WO2022125632A1

WO2022125632A1 - Chimeric antigen receptor-expressing cells targeting microbe-associated molecular patterns

Info

Publication number: WO2022125632A1
Application number: PCT/US2021/062362
Authority: WO
Inventors: Keith Crawford
Original assignee: Miraki Innovation Think Tank LLC
Current assignee: Miraki Innovation Think Tank LLC
Priority date: 2020-12-08
Filing date: 2021-12-08
Publication date: 2022-06-16
Anticipated expiration: 2023-06-08

Abstract

Chimeric antigen receptors (CARs) and CAR-expressing cells that recognize and bind to one or more microbe-associated molecular patterns (MAMPs) are provided.

Description

CHIMERIC ANTIGEN RECEPTOR-EXPRESSING CELLS TARGETING

MICROBE-ASSOCIATED MOLECULAR PATTERNS

SEQUENCE LISTING

[0001] 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

“2021_201 lA_ST25.txt”; the file was created on December 8, 2021; the size of the file is 132 KB.

FIELD OF THE INVENTION

[0002] 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.

BACKGROUND

[0003] The development of 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. 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. Acad. Sci. USA 86, 10024-10028 (1989); Ertl et al. Cancer Res. 71, 3175-3181 (2011)). The so-called second and third generation CARs include additional activation domains from co- stimulatory molecules such as CD28 and CD137 (41BB) which serve to enhance T cell survival and proliferation. 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)). Various preclinical and early-phase clinical trials highlight the efficacy of CAR T cells to treat cancer patients having solid tumors and hematopoietic malignancies (Kershaw et al. Clin. Cancer Res. 12, 6106-6115 (2006); Larners et al. J. Clin. Oncol. 24, e20-e22 (2006); Morgan et aC Mol. Ther. 18, 843-851 (2010); Pule et al. Nat. Med. 14, 1264-1270 (2008); Till et al. Blood 112, 2261-2271 (2008)).

[0004] The promising early results achieved through the use of CAR-based T cells suggests engineered CAR-based cells might find applications in related fields. For example, while CARbased 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. Indeed, there is an increasing need in the fields of health care, food safety, agricultural testing, and biodefense for affordable and highly sensitive assays that rapidly and accurately identify the presence of microbes and microbial components in samples of interest. Important microbes and their components include, for example, environmental and pathogenic agents, including bacteria, viruses, toxins, and antigens. To meet this need, a variety of cellbased biosensor products have been commercially developed and released.

[0005] 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. In this system, when 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 Ca²⁺ within the B cells. In the CANARY® system, the B cells express aequorin, a Ca²⁺-sensitive photoprotein, which results in cell luminescence in the presence of elevated intracellular Ca²⁺ levels. Thus, the luminescence can be used to indicate antigen binding.

[0006] The CANARY® system can be used to efficiently identify a number of specific antigens, including those from bacteria, viruses, and toxins. However, 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.

[0007] Thus, the need remains for the development of a universal and near-universal cellbased biosensor that can be adapted for use in multiple testing platforms across a broad range of environmental and pathogenic agents. The present invention is directed to this and other important goals.

BRIEF SUMMARY

[0008] 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.

[0009] While described in more detail below, 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. The optional secondary target-binding domains do not comprise an MTM or engineered MTM. In some embodiments, the optional secondary target-binding domains help to stabilize interactions between the CAR and a microbe bound by the protein.

[0010] These 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.

[0011] As used herein, “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. For the sake of convenience, and unless otherwise indicated by context, reference to “MTM-based CARs” should be understood to include both CARs comprising a naturally-occurring MTM and CARs comprising an engineered MTM.

[0012] Given that the 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. In some aspects, a microbe-binding domain can be a recombinant molecule. In addition to the (i) microbe-binding domain and (ii) the optional one or more secondary target binding domains, 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.

[0013] 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. [0014] As indicated above, 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. Alternatively, or in addition, 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.

Alternatively, or in addition, 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. As a non-limiting example, the secondary binding domain may be a portion of an antibody, such as a single-chain variable fragment (scFv).

[0015] 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_xSer (where x is 1-5 Gly), Gly_xSerGly_xSer (where x is 1-5 Gly), Gly4Ser, and Gly4Ser repeats.

