary target-binding domains may bind to a molecule expressed by a cell infected by a microbe. In such instances, binding by the secondary target-binding domain may serve to stabilize and/or enhance binding between the primary target-binding domain and a MAMP.
  • the CARs of the invention may have 1, 2, 3 or more primary target-binding domains and 1, 2, 3 or more secondary target-binding domains.
  • these primary domains may bind to the same or different MAMP.
  • these secondary domains may bind to the same or different target molecule.
  • the CARs of the invention may be bi-specific, tri-specific, quadra-specific, quinta-specific, sexta-specific, septa-specific, octospecific, etc.
  • each of the MTM-based CARs of the invention binds to at least one microbe-associated molecular pattern (MAMP).
  • MAMP microbe-associated molecular pattern
  • microbe-associated molecular patterns refers to molecules, components or motifs associated with or secreted or released by microbes or groups of microbes (whole and/or lysed and/or disrupted) that are generally recognized by corresponding pattern recognition receptors (PRRs) of the MTM microbe-binding domains defined herein.
  • PRRs pattern recognition receptors
  • the MAMPs encompass molecules associated with cellular components released during cell damage or lysis.
  • MAMPs include, but are not limited to, microbial carbohydrates (e.g., lipopolysaccharide or LPS, mannose), endotoxins, microbial nucleic acids (e.g., bacterial, fungal or viral DNA or RNA; e.g., nucleic acids comprising a CpG site), microbial peptides (e.g., flagellin), peptidoglycans, lipoteichoic acids, N-formylmethionine, lipoproteins, lipids, phospholipids or their precursors (e.g., phosphochloline), and fungal glucans.
  • microbial carbohydrates e.g., lipopolysaccharide or LPS, mannose
  • endotoxins e.g., bacterial, fungal or viral DNA or RNA; e.g., nucleic acids comprising a CpG site
  • microbial peptides e.g., flagellin
  • microbe components comprise cell wall or membrane components known as pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS) endotoxin, lipoteichoic acid, and attached or released outer membrane vesicles.
  • PAMPs pathogen-associated molecular patterns
  • a microbe comprises a host cell membrane and a pathogen component or a PAMP.
  • microbe components comprise damage-associated molecular patterns (DAMPs), also known as danger-associated molecular patterns, danger signals, and alarmin.
  • DAMPs damage-associated molecular patterns
  • PAMPs bacterium-associated molecular patterns
  • PRRs pattern recognition receptors
  • DAMPs are recognized by immune receptors, such as toll-like receptors (TLRs) and NOD-like receptor family, pyrin domain containing 3 (NLRP3), expressed by sentinel cells of the immune system.
  • TLRs toll-like receptors
  • NLRP3 pyrin domain containing 3
  • DAMPs include portions of nuclear and cytosolic proteins, ECM (extracellular matrix), mitochondria, granules, ER (endoplasmic reticulum), and plasma membrane.
  • MAMPs include carbohydrate recognition domain (CRD)-binding motifs.
  • CRD carbohydrate recognition domain
  • CRD-binding motifs refers to molecules or motifs that are bound by a molecule or composition comprising a CRD (i.e. CRDs recognize and bind to CRD-binding motifs).
  • the term “carbohydrate recognition domain” or “CRD” refers to one or more regions, at least a portion of which, can bind to carbohydrates on a surface of microbes or pathogens.
  • the CRD can be derived from a lectin, as described herein.
  • the CRD can be derived from a mannan-binding lectin (MBL).
  • MAMPs are molecules, components or motifs associated with microbes or groups of microbes that are recognized by lectin-based MTMs (collectin-based MTMs) described herein that have a CRD domain.
  • MAMPs are molecules, components, or motifs associated with microbes or groups of microbes that are recognized by mannan-binding lectin (MBL).
  • MAMPs are molecules, components or motifs associated with microbes or groups of microbes that are recognized by a C-reactive protein (CRP)-based MTMs (collectin-based MTMs).
  • CRP C-reactive protein
  • MAMPs as used herein includes microbe components such as MAMPs, PAMPs and DAMPs as defined above.
  • MAMPs can be exposed, released or generated from microbes in a sample by various sample pretreatment methods.
  • the MAMPs can be exposed, released or generated by lysing or killing at least a portion of the microbes in the sample.
  • any means known or available to the practitioner for lysing or killing microbe cells can be used. Exemplary methods for lysing or killing the cells include, but are not limited to, physical, mechanical, chemical, radiation, biological, and the like.
  • pre-treatment for lysing and/or killing the microbe cells can include application of one or more of ultrasound waves, vortexing, centrifugation, vibration, magnetic field, radiation (e.g., light, UV, Vis, IR, X-ray, and the like), change in temperature, flash-freezing, change in ionic strength, change in pH, incubation with chemicals (e.g. antimicrobial agents), enzymatic degradation, and the like.
  • ultrasound waves e.g., ultrasound waves, vortexing, centrifugation, vibration, magnetic field, radiation (e.g., light, UV, Vis, IR, X-ray, and the like), change in temperature, flash-freezing, change in ionic strength, change in pH, incubation with chemicals (e.g. antimicrobial agents), enzymatic degradation, and the like.
  • microbe generally refers to microorganism(s), including bacteria, virus, fungi, parasites, protozoan, archaea, protists, e.g., algae, and a combination thereof.
  • microbe encompasses both live and dead microbes.
  • microbe also includes pathogenic microbes or pathogens, e.g., bacteria causing diseases such as sepsis, plague, tuberculosis and anthrax; protozoa causing diseases such as malaria, sleeping sickness and toxoplasmosis; and fungi causing diseases such as ringworm, candidiasis or histoplasmosis.
  • the microbe is a human pathogen, in other words a microbe that causes at least one disease in a human.
  • the microbe is a Gram-positive bacterial species, a Gram-negative bacterial species, a mycobacterium, a fungus, a parasite, protozoa, or a virus.
  • the Gram-positive bacterial species comprises bacteria from the class Bacilli.
  • the Gram-negative bacterial species comprises bacteria from the class Gammaproteobacteria.
  • the mycobacterium comprises bacteria from the class Actinobacteria.
  • the fungus comprises fungus from the class Saccharomycetes.
  • the microbe is Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida albicans, ox Escherichia coli.
  • the microbe is S. aureus strain 3518, A pyogenes strain 011014, K. pneumoniae strain 631, E. coli strain 41949, P. aeruginosa strain 41504, C. albicans strain 1311, oxM. tuberculosis strain H37Rv.
  • the microbe is Bartonella henselae, Borrelia burgdorferi, Campylobacter jejuni, Campylobacterfetus, Chlamydia trachomatis, Chlamydia pneumoniae, Chylamydia psittaci, Simkania negevensis, Escherichia coli (e.g., 0157:H7 and K88), Ehrlichia chafeensis, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Enterococcus faecalis, Haemophilius influenzae, Haemophilius ducreyi, Coccidioides immitis, Bordetella pertussis, Coxiella burnetii, Ureaplasma urealyticum, Mycoplasma genitalium, Trichomatis vaginalis, Helicobacter pylori, Helicobacter
  • microbe component and “microbial component” refer to any part of a microbe such as cell wall components, cell membrane components, cell envelope components, cytosolic components, intracellular components, nucleic acid (DNA or RNA), or organelles in the case of eukaryotic microbes.
  • the terms “microbe component” and “microbial component” have the same meaning and they are used interchangeably herein.
  • the microbial component comprises a component from a Gram-positive bacterial species, a Gram-negative bacterial species, a mycobacterium, a fungus, a parasite, a virus, or any microbe described herein or known in the art.
  • the MTM-based CARs and MTM-based CAR expressing cells defined herein can be used to detect the MAMP of a microbe or a microbial component in a sample.
  • a sample can include but is not limited to, a patient sample, an animal or animal model sample, an agricultural sample, a food and beverage sample, an environmental sample, a pharmaceutical sample, a biological sample, and a non-biological sample.
  • a biological sample can include but is not limited to, cells, tissue, peripheral blood, and a bodily fluid.
  • Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc.
  • the biological sample can be collected from any source, including, e.g., human or animal suspected of being infected or contaminated by a microbe(s).
  • Biological fluids can include a bodily fluid and may be collected in any clinically acceptable manner.
  • Biological fluids can include, but are not limited to, mucous, phlegm, saliva, sputum, blood, plasma, serum, serum derivatives, bile, sweat, amniotic fluid, menstrual fluid, mammary fluid, peritoneal fluid, interstitial fluid, urine, semen, synovial fluid, interocular fluid, a joint fluid, an articular fluid, and cerebrospinal fluid (CSF).
  • a fluid may also be a fine needle aspirate or biopsied tissue.
  • Blood fluids can be obtained by standard phlebotomy procedures and may be separated into components such as plasma for analysis. Centrifugation can be used to separate out fluid components to obtain plasma, buffy coat, erythrocytes, cells, pathogens and other components.
  • the sample such as a fluid
  • the sample may be purified before introduction to a device or a system of the invention.
  • filtration or centrifugation to remove particulates and chemical interference may be used.
  • Various filtration media for removal of particles includes filter paper, such as cellulose and membrane filters, such as regenerated cellulose, cellulose acetate, nylon, PTFE, polypropylene, polyester, polyethersulfone, polyarylethersulfone, polycarbonate, and polyvinylpyrrolidone.
  • Environmental samples include, but are not limited to, air samples, liquid and fluid samples, and dry samples.
  • Suitable air samples include, but are not limited to, an aerosol, an atmospheric sample, and a ventilator discharge.
  • Suitable dry samples include, but are not limited to, soil.
  • Environmental fluids include, for example, saturated soil water, groundwater, surface water, unsaturated soil water; and fluids from industrialized processes such as waste water.
  • Agricultural fluids can include, for example, crop fluids, such as grain and forage products, such as soybeans, wheat, and corn.
  • Pharmaceutical samples include, but are not limited to, drug material samples and therapeutic fluid samples, for example, for quality control or detection of endotoxins.
  • Suitable therapeutic fluids include, but are not limited to, a dialysis fluid.
  • the MTM-based CARs and MTM-based CAR expressing cells of the invention can used to detect the MAMP of a microbe or a microbial component in vivo.
  • the present invention including the MTM-based CARs, MTM-based CAR expressing cells, compositions, and methods, may be used in conjunction with a subject.
  • a “subject” is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • MTMs microbe-targeting molecules
  • engineered MTMs that have the shared characteristic of binding to one or more MAMPs. These MTMs are used as the microbe-binding domain of the CARs of the invention.
  • MTM-based CARs can be used to contact, and optionally isolate, microbes and microbial components from a sample based on the identity of the MAMP produced by the microbe, rather than the identity of microbe itself. While some MAMPs are produced by only a single species of microbe, other MAMPs are shared across species.
  • MTM-based CARs of the invention bind to only MAMPs of a particular species of microbe
  • other MTM-based CARs of the invention can bind to MAMPs produced by all members of a particular class, order, family, genus or sub-genus of microbe.
  • MTM-based CAR and “engineered MTM-based CAR” refers to any one of the molecules described herein (or described in patents or patent application incorporated by reference) that can bind to microbes or microbe components.
  • MTMs used in the CARs of the invention include naturally-occurring molecules and proteins
  • engineered MTMs of the invention are those have been manipulated in some manner by the hand of man.
  • engineered MTM includes any non-naturally-occurring MTM.
  • Engineered MTMs of the invention retain the binding specificity to a MAMP of the wild-type (i.e. naturally-occurring) molecule on which the engineered MTM is based.
  • the MTMs of the invention are defined based on their binding activity, therefore both naturally-occurring and engineered MTMs will comprise at least one microbe-binding domain, i.e. a domain that recognizes and binds to one or more MAMPs (including, at least two, at least three, at least four, at least five, or more) as described herein.
  • a microbe-binding domain can be a naturally-occurring or a synthetic molecule.
  • a microbe-binding domain can be a recombinant molecule.
  • the microbe-binding domain may comprise some or all of a peptide; polypeptide; protein; peptidomimetic; antibody; antibody fragment; antigen-binding fragment of an antibody; carbohydrate-binding protein; lectin; glycoprotein; glycoprotein-binding molecule; amino acid; carbohydrate (including mono-; di-; tri- and poly-saccharides); lipid; steroid; hormone; lipid- binding molecule; cofactor; nucleoside; nucleotide; nucleic acid; DNA; RNA; analogues and derivatives of nucleic acids; peptidoglycan; lipopolysaccharide; small molecule; endotoxin; bacterial lipopolysaccharide; and any combination thereof.
  • the microbe-binding domain can be a microbe-binding domain of a lectin.
  • An exemplary lectin is mannan binding lectin (MBL) or other mannan binding molecules.
  • MBL mannan binding lectin
  • acceptable microbe-binding domains also include microbebinding domains from toll-like receptors, nucleotide oligomerization domain-containing (NOD) proteins, complement receptors, collectins, fi colins, pentraxins such as serum amyloid and C- reactive protein, lipid transferases, peptidoglycan recognition proteins (PGRs), and any combinations thereof.
  • microbe-binding domains can be microbe-binding molecules described in the International Patent Application No.
  • the MTMs of the invention will typically have one or more domains in addition to a microbe-binding domain.
  • domains include, but are not limited to, an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain, and an immunoglobulin-like domain.
  • Engineered MTMs of the invention include, but are not limited to, MTMs identical to a naturally-occurring MTM but having at least one amino acid change in comparison to the wildtype molecule on which they are based.
  • Such “sequence-variant engineered MTMs” have at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 sequence identity, though in all cases less than 100% sequence identity, to the wild-type molecule on which they are based.
  • the changes may be any combination of additions, insertions, deletions and substitution
  • Engineered MTMs of the invention also include, but are not limited to, MTMs that comprise domains from two or more different MTMs, i.e. fusion proteins.
  • Such “domain-variant engineered MTMs” have domains from 2, 3, 4, 5 or more different proteins.
  • MTMs can be a fusion protein comprising a microbe-binding domain and an oligomerization domain, or a fusion protein comprising a microbe-binding domain and a signal domain, or a fusion protein comprising a microbe-binding domain, an oligomerization domain, and signal domain, to name a few examples.
  • the domains within a domain-variant engineered MTM are from at least two different proteins.
  • MTMs include fusion proteins comprising at least the microbe-binding domain of a lectin and at least a part of a second protein or peptide, e.g., but not limited, to an Fc portion of an immunoglobulin.
  • Engineered MTMs of the invention further include, but are not limited to, MTMs that comprise domains from two or more different MTMs, wherein at least one of the domains is a sequence variant of the wild-type domain upon which it is based, i.e. having at least one amino acid change in comparison to the wild-type molecule on which it is based.
  • sequence- and domain-variant engineered MTMs have domains from 2, 3, 4, 5 or more different proteins, and at least one of the domains has at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 sequence identity, though in all cases less than 100% sequence identity, to the wild-type domain on which it is based.
  • the changes may be any combination of additions, insertions, deletions and substitutions, where the altered amino acids may be naturally-occurring or non-naturally-occurring amino acids, and conservative or nonconservative changes.
  • MTMs of the invention three broad categories of suitable MTMs are defined in the following paragraphs, namely: (i) collectin-based MTMs, (ii) ficolin-based MTMs, and (iii) toll-like receptor-based MTMs. It should be understood that these three categories are not the only categories of MTMs encompassed by the invention.
  • MTMs may also be produced using the lectin binding domains of one or more of the following C-type lectins: Lecticans, Asialoglycoprotein and DC receptors, Selectins, NK cell receptors, Multi-CTLD endocytic receptors, Reg group, Chondrolectin, Layilin, Tetranectin, Polycystin, Attractin (ATRN), Eosinophil major basic protein (EMBP), DGCR2, Thrombomodulin, CD93, CD248, CLEC14A, Bimlec, SEEC, and CBCP/F rem 1/QB RICK.
  • C-type lectins Lecticans, Asialoglycoprotein and DC receptors, Selectins, NK cell receptors, Multi-CTLD endocytic receptors, Reg group, Chondrolectin, Layilin, Tetranectin, Polycystin, Attractin (ATRN), Eosinophil major basic protein (EMBP), DGCR2, Thro
  • the MTMs of the invention include collectin-based MTMs. These MTMs are either naturally-occurring collectin proteins or engineered MTM fusion proteins that comprise at least one collectin microbe-binding domain, such as the lectin carbohydrate-recognition domain (CRD), and at least one additional domain
  • collectin-based MTMs are either naturally-occurring collectin proteins or engineered MTM fusion proteins that comprise at least one collectin microbe-binding domain, such as the lectin carbohydrate-recognition domain (CRD), and at least one additional domain
  • CCD lectin carbohydrate-recognition domain
  • Collectins are a family of collagenous calciumdependent lectins that function in defense, thus playing an important role in the innate immune system. They are soluble molecules comprising pattern recognition receptors (PRRs) within the microbe-binding domain that recognize and bind to particular oligosaccharide structures or lipids displayed on the surface of microbes, i.e. MAMPs of oligosaccharide origin.
  • PRRs pattern recognition receptors
  • Members of the family have a common structure, characterized by four parts or domains arranged in the following N- to C-terminal arrangement: (i) a cysteine-rich domain, (ii) a collagen-like domain, (iii) a coiled-coil neck domain, and (iv) a microbe-binding domain which includes a C-type lectin domain, also termed the carbohydrate recognition domain (CRD).
  • CRD carbohydrate recognition domain
  • the functional form of the molecule is a trimer made up of three identical chains. MAMP recognition is mediated by the CRD in presence of calcium. See FIG. 5.
  • MBL mannose-binding lectin
  • SP-A surfactant protein A
  • SP-D surfactant protein D
  • iv collectin liver 1 (CL-L1; SEQ ID NO: 15)
  • v collectin placenta 1 (CL-P1; SEQ ID NO: 16)
  • conglutinin collectin of 43 kDa (CL-43; SEQ ID NO: 17)
  • vii collectin of 46 kDa (CL-46; SEQ ID NO: 18)
  • (viii) collectin kidney 1 (CL-K1; SEQ ID NO: 19)
  • ix conglutinin (SEQ ID NO:20).
  • MTM mannose-binding lectin
  • MBL Homo sapiens; GenBank: AAH69338.1; SEQ ID NO: 1:
  • Surfactant protein A (SP-A; Homo sapiens; GenBank: AAA36632.1; SEQ ID NO: 13): MTFKNNTTLS LFRLFNSFPL HKGYITTREL HQLALANLI F MFNCADLHFP
  • Surfactant protein D (SP-D; Homo sapiens; GenBank: AAB59450.1; SEQ ID NO: 14):
  • Collectin liver 1 (CL-L1; also known as collectin- 10; Homo sapiens; NCBI Reference
  • Conglutinin collectin (CL-43; Bos taurus; GenBank: AAH6148; SEQ ID NO: 17):
  • Collectin kidney 1 (CL-K1; also known as collectin-11; Homo sapiens; GenBank:
  • Conglutinin (Bos taurus; GenBank: BAA04983; SEQ ID NO:20):
  • the MTMs of the invention also include other collectin-based molecules that bind to one or more MAMPs, e.g. those MTMs comprising at least a portion (e.g. domain) of a lectin- based molecule in the case of an engineered MTM.
  • collectin-based molecule refers to a molecule comprising a microbe-binding domain derived from a collectin, such as a lectin.
  • lectin refers to any molecule including proteins, natural or genetically modified (e.g., recombinant), that interacts specifically with saccharides (e.g., carbohydrates).
  • lectin can also refer to lectins derived from any species, including, but not limited to, plants, animals (e.g. mammals, such as human), insects and microorganisms, having a desired carbohydrate binding specificity.
  • plant lectins include, but are not limited to, the Leguminosae lectin family, such as ConA, soybean agglutinin, peanut lectin, lentil lectin, and Galanthus nivalis agglutinin (GNA) from the Galanthus (snowdrop) plant.
  • Other examples of plant lectins are the Gramineae and Solanaceae families of lectins.
  • animal lectins include, but are not limited to, any known lectin of the major groups S-type lectins, C-type lectins, P-type lectins, and I-type lectins, and galectins.
  • the carbohydrate recognition domain can be derived from a C-type lectin, or a fragment thereof.
  • C-type lectin can include any carbohydrate-binding protein that requires calcium for binding (e.g., MBL).
  • the C-type lectin can include, but are not limited to, collectin, DC-SIGN, and fragments thereof.
  • DC-SIGN can generally bind various microbes by recognizing high-mannose-containing glycoproteins on their envelopes and/or function as a receptor for several viruses such as HIV and Hepatitis C.
  • Collectin-based engineered MTMs of the invention are MTMs that comprise at least a microbe-binding domain of a collectin. These MTMs may also include one or more of the other domains of a collectin, e.g.
  • a cysteine-rich domain a collagen-like domain, and/or a coiled-coil neck domain, as well as one or more domains not typically found in a collectin, such as an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain, and/or an immunoglobulin-like domain.
  • the MTM When a collectin-based engineered MTM has each of the domains of a wild-type collectin, the MTM will be a sequence-variant engineered MTM as defined above.
  • a collectin-based engineered MTM has fewer that all of the domains of a wild-type collectin, the MTM will be a domain-variant engineered MTM or a sequence- and domain-variant engineered MTM as defined above.