[0016] Non-limiting examples of the transmembrane domain of the CARs of the invention include the transmembrane regions of the human CD8a chain.

[0017] 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.

[0018] 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.

[0019] Thus, and in a first embodiment, 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.

[0020] 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).

[0021] In one aspect of this embodiment, 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).

[0022] In a specific aspect of this embodiment, 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.

[0023] In another specific aspect of this embodiment, 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. [0024] 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).

[0025] 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)-(xxiv).

[0026] In certain aspects of this embodiment, the at least one additional domain is an immunoglobulin domain. For example, 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.

[0027] In certain aspects of this embodiment, 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. For example, 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.

[0028] In certain aspects of this embodiment, 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. For example, 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. [0029] In certain aspects of this embodiment, 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).

[0030] In certain other aspects of this embodiment, 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. [0031] 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. Non-limiting examples of suitable transmembrane domains include, but are not limited to, the hinge and transmembrane regions of the human CD8a chain. Non-limiting examples of 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.

[0032] In a second embodiment, 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.

[0033] 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).

[0034] 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).

[0035] 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).

[0036] In certain aspects of this embodiment, the at least one additional domain is an immunoglobulin domain. For example, 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.

[0037] In certain aspects of this embodiment, 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.

[0038] In certain aspects of this embodiment, 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.

[0039] 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. Non-limiting examples of suitable transmembrane domains include, but are not limited to, the hinge and transmembrane regions of the human CD8a chain. Non-limiting examples of 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.

[0040] In a third embodiment, the invention is directed to CARs comprising at least one tolllike receptor (TLR)-based MTM as a primary target-binding domain. The 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.

[0041] 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). [0042] 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). [0043] 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).

[0044] In certain aspects of this embodiment, the at least one additional domain is an immunoglobulin domain. For example, 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.

[0045] In certain aspects of this embodiment, 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.

[0046] In certain aspects of this embodiment, 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

[0047] 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. Non-limiting examples of suitable transmembrane domains include, but are not limited to, the hinge and transmembrane regions of the human CD8a chain. Non-limiting examples of 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.

[0048] In a fourth embodiment, 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.

Thus, and in various non-limiting aspects, 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.

[0049] In a fifth embodiment, the invention is directed to a composition comprising one or more of the MTM-based CARs of the invention. In certain aspects of this embodiment, the composition further comprises at least one naturally-occurring MTM. In certain aspects of this embodiment, the composition further comprises one or more antimicrobial agents.

[0050] In a sixth embodiment, the invention is directed to a composition comprising one or more of the MTM-based CAR-expressing cells of the invention.

[0051] In a seventh embodiment, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1. Diagram showing the structure of exemplary collectins and ficolins.

[0053] Figure 2. Diagram showing the structure of an exemplary CAR comprising a collectin-based engineered MTM.

[0054] Figure 3. Diagram showing a portion of a CAR having four linked target-binding domains.

[0055] Figure 4. Diagram showing a portion of a CAR having three linked target-binding domains, wherein the domains bind three different targets. [0056] 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).

DETAILED DESCRIPTION

I. Definitions

[0057] As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0058] As used herein, “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.

II. The Present Invention

[0059] As summarized above, 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).

[0060] While described in more detail below, 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. The optional secondary target-binding domains do not comprise an MTM or engineered MTM. In some embodiments, the secondary target-binding domains help to stabilize interactions between the CAR and a microbe bound by the protein. [0061] 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.

[0062] As used herein, “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). For the sake of convenience, and unless otherwise indicated by context, reference to “MTM-based CARs” should be understood to include both CARs comprising a naturally-occurring MTM and CARs comprising an engineered MTM. The terms “MTM-based CARs” and “CARs” are used interchangeably, unless indicated otherwise by context.

[0063] In addition to binding a microbe or microbe component, 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. For example, 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.