  • Collectin-based engineered MTMs comprise a microbe-binding domain derived from at least one carbohydrate-binding protein selected from the group consisting of: MBL; SP-A; SP- D; CL-L1, CL-P1; CL-34; CL-46; CL-K1, conglutinin; maltose-binding protein; arabinose- binding protein; glucose-binding protein; Galanthus nivalis agglutinin; peanut lectin; lentil lectin; DC-SIGN; and C-reactive protein; and any combinations thereof.
  • carbohydrate-binding protein selected from the group consisting of: MBL; SP-A; SP- D; CL-L1, CL-P1; CL-34; CL-46; CL-K1, conglutinin; maltose-binding protein; arabinose- binding protein; glucose-binding protein; Galanthus nivalis agglutinin; peanut lectin; lentil lectin; DC-SIGN; and C-
  • the MTM may be MBL, whether full-length human MBL (SEQ ID NO: 1), mature human MBL without the signal sequence (e.g. SEQ ID NO:2), a truncated human MBL that retains microbe surface-binding (e.g. SEQ ID NO:3), the carbohydrate recognition domain (CRD) of human MBL (e.g. SEQ ID NO:4), or the neck and carbohydrate recognition domain of human MBL (e.g. SEQ ID NO:5), whether used alone or in combination with a second protein in the form of a fusion protein, such as a FcMBL protein as defined herein.
  • SEQ ID NO: 1 full-length human MBL
  • mature human MBL without the signal sequence e.g. SEQ ID NO:2
  • a truncated human MBL that retains microbe surface-binding e.g. SEQ ID NO:3
  • CCD carbohydrate recognition domain
  • SEQ ID NO:5 the neck and carbohydrate recognition domain of human
  • amino acid sequence of full-length human MBL (SEQ ID NO: 1; GenBank: AAH69338.1) is: MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSS PGINGFPGKD
  • NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI The amino acid sequence of mature human MBL without the signal sequence (SEQ ID NO:2) is: ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI
  • amino acid sequence of a truncated MBL that retains microbe surface-binding is:
  • the amino acid sequence of the carbohydrate recognition domain (CRD) of human MBL (SEQ ID NO:4) is: VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGA IQNLIKEEAF LGITDEKTEG QFVDLTGNRL TYTNWNEGEP NNAGSDEDCV LLLKNGQWND
  • amino acid sequence of the neck and carbohydrate recognition domain of MBL is: PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK
  • the truncated forms of the naturally-occurring protein include portions of any one of SEQ ID NOs: 1-5 lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more amino acids from the amino-terminus of the protein, or the carboxy-terminus of the protein, or internally within the protein, or any combination thereof.
  • the truncated forms of the naturally-occurring protein of any one of SEQ ID NOs: 1-5 have a deletion of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the amino acids from the amino-terminus of the protein, or the carboxy -terminus of the protein, or internally within the protein, or any combination thereof.
  • particularly useful truncated forms of the protein one example is the full-length amino acid sequence of the carbohydrate recognition domain (CRD) of MBL, shown in SEQ ID NO:4.
  • suitable CRDs include CRDs having an amino acid sequence of about 10 to about 110 amino acid residues, or about 50 to about 100 amino acid residues, of SEQ ID NO:4.
  • the microbe-binding domain can have an amino acid sequence of at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110 amino acid residues or more, of SEQ ID NO:4.
  • the carbohydrate recognition domain of an engineered MBL protein can comprise SEQ ID NO:4.
  • the carbohydrate recognition domain of an engineered MBL protein can comprise a fragment of SEQ ID NO:4 as defined above.
  • exemplary amino acid sequences of such fragments include, but are not limited to, ND, EZN (where Z is any amino acid, e.g., P), NEGEPNNAGS (SEQ ID NO: 10) or a fragment thereof comprising EPN, GSDEDCVLL or a fragment thereof comprising E, and LLLKNGQWNDVPCST (SEQ ID NO: 11) or a fragment thereof comprising ND.
  • Modifications to such CRD fragments e.g., by conservative substitution (i.e., where an amino acid is replace by an amino acid within the same class of amino acids, where the classes are: aliphatic amino acids (G, A, L, V, I); hydroxyl or sulfur/selenium-containing amino acids (S, C, U, T, M); aromatic amino acids (F, Y, W); basic amino acids (H, K, R); and acidic amino acids (D, E, N, Q)), are also within the scope described herein.
  • the MBL or a fragment thereof used in the microbe surface-binding domain of the engineered MBLs described herein can be a wild-type molecule or a recombinant molecule.
  • sequence variants of the naturally-occurring protein and the truncated forms thereof include proteins having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs: 1-5, or truncated forms thereof, that retain the ability to reduce platelet activation in blood of the protein upon which they are based.
  • Engineered MTM comprising MBL
  • the MTM is an engineered MTM comprising MBL, as defined above, and a second protein in the form of a fusion protein.
  • An exemplary fusion protein comprises some or all of naturally-occurring MBL, such as the carbohydrate recognition domain (CRD) of MBL, and a portion of an immunoglobulin, such as the Fc domain. In use, the Fc domain dimerizes and strengthens the avidity and affinity of the binding by MBLs to monomeric sugars.
  • the N- terminus of fusion proteins can further comprise an oligopeptide linker adapted to bind a solid substrate and orient the CRD of the MBL domain away from a substrate to which it is immobilized.
  • engineered forms of MBLs are known in the art and include each of the forms of MBL disclosed in U.S. Patent No. 9,150,631, U.S. Patent Pub.
  • FcMBL is a specific engineered form of MBL of the invention that comprises the neck and CRD domains of MBL linked to an IgG Fc domain.
  • Proline 81 of mature MBL (SEQ ID NO:2) is a convenient N-terminal point at which to begin the sequence of this engineered construct.
  • the neck and CRD domains (SEQ ID NO:5) of MBL are fused downstream (C-terminal) to the Fc domain of human IgG (Fey).
  • the Fc domain may include the CH2-CH3 interface of the IgG Fc domain, which contains the binding sites for a number of Fc receptors including Staphylococcal protein A.
  • Fc domain dimerizes and strengthens the avidity and affinity of the binding by MBLs to monomeric sugars.
  • FcMBL is described in detail in U.S. Patent No. 9,150,631, the entire disclosure of which is hereby incorporated by reference in its entirety.
  • FcMBLs that may be used in each of the aspects and embodiments of the invention include, but are not limited to, proteins where the neck and CRD domains of MBL are linked to an Fc component of human IgGl, with examples of the resulting constructs set forth in SEQ ID NOs:6, 7 and 9, and proteins where the CRD domain alone of MBL is linked to an Fc component of human IgGl, with an example of the resulting construct set forth in SEQ ID NO: 8.
  • FcMBL.81 SEQ ID NO:6
  • EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GAPDGDSSLA ASERKALQTE MARIKKWLTF SLGKQVGNKF FLTNGEINIT FEKVKALCVK FQASVATPRN AAENGAIQNL IKEEAFLGIT DEKTEGQFVD LTGNRLTYTN WNEGEPNNAG SDEDCVLLLK NGQWNDVPCS TSHLAVCEFP I
  • AKT-FcMBL (SEQ ID NO:7):
  • FcMBL.111 (SEQ ID NO: 8):
  • EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GATSKQVGNK FFLTNGEEVI TFEKVKALCV KFQASVATPR NAAENGAIQN LIKEEAFLGI TDEKTEGQFV DLTGNRLTYT NWNEGEPNNA GSDEDCVLLL KNGQWNDVPC STSHLAVCEF PI
  • FcMBL (SEQ ID NO:9):
  • the Fc region or a fragment thereof can comprise at least one mutation, e.g., to modify the performance of the engineered MBL.
  • the half-life of the engineered MBL described herein can be increased, e.g., by mutating the lysine (K) at the residue 232 to alanine (A) as shown in the Fc domain sequence provided in SEQ ID NO: 12.
  • Other mutations e.g., located at the interface between the CH2 and CH3 domains shown in Hinton et al (2004) J Biol Chem. 279:6213-6216 and Vaccaro C. et al. (2005) Nat Biotechnol. 23: 1283-1288, can be also used to increase the half-life of the IgGl and thus the engineered MBL.
  • the FcMBL of the invention comprises or consists of an amino acid sequence having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs:6-9, that retain the active of protein upon which they are based.
  • the exemplary MBL sequences provided herein are not construed to be limiting.
  • the exemplary sequences provided herein are derived from a human species, amino acid sequences of the same carbohydrate recognition domain in plants and other animal species such as mice, rats, porcine, bovine, feline, and canine are known in the art and within the scope described herein.
  • the present invention encompasses use of any other collectin in an MTM that binds MAMPs, when used in conjunction with the methods and compositions of the invention.
  • any of the following additional collectins may also be used in the aspects and embodiments of the invention as defined herein: (i) surfactant protein A (SP-A; SEQ ID NO: 13), (ii) surfactant protein D (SP-D; SEQ ID NO: 14), (iii) collectin liver 1 (CL-L1; SEQ ID NO: 15), (iv) collectin placenta 1 (CL-P1; SEQ ID NO: 16), (v) conglutinin collectin of 43 kDa (CL-43; SEQ ID NO: 17), (vi) collectin of 46 kDa (CL-46; SEQ ID NO: 18), (vii) collectin kidney 1 (CL-K1; SEQ ID NO: 19), and (viii) conglutinin (SEQ ID NO:20).
  • both naturally-occurring collectins and engineered forms of the proteins may be used in the invention.
  • Engineered forms of the proteins include, but are not limited to, truncated forms of the naturally-occurring proteins, sequence variants of the naturally- occurring proteins, sequence variants of the truncated forms of the proteins, fusion proteins comprising the naturally-occurring protein, fusion proteins comprising the truncated forms of the proteins, and fusion proteins comprising the sequence variants.
  • the truncated forms of the naturally-occurring collectins include portions of any one of SEQ ID NOs: 13-20 lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more amino acids from the amino-terminus of the protein, or the carboxyterminus of the protein, or internally within the protein, or any combination thereof.
  • the truncated forms of the naturally-occurring protein of any one of SEQ ID NOs: 13-20 have a deletion of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the amino acids from the amino-terminus of the protein, or the carboxy -terminus of the protein, or internally within the protein, or any combination thereof.
  • sequence variants of the naturally-occurring protein and the truncated forms thereof include proteins having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs: 13-20, or truncated forms thereof, that retain the activity of the protein upon which they are based.
  • Ficolins are a family of lectins that activate the lectin pathway of complement activation upon binding to a pathogen. Ficolins are soluble molecules comprising pattern recognition receptors (PRRs) within a microbe-binding domain that recognize and selectively bind acetylated compounds, typically N-acetylglucosamine (GlcNAc), produced by pathogens.
  • PRRs pattern recognition receptors
  • the lectin pathway is activated by binding of a ficolin to an acetylated compound on the pathogen surface, which activates the serine proteases MASP-1 and MASP-2, which then cleave C4 into C4a and C4b, and cleave C2 into C2a and C2b. C4b and C2b then bind together to form C3-convertase of the classical pathway, leading to the eventual lysis of the target cell via the remainder of the steps in the classical pathway.