[0064] 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. When the CAR has two or more primary target-binding domains, these primary domains may bind to the same or different MAMP. Similarly, when the CAR has two or more secondary target-binding domains, these secondary domains may bind to the same or different target molecule. Thus, the CARs of the invention may be bi-specific, tri-specific, quadra-specific, quinta-specific, sexta-specific, septa-specific, octospecific, etc. MAMPs

[0065] Before discussing the MTM-based CARs of the invention, it will be helpful to understand the molecules to which the MTM portion of the CARs bind. As indicated above, each of the MTM-based CARs of the invention binds to at least one microbe-associated molecular pattern (MAMP). Some MTM-based CARs bind at least two, at least three, at least four, at least five, or more than five MAMPs.

[0066] As used herein and throughout the specification, the term “microbe-associated molecular patterns” or “MAMPs” 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. In some aspects, the MAMPs encompass molecules associated with cellular components released during cell damage or lysis. Examples of 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.

[0067] In some aspects, 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. In some aspects, a microbe comprises a host cell membrane and a pathogen component or a PAMP.

[0068] In some aspects, microbe components comprise damage-associated molecular patterns (DAMPs), also known as danger-associated molecular patterns, danger signals, and alarmin. These biomolecules can initiate and sustain a non-infectious inflammatory response in a subject, in contrast to PAMPs which initiate and sustain an infectious pathogen-induced inflammatory response. Upon release from damaged or dying cells, DAMPs activate the innate immune system through binding to pattern recognition receptors (PRRs). 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. DAMPs include portions of nuclear and cytosolic proteins, ECM (extracellular matrix), mitochondria, granules, ER (endoplasmic reticulum), and plasma membrane. [0069] In some aspects, MAMPs include carbohydrate recognition domain (CRD)-binding motifs. As used herein, the term “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). As used herein, 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. In some aspects, the CRD can be derived from a lectin, as described herein. In some aspects, the CRD can be derived from a mannan-binding lectin (MBL). Accordingly, in some aspects, 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. In one embodiment, MAMPs are molecules, components, or motifs associated with microbes or groups of microbes that are recognized by mannan-binding lectin (MBL).

[0070] In some aspects, 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).

[0071] For clarity, MAMPs as used herein includes microbe components such as MAMPs, PAMPs and DAMPs as defined above.

[0072] When necessary, and unless otherwise detectable without pre-treatment, MAMPs can be exposed, released or generated from microbes in a sample by various sample pretreatment methods. In some aspects, the MAMPs can be exposed, released or generated by lysing or killing at least a portion of the microbes in the sample. Without limitations, 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. Accordingly, 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. Microbes

[0073] As used herein, the term “microbe”, and the plural “microbes”, generally refers to microorganism(s), including bacteria, virus, fungi, parasites, protozoan, archaea, protists, e.g., algae, and a combination thereof. The term “microbe” encompasses both live and dead microbes. The term “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.

[0074] In some aspects, the microbe is a human pathogen, in other words a microbe that causes at least one disease in a human.

[0075] In some aspects, the microbe is a Gram-positive bacterial species, a Gram-negative bacterial species, a mycobacterium, a fungus, a parasite, protozoa, or a virus. In some aspects, the Gram-positive bacterial species comprises bacteria from the class Bacilli. In some aspects, the Gram-negative bacterial species comprises bacteria from the class Gammaproteobacteria. In some aspects, the mycobacterium comprises bacteria from the class Actinobacteria. In some aspects, the fungus comprises fungus from the class Saccharomycetes.

[0076] In some aspects, the microbe is Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida albicans, ox Escherichia coli. In some aspects, 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.

[0077] In some aspects, 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 hepaticus, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacterium avium, Mycobacterium celatum, Mycobacterium celonae, Mycobacterium fortuitum, Mycobacterium genavense, Mycobacterium haemophilum, Mycobacterium intr acellular e, Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium ulcerans, Mycobacterium xenopi, Corynebacterium diptheriae, Rhodococcus equi, Rickettsia aeschlimannii, Rickettsia africae, Rickettsia conorii, Arcanobacterium haemolyticum, Bacillus anthracia, Bacillus cereus, Lysteria monocytogenes, Yersinia pestis, Yersinia enterocolitica, Shigella dysenteriae, Neisseria meningitides, Neisseria gonorrhoeae, Streptococcus bovis, Streptococcus hemolyticus, Streptococcus mutans, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Staphylococcus saprophyticus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Treponema pallidum, Human rhinovirus, Human coronavirus such as SARS-CoV-2, Dengue virus, Filoviruses (e.g., Marburg and Ebola viruses), Hantavirus, Rift Valley virus, Hepatitis B, C, and E, Human Immunodeficiency Virus (e.g., HIV-1, HIV-2), HHV-8, Human papillomavirus, Herpes virus (e.g., HV-I and HV-II), Human T-cell lymphotrophic viruses (e.g., HTLV-I and HTLV-II), Bovine leukemia virus, Influenza virus, Guanarito virus, Lassa virus, Measles virus, Rubella virus, Mumps virus, Chickenpox (Varicella virus), Monkey pox, Epstein Bahr virus, Norwalk (and Norwalk-like) viruses, Rotavirus, Parvovirus Bl 9, Hantaan virus, Sin Nombre virus, Venezuelan equine encephalitis, Sabia virus, West Nile virus, Yellow Fever virus, causative agents of transmissible spongiform encephalopathies, Creutzfeldt- Jakob disease agent, variant Creutzfeldt-Jakob disease agent, Candida, Cryptcooccus, Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax, Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes, Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoon hellem, or Encephalitozoon cuniculi, among other viruses, bacteria, archaea, protozoa, and fungi. In yet other aspects, the microbe is a bioterror agent (e.g., B. anthracis, and smallpox).

[0078] As used herein, “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. In some aspects, 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.

Sample

[0079] 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.

[0080] In some aspects, the sample, such as a fluid, may be purified before introduction to a device or a system of the invention. For example, 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.

[0081] 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.

[0082] 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.

[0083] 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.

Subi ect

[0084] The present invention, including the MTM-based CARs, MTM-based CAR expressing cells, compositions, and methods, may be used in conjunction with a subject. As used herein, 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

[0085] As summarized above, the invention is directed to “microbe-targeting molecules” (MTMs) and 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. [0086] 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. 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.

[0087] As used herein, “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. [0088] As will be apparent, while the “MTMs” used in the CARs of the invention include naturally-occurring molecules and proteins, the “engineered MTMs” of the invention are those have been manipulated in some manner by the hand of man. As used herein and throughout the specification, the term “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.

[0089] 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. In some aspects, a microbe-binding domain can be a recombinant molecule.

[0090] Acceptable microbe-binding domains for use in the MTMs of the invention are limited only in their ability to recognize and bind at least one MAMP. In some aspects, 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.

[0091] In particular aspects, 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. Non-limiting examples of 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. In some aspects, microbe-binding domains can be microbe-binding molecules described in the International Patent Application No. WO 2013/012924, the contents of which are incorporated by reference in their entirety. [0092] The MTMs of the invention will typically have one or more domains in addition to a microbe-binding domain. Such 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.

[0093] 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 substitutions, where the altered amino acids may be naturally-occurring or non- naturally-occurring amino acids, and conservative or non-conservative changes.

[0094] 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. For example, 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. In each case, the domains within a domain-variant engineered MTM are from at least two different proteins. Other examples of such 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.

[0095] 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. These “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.

[0096] As non-limiting examples of the 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. For example, 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.

Collectin-based MTMs

[0097] 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

[0098] Collectins (collagen-containing C-type lectins) 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. Upon binding of collectins to a microbe, clearance of the microbe is achieved via aggregation, complement activation, opsonization, and activation of phagocytosis.

[0099] 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). 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.

[00100] There are currently nine recognized members of the family: (i) mannose-binding lectin (MBL; mannose-binding lectin; e.g. SEQ ID NO: 1), (ii) surfactant protein A (SP-A; SEQ ID NO: 13), (iii) surfactant protein D (SP-D; SEQ ID NO: 14), (iv) collectin liver 1 (CL-L1; SEQ ID NO: 15), (v) collectin placenta 1 (CL-P1; SEQ ID NO: 16), (vi) 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), and (ix) conglutinin (SEQ ID NO:20). Each of these proteins is an MTM of the invention.