  • Members of the family have a common structure, characterized by three parts or domains arranged in the following N- to C-terminal arrangement: (i) a short N-terminal domain, (ii) a collagen-like domain, and (iii) a fibrinogen-like domain that makes up the microbe-binding domain.
  • the functional form of the molecule is a trimer made up of three identical chains. See FIG. 5.
  • ficolin 1 M-ficolin
  • ficolin 2 L-ficolin
  • H-ficolin H-ficolin
  • amino acid sequences of the human forms of the proteins are provided in the following paragraphs, with the fibrinogen-like domain underlined:
  • Ficolin 1 precursor SEQ ID NO:21 - NP 001994.2 - Homo sapiens
  • LLDRGYFLSG WHTIYLPDCR PLTVLCDMDT DGGGWTVFQR RMDGSVDFYR DWAAYKQGFG
  • Ficolin 2 isoform a precursor - SEQ ID NO:22 - NP 004099.2 - Homo sapiens
  • Ficolin 3 isoform 1 precursor SEQ ID NO:23 - NP 003656.2 - Homo sapiens
  • both naturally-occurring ficolins and engineered forms of the proteins may be used in the invention.
  • Engineered forms of the proteins include, but are not limited to, truncated forms of the naturally-occurring proteins, sequence variants of the naturally- occurring proteins, sequence variants of the truncated forms of the proteins, fusion proteins comprising the naturally-occurring protein, fusion proteins comprising the truncated forms of the proteins, and fusion proteins comprising the sequence variants.
  • the truncated forms of the naturally-occurring ficolins include portions of any one of SEQ ID NOs:21-23 lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more amino acids from the amino-terminus of the protein, or the carboxy-terminus of the protein, or internally within the protein, or any combination thereof.
  • the truncated forms of the naturally-occurring protein of any one of SEQ ID NOs:21-23 have a deletion of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the amino acids from the amino-terminus of the protein, or the carboxy -terminus of the protein, or internally within the protein, or any combination thereof.
  • sequence variants of the naturally-occurring protein and the truncated forms thereof include proteins having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs:21-23, or truncated forms thereof that retain the activity of the protein upon which they are based.
  • the MTMs of the invention also include other ficolin-based molecules that bind to one or more MAMPs (acetylated compounds for the ficolins), e.g. those MTMs comprising at least a portion (e.g. domain) of a ficolin-based molecule in the case of an engineered MTM.
  • ficolin-based molecule refers to a molecule comprising a microbebinding domain derived from a ficolin.
  • the term “ficolin” as used herein refers to any molecule including proteins, natural or genetically modified (e.g., recombinant), that interacts specifically with acetylated compounds (e.g., GlcNAc).
  • the term “ficolin” as used herein can also refer to ficolins derived from any species, including, but not limited to, plants, animals (e.g. mammals, such as human), insects and microorganisms, having the desired binding specificity.
  • Ficolin-based engineered MTMs of the invention are MTMs that comprise at least a microbe-binding domain of a ficolin, e.g. the fibrinogen-like domain of a ficolin. These MTMs may also include one or more of the other domains of a ficolin, e.g.
  • a short N-terminal domain and/or a collagen-like domain as well as one or more domains not typically found in a ficolin, such as a collectin cysteine-rich domain, a collectin collagen-like domain, a collectin coiled-coil neck domain, a TLR transmembrane helix, a TLR C-terminal cytoplasmic signaling domain, an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain, and an immunoglobulin-like domain.
  • a collectin cysteine-rich domain such as a collectin collagen-like domain, a collectin coiled-coil neck domain, a TLR transmembrane helix, a TLR C-terminal cytoplasmic signaling domain, an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain,
  • the MTM When a ficolin-based engineered MTM has each of the domains of a wild-type ficolin, the MTM will be a sequence-variant engineered MTM as defined above. When a ficolin-based engineered MTM has fewer that all of the domains of a wild-type ficolin, the MTM will be a domain-variant engineered MTM or a sequence- and domain-variant engineered MTM as defined above.
  • Ficolin-based engineered MTMs comprise a microbe-binding domain comprising at least one fibrinogen-like domain of a ficolin selected from the group consisting of ficolin 1, ficolin 2, and ficolin 3.
  • the at least one additional domain is an immunoglobulin domain.
  • the immunoglobulin domain may comprise the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the MTMs and engineered MTMs of the invention comprise a microbe-binding domain comprising the fibrinogen-like domain of ficolin 1 of SEQ ID NO:21, optionally with an immunoglobulin domain of SEQ ID NO: 12.
  • the MTMs and engineered MTMs of the invention comprise a microbe-binding domain comprising the fibrinogen-like domain of ficolin 2 of SEQ ID NO:22, optionally with an immunoglobulin domain of SEQ ID NO: 12.
  • the MTMs and engineered MTMs of a microbebinding domain comprising the fibrinogen-like domain of ficolin 3 of SEQ ID NO:23, optionally with an immunoglobulin domain of SEQ ID NO: 12.
  • the engineered MTMs of the invention comprise an microbe-binding domain having an amino acid sequence selected from SEQ ID NO:21 - SEQ ID NO:23, or an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NO:21 - SEQ ID NO:23, but less than 100% identical, and that retains the microbe-binding activity of the wild-type protein.
  • the ficolin-based engineered MTMs comprise a ficolin microbebinding domain comprising the fibrinogen-like domain of any one of SEQ ID NOs:21, 22 and 23 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs:21, 22 and 23 and an immunoglobulin domain comprising the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the microbe-binding domain comprising a fibrinogen-like domain of a ficolin from a primate, mouse, rat, hamster, rabbit, or any other species as described herein.
  • exemplary sequences provided herein for the ficolins are not to be construed as limiting.
  • exemplary sequences provided herein are derived from a human, amino acid sequences of ficolins from other species such as mice, rats, porcine, bovine, feline, and canine are known in the art and within the scope described herein.
  • Toll-like receptors comprise a family of proteins that are integral to the proper functioning of the innate immune system.
  • the proteins are type I integral membrane proteins (i.e. single-pass, membrane-spanning receptors) that are typically found on the surface of sentinel cells, such as macrophages and dendritic cells, but can also be found on the surface of other leukocytes including natural killer cells, T cells and B cells, and non-immune cells including epithelial cell, endothelial cells, and fibroblasts.
  • TLR-expressing cells After microbes have gained entry to a subject, such as a human, through the skin or mucosa, they are recognized by TLR-expressing cells, which leads to innate immune responses and the development of antigen-specific acquired immunity. TLRs thus recognize MAMPs by microbes.
  • N-terminal ligandbinding domain i.e. the microbe-binding domain
  • a single transmembrane helix ⁇ 20 amino acids
  • a C-terminal cytoplasmic signaling domain i.e. the cytoplasmic signaling domain
  • the ligand-binding domain is a glycoprotein comprising 550-800 amino acid residues (depending on the identity of the TLR), constructed of tandem copies of leucine-rich repeats (LRR), which are typically 22-29 residues in length and that contains hydrophobic residues spaced at distinctive intervals.
  • LRR leucine-rich repeats
  • the receptors share a common structural framework in their extracellular, ligand-binding domains.
  • the domains each adopt a horseshoe-shaped structure formed by the leucine-rich repeat motifs.
  • TLR The functional form of a TLR is a dimer, with both homodimers and heterodimers being known. In the case of heterodimers, the different TLRs in the dimer may have different ligand specificities. Upon ligand binding, TLRs dimerize their ectodomains via their lateral faces, forming “m”-shaped structures. Dimerization leads to downstream signaling.
  • a set of endosomal TLRs comprising TLR3, TLR7, TLR8 and TLR9 recognize nucleic acids derived from viruses as well as endogenous nucleic acids in context of pathogenic events. Activation of these receptor leads to production of inflammatory cytokines as well as type I interferons (interferon type I) to help fight viral infection.
  • interferon type I type I interferons
  • TLR1 toll-like receptor 1 precursor
  • NCBI Reference Sequence NP 003254.2
  • TLR2 toll-like receptor 2 precursor
  • NCBI Reference Sequence NP_001305722.1 (SEQ ID NO:25)
  • TLR3 toll-like receptor 3 precursor
  • NCBI Reference Sequence NP 003256.1 (SEQ ID NO:26)
  • TLR4 toll-like receptor 4 isoform D
  • NCBI Reference Sequence NP 612567.1 (SEQ ID NO:27)
  • TLR6 toll-like receptor 6 precursor
  • NCBI Reference Sequence NP 006059.2 (SEQ ID NO:29)
  • TLR7 toll-like receptor 7 precursor
  • NCBI Reference Sequence NP 057646.1 (SEQ ID NO:30)
  • LAELRYLDFS NNRLDLLHST AFEELHKLEV LDISSNSHYF QSEGITHMLN FTKNLKVLQK LMMNDNDISS STSRTMESES LRTLEFRGNH LDVLWREGDN RYLQLFKNLL KLEELDISKN SLSFLPSGVF DGMPPNLKNL SLAKNGLKSF SWKKLQCLKN LETLDLSHNQ LTTVPERLSN CSRSLKNLIL KNNQIRSLTK YFLQDAFQLR YLDLSSNKIQ MIQKTSFPEN VLNNLKMLLL HHNRFLCTCD AVWFVWWVNH TEVTIPYLAT DVTCVGPGAH KGQSVISLDL YTCELDLTNL ILFSLSISVS LFLMVMMTAS HLYFWDVWYI YHFCKAKIKG YQRLISPDCC YDAFIVYDTK DPAVTEWVLA ELVAKLEDPR EKHFNLCLEE RDWLPGQPV
  • TLR8 (toll-like receptor 8 isoform 1) - UniProtKB/Swiss-Prot: Q9NR97.1 (SEQ ID NO:
  • TLR9 toll-like receptor 9 precursor
  • NCBI Reference Sequence NP 059138.1
  • both naturally-occurring TLR and engineered forms of the proteins may be used in the invention.
  • Engineered forms of the proteins include, but are not limited to, truncated forms of the naturally-occurring proteins, sequence variants of the naturally- occurring proteins, sequence variants of the truncated forms of the proteins, fusion proteins comprising the naturally-occurring protein, fusion proteins comprising the truncated forms of the proteins, and fusion proteins comprising the sequence variants.
  • the truncated forms of the naturally-occurring TLRs include portions of any one of SEQ ID NOs:24-33 lacking 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more amino acids from the amino-terminus of the protein, or the carboxy-terminus of the protein, or internally within the protein, or any combination thereof.
  • the truncated forms of the naturally-occurring protein of any one of SEQ ID NOs:24-33 have a deletion of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the amino acids from the amino-terminus of the protein, or the carboxy -terminus of the protein, or internally within the protein, or any combination thereof.
  • sequence variants of the naturally-occurring protein and the truncated forms thereof include proteins having at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs:24-33, or truncated forms thereof that retain the activity of the protein upon which they are based.
  • the MTMs of the invention also include other TLR-based molecules that bind to one or more MAMPs, e.g. those MTMs comprising at least a portion (e.g. domain) of a TLR-based molecule in the case of an engineered MTM.
  • TLR-based molecule refers to a molecule comprising a microbe-binding domain (i.e. an N-terminal ligand-binding domain) derived from a TLR.
  • TLR refers to any molecule including proteins, natural or genetically modified (e.g., recombinant), that interacts specifically with an MAMP and that has a Toll IL-1 receptor (TIR) domain in their signaling domain.
  • TIR Toll IL-1 receptor
  • TLR can also refer to TLR derived from any species, including, but not limited to, plants, animals (e.g. mammals, such as human), insects and microorganisms, having the desired binding specificity.
  • TLR-based engineered MTMs of the invention are MTMs that comprise at least a microbe-binding domain of a TLR, e.g. the N-terminal ligand-binding domain of a TLR. These MTMs may also include one or more of the other domains of a TLR, e.g.
  • transmembrane helix and/or a C-terminal cytoplasmic signaling domain as well as one or more domains not typically found in a TLR, such as a ficolin short N-terminal domain, a ficolin collagen-like domain, a collectin cysteine-rich domain, a collectin collagen-like domain, a collectin coiled-coil neck domain, an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain, and an immunoglobulin-like domain.
  • a ficolin short N-terminal domain such as a ficolin collagen-like domain, a collectin cysteine-rich domain, a collectin collagen-like domain, a collectin coiled-coil neck domain, an oligomerization domain, a signal domain, an anchor domain, a collagen-like domain, a fibrinogen-like domain, an immunoglobulin domain, and an immunoglobulin-like
  • the MTM When a TLR-based engineered MTM has each of the domains of a wild-type TLR, the MTM will be a sequence-variant engineered MTM as defined above. When a TLR-based engineered MTM has fewer that all of the domains of a wild-type TLR, the MTM will be a domain-variant engineered MTM or a sequence- and domain-variant engineered MTM as defined above.
  • TLR-based engineered MTMs comprise a microbe-binding domain comprising at least one N-terminal ligand-binding domain of a TLR selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.
  • the at least one additional domain is an immunoglobulin domain.
  • the immunoglobulin domain may comprise the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the MTMs and engineered MTMs of the invention comprise a microbe-binding domain comprising the N-terminal ligand-binding domain of TLR1 of SEQ ID NO:24, or the N-terminal ligand-binding domain of TLR2 of SEQ ID NO:25, or the N-terminal ligand-binding domain of TLR3 of SEQ ID NO:26, or the N-terminal ligand-binding domain of TLR4 of SEQ ID NO:27, or the N-terminal ligand-binding domain of TLR5 of SEQ ID NO:28, or the N-terminal ligand-binding domain of TLR6 of SEQ ID NO:29, or the N-terminal ligandbinding domain of TLR7 of SEQ ID NO:30, or the N-terminal ligand-binding domain of TLR8 of SEQ ID NO:31, or the N-terminal ligand-binding domain of TLR9 of SEQ ID NO:32,
  • the engineered MTMs of the invention comprise an microbe-binding domain having an amino acid sequence selected from SEQ ID NO:24 - SEQ ID NO:33, or an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NO:24 - SEQ ID NO:33, but less than 100% identical, and that retains the microbe-binding activity of the wild-type protein.
  • the TLR-based engineered MTMs comprise a TLR microbe-binding domain comprising the N-terminal ligand-binding domain of any one of SEQ ID NOs:24-33 or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs:24-33 and an immunoglobulin domain comprising the amino acid sequence of SEQ ID NO: 12 or a sequence variant thereof having at least 85% sequence identity to SEQ ID NO: 12.
  • the microbe-binding domain comprising a N-terminal ligand-binding domain of a TLR from a primate, mouse, rat, hamster, rabbit, or any other subject as described herein.
  • TLRs are not to be construed as limiting.
  • the exemplary sequences provided herein are derived from a human, amino acid sequences of TLRs from other species such as mice, rats, porcine, bovine, feline, and canine are known in the art and within the scope described herein.
  • the MTMs of the invention are those described in at least one of the following: US provisional application numbers 61/296,222, 61/508,957, 61/604,878, 61/605,052, 61/605,081, 61/788,570, 61/846,438, 61/866,843, 61/917,705, 62/201,745, 62/336,940, 62/543, 614; PCT application numbers PCT/US2011/021603, PCT/US2012/047201, PCT/US2013/028409 , PCT/US2014/028683, PCT/US2014/046716, PCT/US2014/071293, PCT/US2016/045509, PCT/US2017/032928; US patent application numbers 13/574,191, 14/233,553, 14/382,043, 14/766,575, 14/831,480, 14/904,583, 15/105,298, 15/415,352, 15/483,216,
  • the present invention is directed to chimeric antigen receptors (CARs) that recognize and bind to at least one target, where at least one of the targets is a MAMP.
  • CARs are integral membrane proteins that comprise (i) one or more primary targetbinding domains, (ii) optionally one or more secondary target binding domains, (iii) a transmembrane domain, and (iv) an intracellular activation domain.
  • the CARs may optionally further comprise a hinge domain between the microbe-binding domain and the transmembrane domain.
  • the first domain of the CARs of the invention is an extracellular domain comprising the target-binding domains (both primary and secondary), where the target-binding domains are joined to each other by short peptide linkers (see Figures 3 and 4).
  • this first domain includes at least one primary target-binding domain and optionally at least one secondary target-binding domain.
  • the CARs of the invention can vary based on the number of primary and secondary target-binding domains present in the protein. However, each CAR of the invention comprises at least one primary target-binding domain. Thus, the CARs of the invention have 1, 2, 3 or more primary target-binding domains and optionally 1, 2, 3 or more secondary target-binding domain.
  • the primary target-binding domain is the microbe-binding domain.
  • the microbebinding domain is an MTM or engineered MTM as defined herein (generally referred to as “an MTM” for the sake of brevity in the following paragraphs).
  • the MAMP bound by the primary binding domain may be the target of the CAR.
  • the MAMP may simply be a molecular anchor for the CAR that stabilizes the secondary target-binding domains, which bind to the targets of interest for the particular CAR being used.
  • the optional secondary target-binding domain differs from the primary target-binding domain in that it does not comprise an MTM or engineered MTM, otherwise the identity of the secondary target-binding domain can vary widely.
  • the secondary domain could comprise a universal capture site, such a binding domain common to all CARs that allows the protein to be isolated from a sample after it has bound a MAMP.
  • the secondary domain could bind a target that stabilizes the binding between the microbe binding domain (i.e. the primary target-binding domain) and a MAMP. Such stabilization could serve to maintain the binding over longer periods of time and/or increase the affinity of the CAR for the MAMP.
  • the secondary domain could bind a non-mutable target of a pathogen, thus preventing the pathogen from escaping immune surveillance.
  • the secondary domain could bind to one or more targets expressed by the virus that do not change over time and well as easily accessible targets, such as spike proteins.
  • targets expressed by the virus that do not change over time and well as easily accessible targets, such as spike proteins.
  • the CAR should maintain the ability to bind to the virus based on the presence of the secondary domain that binds to a non-mutable target.
  • such an CAR would offer broad-spectrum protection and avoid the need to generate new antibody cocktails for each new emergent infectious disease.
  • the target of the secondary domain may be, but is not limited to, a peptide, a polypeptide, a sugar, DNA, RNA, or other nucleic acid molecule.
  • the secondary binding domain may be a portion of an antibody, such as a single-chain variable fragment (scFv).
  • the target-binding domains are linked by short peptides of 1-50 amino acids in length (see Figures 3 and 4).
  • the peptide linkers will vary widely based on the identity of the primary and secondary target-binding domains. Suitable linkers include Gly x , wherein x is between 2 and 49 Gly, and where one or more of the Gly may be substituted by Ser. Specific linkers include Alas, Gly x Ser (where x is 1-5 Gly), Gly x SerGly x Ser (where x is 1-5 Gly), Gly4Ser, and Gly4Ser repeats.
  • the primary and secondary target-binding domains may be arranged in any order and in any sequence.
  • Non-limiting examples include, from the amino terminus of the proteins, primary-secondary; secondary-primary; primary-secondary-primary; primary-primary- secondary; secondary-primary-secondary; secondary-secondary-primary; primary-secondary- primary-secondary; primary-primary-secondary-secondary; primary-primary-primary-secondary; primary-secondary-secondary-secondary.
  • the target-binding domain closest to the surface of the cell (whether primary or secondary) is joined to the transmembrane domain which in turn, is joined to the intracellular cellular activation domain to make a complete polypeptide that is the CAR of the invention.
  • a hinge domain is inserted between the target-binding domain closest to the surface of the cell and the transmembrane domain (see Figure 2).
  • the second domain of the CARs of the invention is the transmembrane domain.
  • the transmembrane domain allows the CAR to be anchored into the cell membrane via a hydrophobic alpha helix that spans the cell membrane. It is essential for the stability of the receptor as a whole, and transfers binding signals from the microbial-binding domain and/or other target-binding domain(s) into the cell to the activation domain. Different transmembrane domains result in different receptor stability.
  • Exemplary transmembrane domains include, but are not limited to, the transmembrane region of the human CD8a chain.
  • an exemplary hinge and transmembrane domain includes, but is not limited to, the hinge and transmembrane regions of the human CD8a chain.
  • the third domain of the CARs of the invention is an intracellular activation domain. This domain aids in cellular activation upon binding of the CAR to an MAMP. Cell activation either provides a signal that can be detected (thus indicating the presence of a MAMP) or functional response by the cell, such as cytolytic activity, or both, and depends on the identity of the cell and the identity of the activation domain.
  • suitable activation domains include, but are not limited to, the cytoplasmic regions of CD28, CD28, CD137 (41BB), 0X40 and HVEM which serve to enhance T cell survival and proliferation; and CD3( ⁇ and FcRs which induce T cell activation.
  • One or more than one T cell activation domain may be included in the CAR, such as two, three, four or more T cell activation domains.
  • the hinge domain is a short structural domain that links the targetbinding domains and the transmembrane domain. In proteins comprising the hinge domain, this domain is located between the target-binding domain closest to the cell and the outer membrane of the cell. Hinge domains enhance the flexibility of the target-binding domains, thus reducing the spatial constraints between the CAR and the target MAMP. This promotes target binding. Suitable hinge domains are often based on membrane-proximal regions from other immune molecules including IgG, CD8, and CD28.
  • An exemplary CAR of the invention is an CAR that comprises a primary targetbinding domain that binds to a viral MAMP (e.g. glycans on the viral envelope), and one or two secondary target-binding domains that bind to a viral coat or spike proteins (see Figure 5), that targets a virus, such as SARS-CoV-2 or HIV.
  • a viral MAMP e.g. glycans on the viral envelope
  • a viral coat or spike proteins see Figure 5
  • the present invention is also directed to cells expressing the CARs of the invention.
  • the cells used as the CAR-expressing cells of the invention are not unduly limited in identity. However, such cells will typically be those cells that can readily express and display a CAR of the invention in the surface of the cell.
  • These cells include immune cells, such as T cells, B cells, neutrophils, natural killer (NK) cells, monocytes, macrophages, and dendritic cells.
  • Suitable cells include those having an effector function, e.g. a cell with cytologic activity.
  • Selected cells may be engineered to express CARs by means readily known to the skilled artisan.
  • a polynucleotide vector is constructed that encodes the CAR and the vector is transfected into a population of cells. The cells are then grown under conditions promoting expression of the CAR by the cells. Successful transfection (or transduction which refers to viral-mediated gene integration) and display of CARs by cells is conducted via conventional means.
  • cells may be engineered to produce CARs by first constructing a retroviral vector encoding a selected CAR.
  • An exemplary retroviral vector includes, but is not limited to, the vector backbone pMSGVl-CD8-28BBZ, which is derived from pMSGV (murine stem cell virus-based splice-gag vector). DNA sequencing can be used to confirm proper construction of the vector before transfection of cells. Retroviral transduction may be performed using known techniques, such as that of Johnson et al. (Blood 114, 535-546 (2009)). The surface expression of CAR on transduced cells may be determined, for example, by flow cytometry after staining cells.
  • compositions Comprising MTM-based CARs
  • the invention also includes compositions comprising one or more of types of MTM- based CARs defined herein.
  • types of MTM-based CARs vary based on the identity of the primary and/or secondary target binding domains.
  • particular MTMs used in the primary target-binding domains (i.e. microbe-binding domains) of the CARs can be defined based on (i) structural terms (e.g. based on the components of the MTM; the amino acid sequence of the MTM; the nucleic acid sequence of the MTM; etc.), (ii) functional terms (e.g.
  • the different “types” of MTM-based CARs may be defined based on the “types” of MTMs used in the primary target-binding domain(s) of the CARs of the composition, where the MTMs may differ based on structural and/or functional terms from each other.
  • the composition When there is more than one type of MTM-based CAR in a composition, defined based on the identity of the MTM, the composition is said to comprise a mixture of different types of MTMs within the composition. In another instance, different “types” of MTM-based CARs may be defined based on the identity of the secondary target-binding domain, when it is present.
  • compositions may comprise different types of MTM-based CARs within one category of MTMs, and/or within one category of secondary target-binding domain, as defined herein, or the compositions may comprise different types of MTM-based CARs within two or more different categories of MTMs, and/or two or more different categories of secondary targetbinding domains, as defined herein.
  • the compositions of the invention include “cocktails” of different types of MTM-based CARs, wherein the composition can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of MTMs within a single composition.
  • compositions of the invention may comprise mixtures of MTM-based CARs comprising naturally-occurring MTMs (e.g. MBLs), mixture of both naturally-occurring MTMs (e.g. MBLs) and the engineered MTMs defined herein (e.g. FcMBLs), or mixtures of only engineered MTMs (e.g., FcMBLs).
  • MBLs naturally-occurring MTMs
  • FcMBLs engineered MTMs defined herein
  • FcMBLs engineered MTMs defined herein
  • FcMBLs mixtures of only engineered MTMs
  • compositions Comprising MTM-based CAR-expressing Cells
  • compositions comprising one or more of types of MTM- based CAR-expressing cells as defined herein.
  • compositions of the invention may comprise mixtures of cells expressing MTM- based CARs comprising naturally-occurring MTMs (e.g. MBLs), mixture of cells expressing both naturally-occurring MTMs (e.g. MBLs) and the engineered MTMs defined herein (e.g. FcMBLs), or mixtures of cells expressing only engineered MTMs (e.g., FcMBLs).
  • MBLs naturally-occurring MTMs
  • MBLs naturally-occurring MTMs
  • FcMBLs engineered MTMs defined herein
  • FcMBLs engineered MTMs defined herein
  • compositions comprising one or more different types of MTM-based CARs and cells expressing MTM-based CARs may include suitable carriers and diluents. Suitable carriers and diluents are commonly known and will vary depending on the MTM being used and the mode of use.
  • suitable carriers and diluents include water, buffered water, saline, buffered saline, dextrose, glycerol, ethanol, and combinations thereof, propylene glycol, polysorbate 80 (Tween-80TM), poly (ethyl ene)gly col 300 and 400 (PEG 300 and 400), hydrophilic and hydrophobic carriers, and combinations thereof.
  • Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes, other stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.
  • the composition comprises as a carrier or diluent, an aqueous solution comprising sodium acetate having a pH of about 3.2.
  • compositions of the invention may also comprise one or more antimicrobial agents.
  • MTM-based CARs and MTM-based CAR-expressing cells bind to and isolate one or more microbes or microbe components.
  • An antimicrobial agent can optionally be included to treat (e.g. kill or inactivate) one or more known or suspected pathogens.
  • compositions comprise one or more antimicrobial agents
  • suitable agents include, but are not limited to, antibiotics, antivirals and antifungals.
  • Antibiotics can be from classes including but not limited to Cephalosporin, Glycopeptide, Cyclic lipopeptide, Aminoglycoside, Macrolide, Oxazolidinone, Fluoroquinolones, Lincosamides, or Carbapenem.
  • Antifungals can be from classes including but not limited to Polyenes, Azoles, Nucleoside Analog, Echinocandin, or Allylamine.
  • Antivirals can be from classes including but not limited to CCR5 anatonists, Fusion inhibitors, Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Nucleotide reverse transcriptase inhibitors (NtRTIs), Integrase inhibitors, Protease inhibitors, DNA polymerase inhibitors, Guanosine analogs, Interferon-alpha, M2 ion channel blockers, Nucleoside inhibitors, NS5A polymerase inhibitors, NS3/4A protease inhibitors, Neuraminidase inhibitors, Nucleoside analogs, and Direct acting antivirals (DAAs).
  • CCR5 anatonists Fusion inhibitors, Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Nucleotide reverse transcriptase inhibitors (N
  • antimicrobials include but are not limited to aminoglycosides, ansamycins, beta-lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron- sequestering glycoproteins, linosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combinations thereof.
  • antibiotics that may be included in the compositions of the invention include, but are not limited to, broad penicillins, amoxicillin (e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin), Penicillins and Beta Lactamase Inhibitors (e.g., Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam, Benzylpenicillin, Cioxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin), Cephalosporins (e.g., Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine), Cephalosporin II Generation (e.g., Cefaclor, Cef
  • Some exemplary antifungals that may be included in the compositions of the invention include, but are not limited to, polyene antifungals, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, imidazole antifungals, triazole antifungals, thiazole antifungals, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Triazolesfedit], Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propi conazole, Ravuconazole, Terconazole, Voriconazole, Abafungin,
  • Some exemplary antivirals that may be included in the compositions of the invention include, but are not limited to, Abacavir, Acyclovir, Adefovir, Amantadine, Ampligen, Amprenavir, antiretroviral, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir, Combivir, Daclatasvir, Darunavir, Delavirdine, Dasabuvir, Didanosine, Docosanol, Dolutegravir, Doravirine, Ecoliever, Edoxudine, Efavirenz, Elbasvir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine, Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ganciclovir, Gemcitabine, Glecaprevir, Grazoprevir, Ibacitabine, Idoxuridine, Imiquimod,
  • compositions of the invention can take many different forms, varying widely based on (i) the identity of the CARs and cells expressing the CARs in the composition, (ii) the identity of the primary target-binding domain in the CARs, (iii) the identity of the secondary target-binding domain in the CARs, (iv) the identity of other components in the composition, and (v) the intended use of the composition, to name only a few of the relevant factors.
  • compositions of the invention may further comprise pharmaceutically acceptable carriers and diluents when administered to or used on a living subject.
  • suitable carriers and diluents are commonly known and will vary depending on the MTM-based CARs and the MTM- based CAR-expressing cells being used or administered and the mode of use or administration.
  • suitable carriers and diluents include saline, buffered saline, dextrose, water-for- inj ection, glycerol, ethanol, and combinations thereof, propylene glycol, polysorbate 80 (Tween- 80TM), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g.
  • Cremophor EL Cremophor EL
  • pol oxamer 407 and 188 a cyclodextrin or a cyclodextrin derivative (including HPCD ((2-hydroxypropyl)-cyclodextrin) and (2-hydroxyethyl)-cyclodextrin), hydrophilic and hydrophobic carriers, and combinations thereof.
  • Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes, other stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.
  • an MTM-based CAR composition or MTM-based CAR-expressing cell composition of the present invention comprises an aerosolized composition.
  • the composition can comprise a solution, e.g., a liquid, of MTM-based CARs or MTM-based CAR- expressing cells that can be aerosolized by any aerosol generating/delivery device as known in the art (described below).
  • the aerosolized particles can be of a suitable size for deposition into the lungs, including the smaller airways and alveoli, or onto a carrier or inert surface.
  • An exemplary sized particle can range from about 1 micron to about 5 microns, for example, from about 1 micron to about 3 microns.
  • An aerosolized composition can be delivered to a subject, e.g., via the nose, eyes or mouth, by any aerosol generating/delivery device as known in the art.
  • the delivery device comprises a nebulizer where the nebulizer can comprise a small volume nebulizer, a large volume nebulizer, or an ultrasound nebulizer.
  • the delivery device comprises a metered-dose inhaler.
  • the inhaler can include spacers or holding chambers.
  • the delivery device comprises a dry-powder inhaler.
  • the aerosolized composition can be administered to a subject preventatively to prevent one or more pathogens from entering a subject, e.g., into the eye(s), nose, mouth, and/or respiratory system including the airway, lungs and blood vessels, and blood.
  • the MTM-based CARs or MTM-based CAR-expressing cells of the composition act by binding to one or more microbes or microbe components and preventing the one or more microbe or microbe components, thus immobilizing it and preventing it from entering the eye(s), nose, mouth, and/or respiratory system including the airway, lungs and blood vessels, and ultimately the blood.
  • a MTM-based CAR or MTM-based CAR-expressing cellular composition of the present invention comprises an ocular rinse or ointment.
  • the MTM-based CAR or MTM-based CAR-expressing cellular composition can be mixed with any suitable biocompatible ocular rinses that are known in the art.
  • the composition can be administered to a subject by dropping the rinse into the eye, or applying an ointment proximate to the eye, e.g., the eye-lids.
  • a MTM-based CAR or MTM-based CAR-expressing cellular composition of the present invention comprises a mouth rinse.
  • the MTM-based CAR or MTM-based CAR-expressing cellular composition can be mixed with any suitable biocompatible mouth rinses that are known in the art.
  • the composition can be administered to a subject’s mouth by swooshing, gargling and/or swallowing the rinse.
  • a MTM-based CAR or MTM-based CAR-expressing cellular composition of the present invention comprises a nasal rinse, spray or ointment.
  • the MTM- based CAR or MTM-based CAR-expressing cellular composition can be mixed with any suitable biocompatible nasal rinses or spray that are known in the art.
  • the composition can be administered to a subject by dropping or spraying into the nose, or applying an ointment in or proximate to the nose, e.g., the nostrils.
  • any of the MTM-based CAR or MTM-based CAR-expressing cellular compositions described herein can be administered to a subject after exposure to a pathogen, for example, to decrease the pathogen load on the subject and/or to treat a subject having one or more infections.
  • a pathogen for example, to decrease the pathogen load on the subject and/or to treat a subject having one or more infections.
  • a particular antimicrobial based on the pathogen, can be selected and added to the composition.
  • MTM-based CAR or MTM-based CAR-expressing cellular compositions are further described in at least one of the following: US provisional application numbers 61/296,222, 61/508,957, 61/604,878, 61/605,052, 61/605,081, 61/788,570, 61/846,438, 61/866,843, 61/917,705, 62/201,745, 62/336,940, 62/543, 614; PCT application numbers PCT/US2011/021603, PCT/US2012/047201, PCT/US2013/028409 , PCT/US2014/028683, PCT/US2014/046716, PCT/US2014/071293, PCT/US2016/045509, PCT/US2017/032928; US patent application numbers 13/574,191, 14/233,553, 14/382,043,
  • the MTM-based CARs and MTM-based CAR-expressing cells defined herein can be used to contact, and optionally detect, identify and/or isolate, microbes and microbial components from a sample. It will be apparent to the skilled artisan that the MTM- based CARs and MTM-based CAR-expressing cells defined herein can be used in a large number of industries, including but not limited to the fields of health care, food safety, agricultural testing, and biodefense. The MTM-based CARs and MTM-based CAR-expressing cells of the invention can be used in any industry where an affordable and highly sensitive tools are desired, for use in the rapid and accurate identification of the presence of a microbe in a sample of interest.
  • MTM-based CARs and MTM-based CAR-expressing cells can be used in diagnostic and/or therapeutic health care applications.
  • the MTM-based CARs and MTM-based CAR-expressing cells can be used to prevent one or more pathogens from entering a subject, e.g., into the eye(s), nose, mouth, and/or respiratory system, including the airway, lungs and blood vessels, and blood; to decrease the pathogen load on the subject; and/or to treat a subject having one or more infections.
  • the MTM-based CARs and MTM-based CAR- expressing cells can be used in methods of treating a subject infected by a microbial pathogen.
  • the invention includes methods of treating a microbial infection in a subject comprising administering to a subject in need thereof a therapeutically-effective amount of a MTM-based CAR or MTM-based CAR-expressing cell of the invention to the subject, thereby treating the microbial infection in the subject.
  • the MTM-based CARs and MTM-based CAR- expressing cells can be used in methods of detecting a microbe in a sample.
  • the invention includes methods of detecting a microbe or a portion of a microbe in a sample comprising contact a sample suspected of containing a microbe with a MTM-based CAR or MTM-based CAR-expressing cell of the invention under conditions promoting binding of the microbe or portion thereof by the MTM-based CAR or MTM-based CAR-expressing cell, and detecting binding of the MTM-based CAR or MTM-based CAR-expressing cell, thereby detecting a microbe in a sample.
  • the MTM-based CARs and MTM-based CAR- expressing cells can be used in methods of detecting a microbe or microbial pathogen in a subject.
  • the invention includes methods of detecting a microbe or microbial infection in a subject comprising administering to a subject suspect of having a microbe or microbial infection a MTM-based CAR or MTM-based CAR-expressing cell of the invention to the subject and detecting binding of the MTM-based CAR or MTM-based CAR-expressing cell, thereby detecting a microbe or microbial infection in the subject.
  • the microbial infection may be caused by a microorganism, including bacteria, virus, fungi, parasites, protozoan, archaea, protists, e.g., algae, and a combination thereof.
  • the microorganism or microbe causing the infection includes pathogenic microbes or pathogens, e.g., bacteria causing diseases such as sepsis, plague, tuberculosis and anthrax; protozoa causing diseases such as malaria, sleeping sickness and toxoplasmosis; and fungi causing diseases such as ringworm, candidiasis or histoplasmosis.
  • the microbe is a human pathogen, in other words a microbe that causes at least one disease in a human.
  • the microbe is a Gram -positive bacterial species, a Gram -negative bacterial species, a mycobacterium, a fungus, a parasite, protozoa, or a virus.
  • the Gram-positive bacterial species comprises bacteria from the class Bacilli.
  • the Gram-negative bacterial species comprises bacteria from the class Gammaproteobacteria.
  • the mycobacterium comprises bacteria from the class Actinobacteria.
  • the fungus comprises fungus from the class Saccharomycetes.
  • the microbe is Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida albicans, ox Escherichia coli.
  • the microbe is S. aureus strain 3518, A pyogenes strain 011014, K. pneumoniae strain 631, E. coli strain 41949, P. aeruginosa strain 41504, C. albicans strain 1311, or M. tuberculosis strain H37Rv.
  • the microbe is Bartonella henselae, Borrelia burgdorferi, Campylobacter jejuni, Campylobacterfetus, Chlamydia trachomatis, Chlamydia pneumoniae, Chylamydia psittaci, Simkania negevensis, Escherichia coli (e.g., 0157:H7 and K88), Ehrlichia chafeensis, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Enterococcus faecalis, Haemophilius influenzae, Haemophilius ducreyi, Coccidioides immitis, Bordetella pertussis, Coxiella burnetii, Ureaplasma urealyticum, Mycoplasma genitalium, Trichomatis vaginalis, Helicobacter pylori, Helicobacter
  • the terms “treat”, “treating”, and “treatment” have their ordinary and customary meanings, and include one or more of: blocking, ameliorating, or decreasing in severity and/or frequency a symptom of a microbial infection in a subject, and/or inhibiting the growth, division, spread, or proliferation of microbial cells in a subject.
  • Treatment means blocking, ameliorating, decreasing, or inhibiting by about 1% to about 100% versus a subject in which the methods of the present invention have not been practiced.
  • the blocking, ameliorating, decreasing, or inhibiting is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% versus a subject in which the methods of the present invention have not been practiced.
  • Administration frequencies of formulations comprising MTM-based CARs or populations of MTM-based CAR-expressing cells will vary depending on factors that include the disease being treated, the elements comprising the MTM-based CARs, and the modes of administration.
  • Each formulation may be independently administered 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly.
  • duration of treatment will be based on the disease being treated and will be best determined by the attending physician. However, continuation of treatment is contemplated to last for a number of days, weeks, or months.
  • the subject receiving treatment is a human or non-human animal, e.g., a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • a human or non-human animal e.g., a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • the invention also provides a kit comprising one or more containers filled with one or more MTM-based CARs or populations of MTM-based CAR-expressing cells.
  • the kit may also include instructions for use.
  • Associated with the kit may further be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Labels
  • the MTMs of the present invention may be labeled to allow them to be detected after binding to microbes or microbial components.
  • the identity of the detectable label is limited only in that it can be discerned by the human eye or via a detector in the context of the detection device.
  • Suitable detectable labels include colored or fluorescent particles, such a Europium particles or colloidal gold.
  • Other acceptable labels include latex, which may itself be tagged with colored or fluorescent dyes, and magnetic or paramagnetic components.
  • a further detectable label is a plasmonic fluor, wherein instead of assaying for a color change, one detects fluorescence.
  • Ultrabright fluorescent nanolabels can also be used to improve the limit of detection in the detection devices of the invention, compared with conventional fluorophores.
  • detectable labels include, but are not limited to, an enzyme (e.g., peroxidase, alkaline phosphatase, glucose oxidase), a metal (e.g., gold for electron microscopy applications), a fluorescent marker (e.g., for immunofluorescence and flow cytometry applications, including CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine), a fluorescence-emitting metals (e.g., 152 Eu), a radioactive marker (e.g., radioisotopes for diagnostic purposes, including 3 H, 131 I, 35 S, 14 C, and 125 I), a chemiluminescent marker (e.g., luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole,
  • an enzyme e
  • the MTMs of the invention can be engineered to display side groups that augment, enhance or otherwise alter selected characteristics of the MTMs.
  • the MTMs of the invention may be engineered to display polyfluoro groups on any portion of the molecule.
  • Such groups include fluoropolymers comprising terminal polyfluoro-oligomeric groups. These groups can aid in reducing thrombosis that may result, for example, when blood comes into contact with non-self surfaces. Coating of such surfaces with MTMs displaying polyfluoro-groups can reduce coagulation. Incorporation by Reference

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oncology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des récepteurs antigéniques chimériques (CAR) et des cellules exprimant un CAR qui reconnaissent et se lient à un ou plusieurs motifs moléculaires associés aux microbes (MAMP).
PCT/US2021/062362 2020-12-08 2021-12-08 Cellules exprimant un récepteur antigénique chimérique pour cibler des motifs moléculaires associés aux microbes WO2022125632A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202063122732P 2020-12-08 2020-12-08
US63/122,732 2020-12-08
US202163147882P 2021-02-10 2021-02-10
US63/147,882 2021-02-10

Publications (1)

Publication Number Publication Date
WO2022125632A1 true WO2022125632A1 (fr) 2022-06-16

Family

ID=79259455

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/062362 WO2022125632A1 (fr) 2020-12-08 2021-12-08 Cellules exprimant un récepteur antigénique chimérique pour cibler des motifs moléculaires associés aux microbes

Country Status (1)

Country Link
WO (1) WO2022125632A1 (fr)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US614A (en) 1838-02-22 Discovery in the manufacture of brown paper from a new material called
WO2011090954A2 (fr) 2010-01-19 2011-07-28 President And Fellows Of Harvard College Opsonine obtenue par génie génétique, pour la détection et le traitement de micro-organismes pathogènes
WO2013012924A2 (fr) 2011-07-18 2013-01-24 President And Fellows Of Harvard College Molécules manipulées ciblant un microbe et leurs utilisations
WO2013130875A1 (fr) 2012-02-29 2013-09-06 President And Fellows Of Harvard College Test rapide de sensibilité aux antibiotiques
US20150173883A1 (en) 2012-07-18 2015-06-25 President And Fellows Of Harvard College Modification of surfaces for simultaneous repellency and targeted binding of desired moieties
US20160311877A1 (en) 2013-12-18 2016-10-27 President And Fellows Of Harvard College Crp capture/detection of gram positive bacteria
US9791440B2 (en) 2013-07-15 2017-10-17 President And Fellows Of Harvard College Assays for antimicrobial activity and applications thereof
US20180371057A1 (en) * 2017-06-02 2018-12-27 Regents Of The University Of Minnesota Compositions and methods for improving immunotherapy
US20190077850A1 (en) 2017-08-10 2019-03-14 President And Fellows Of Harvard College Pathogen binding methods and compositions
US10435457B2 (en) 2015-08-06 2019-10-08 President And Fellows Of Harvard College Microbe-binding molecules and uses thereof

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US614A (en) 1838-02-22 Discovery in the manufacture of brown paper from a new material called
WO2011090954A2 (fr) 2010-01-19 2011-07-28 President And Fellows Of Harvard College Opsonine obtenue par génie génétique, pour la détection et le traitement de micro-organismes pathogènes
US9150631B2 (en) 2010-01-19 2015-10-06 President And Fellows Of Harvard College Engineered opsonin for pathogen detection and treatment
WO2013012924A2 (fr) 2011-07-18 2013-01-24 President And Fellows Of Harvard College Molécules manipulées ciblant un microbe et leurs utilisations
US9593160B2 (en) 2011-07-18 2017-03-14 President And Fellows Of Harvard College Engineered microbe-targeting molecules and uses thereof
US9632085B2 (en) 2012-02-29 2017-04-25 President And Fellows Of Harvard College Rapid antibiotic susceptibility testing
WO2013130875A1 (fr) 2012-02-29 2013-09-06 President And Fellows Of Harvard College Test rapide de sensibilité aux antibiotiques
US20150173883A1 (en) 2012-07-18 2015-06-25 President And Fellows Of Harvard College Modification of surfaces for simultaneous repellency and targeted binding of desired moieties
US9791440B2 (en) 2013-07-15 2017-10-17 President And Fellows Of Harvard College Assays for antimicrobial activity and applications thereof
US20160311877A1 (en) 2013-12-18 2016-10-27 President And Fellows Of Harvard College Crp capture/detection of gram positive bacteria
US10435457B2 (en) 2015-08-06 2019-10-08 President And Fellows Of Harvard College Microbe-binding molecules and uses thereof
US20180371057A1 (en) * 2017-06-02 2018-12-27 Regents Of The University Of Minnesota Compositions and methods for improving immunotherapy
US20190077850A1 (en) 2017-08-10 2019-03-14 President And Fellows Of Harvard College Pathogen binding methods and compositions

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. AAH69338.1
"NCBI", Database accession no. NP_001017388.1
ERTL ET AL., CANCER RES., vol. 71, 2011, pages 3175 - 3181
GHANEM MUSTAFA H. ET AL: "Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti-human immunodeficiency virus potency and breadth with minimal immunogenicity", CYTOTHERAPY, vol. 20, no. 3, 1 March 2018 (2018-03-01), GB, pages 407 - 419, XP055902794, ISSN: 1465-3249, DOI: 10.1016/j.jcyt.2017.11.001 *
GROSS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 10024 - 10028
HINTON ET AL., J BIOL CHEM., vol. 279, 2004, pages 6213 - 6216
JOHNSON ET AL., BLOOD, vol. 114, 2009, pages 535 - 546
KERSHAW ET AL., CLIN. CANCER RES., vol. 12, 2006, pages 6106 - 6115
KUMUDHINI PREETHI HARAN ET AL: "Simian Immunodeficiency Virus (SIV)-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication", FRONTIERS IN IMMUNOLOGY, vol. 9, no. 492, 20 March 2018 (2018-03-20), pages 1 - 12, XP055605602, DOI: 10.3389/fimmu.2018.00492 *
LAMERS ET AL., J. CLIN. ONCOL., vol. 24, 2006, pages e20 - e22
MORGAN ET AL., MOL. THER., vol. 18, 2010, pages 843 - 851
PULE ET AL., NAT. MED., vol. 14, 2008, pages 1264 - 1270
RIDER ET AL., SCIENCE, vol. 301, 2003, pages 213 - 215
SADELAIN ET AL., CURR. OPIN. IMMUNOL., vol. 21, 2009, pages 215 - 223
TILL ET AL., BLOOD, vol. 112, 2008, pages 2261 - 2271
VACCARO C. ET AL., NAT BIOTECHNOL., vol. 23, 2005, pages 1283 - 1288

Similar Documents

Publication Publication Date Title
Divyashree et al. Clinical applications of antimicrobial peptides (AMPs): where do we stand now?
US20230270927A1 (en) Compositions, devices and methods for diagnosing and treating infectious disease
Selsted et al. Mammalian defensins in the antimicrobial immune response
da Silva et al. CD14 is critical for TLR2-mediated M1 macrophage activation triggered by N-glycan recognition
Arnold et al. Mannan binding lectin and its interaction with immunoglobulins in health and in disease
Scott et al. The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses
Robinson et al. The sweet-side of leukocytes: galectins as master regulators of neutrophil function
Smith et al. Membrane-targeted complement inhibitors
TW201406959A (zh) 抗發炎胜肽及包含其之成分（三）
CA2475247C (fr) Traitements anti-pathogenes
WO2007001332A2 (fr) Immunoadhesines anti-pathogene
JP2022028646A (ja) 免疫調節化合物
CN110785431A (zh) TGF-β受体II型融合蛋白及其用途
JP2016515121A (ja) ｐ９７のフラグメントおよびその使用
Suzuki et al. Human host defense cathelicidin peptide LL-37 enhances the lipopolysaccharide uptake by liver sinusoidal endothelial cells without cell activation
WO2018202921A2 (fr) Protéines nanostructurées et leurs utilisations
EP3426681B1 (fr) Conformères ouverts de hla-b57
Pal et al. Self-assembling nano-globular peptide from human lactoferrin acts as a systemic enhancer of bone regeneration: A novel peptide for orthopedic application
WO2022125632A1 (fr) Cellules exprimant un récepteur antigénique chimérique pour cibler des motifs moléculaires associés aux microbes
Shang et al. Ovotransferrin exhibits osteogenic activity partially via low-density lipoprotein receptor-related protein 1 (LRP1) activation in MC3T3-E1 cells
US20240019435A1 (en) Compositions, devices and methods comprising microbe-targeting molecules for diagnosing and treating infectious disease
US20230054472A1 (en) Sample preparation and microbial analysis
Rivas-Santiago et al. Antimicrobial peptides for the treatment of pulmonary tuberculosis, allies or foes?
US20240199719A1 (en) Therapeutic compositions comprising microbe-targeting molecules and their use in methods of preventing and treating infectious disease
US20230416319A1 (en) Compositions, devices and methods for identifying, treating and preventing infectious disease using microbe-targeting molecules

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21836711

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21836711

Country of ref document: EP

Kind code of ref document: A1