[00101] MBL (Homo sapiens; GenBank: AAH69338.1; SEQ ID NO: 1):

MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSS PGINGFPGKD

GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKS

PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK

VKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTG

NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI

[00102] Surfactant protein A (SP-A; Homo sapiens; GenBank: AAA36632.1; SEQ ID NO: 13): MTFKNNTTLS LFRLFNSFPL HKGYITTREL HQLALANLI F MFNCADLHFP

PQMRKCFPIP GTSCLFCPKT KTLFPVLHLA NQYLCFLCQH KHCFPQTRFP

DSQPSQATSR CVLS IKFTSS IYINSHNYTL ILTCI IVRVL CPPVGCKSYK

DRGHIHLAHP SVPSTCHGSY QLTSTQYIVW ISKYDSNFQH PKLVYSPTEP

AQVEHMQCFL CMCLQREERE ALLLLPRVTI LTRLSAESTD ERDGDSEPVN

AVCRTALAFV PHESNVMLGI HNLLIWLL

[00103] Surfactant protein D (SP-D; Homo sapiens; GenBank: AAB59450.1; SEQ ID NO: 14):

MLLFLLSALV LLTQPLGYLE AEMKTYSHRT MPSACTLVMC SSVESGLPGR DGRDGREGPR GEKGDPGLPG AAGQAGMPGQ AGPVGPKGDN GSVGEPGPKG DTGPSGPPGP PGVPGPAGRE GALGKQGNIG PQGKPGPKGE AGPKGEVGAP GMQGSAGARG LAGPKGERGV PGERGVPGNT GAAGSAGAMG PQGSPGARGP PGLKGDKGIP GDKGAKGESG LPDVASLRQQ VEALQGQVQH LQAAFSQYKK VELFPNGQSV GEKI FKTAGF VKPFTEAQLL CTQAGGQLAS PRSAAENAAL QQLWAKNEA AFLSMTDSKT EGKFTYPTGE SLVYSNWAPG EPNDDGGSED CVEI FTNGKW NDRACGEKRL WCEF

[00104] Collectin liver 1 (CL-L1; also known as collectin- 10; Homo sapiens; NCBI Reference

Sequence: NP_006429.2; SEQ ID NO: 15):

MNGFASLLRR NQFILLVLFL LQIQSLGLDI DSRPTAEVCA THTISPGPKG

DDGEKGDPGE EGKHGKVGRM GPKGIKGELG DMGDQGNIGK TGPIGKKGDK

GEKGLLGIPG EKGKAGTVCD CGRYRKFVGQ LDIS IARLKT SMKFVKNVIA

GIRETEEKFY YIVQEEKNYR ESLTHCRIRG GMLAMPKDEA ANTLIADYVA KSGFFRVFIG VNDLEREGQY MFTDNTPLQN YSNWNEGEPS DPYGHEDCVE

MLSSGRWNDT ECHLTMYFVC EFIKKKK

[00105] Collectin placenta 1 (CL-P1; Homo sapiens; GenBank: BAB72147.1; SEQ ID

NO: 16):

MKDDFAEEEE VQSFGYKRFG IQEGTQCTKC KNNWALKFS I ILLYILCALL TITVAILGYK WEKMDNVTG GMETSRQTYD DKLTAVESDL KKLGDQTGKK AISTNSELST FRSDILDLRQ QLREITEKTS KNKDTLEKLQ ASGDALVDRQ SQLKETLENN SFLITTVNKT LQAYNGYVTN LQQDTSVLQG NLQNQMYSHN WIMNLNNLN LTQVQQRNLI TNLQRSVDDT SQAIQRIKND FQNLQQVFLQ AKKDTDWLKE KVQSLQTLAA NNSALAKANN DTLEDMNSQL NSFTGQMENI TTISQANEQN LKDLQDLHKD AENRTAIKFN QLEERFQLFE TDIVNI ISNI SYTAHHLRTL TSNLNEVRTT CTDTLTKHTD DLTSLNNTLA NIRLDSVSLR MQQDLMRSRL DTEVANLSVI MEEMKLVDSK HGQLIKNFTI LQGPPGPRGP RGDRGSQGPP GPTGNKGQKG EKGEPGPPGP AGERGPIGPA GPPGERGGKG SKGSQGPKGS RGSPGKPGPQ GPSGDPGPPG PPGKEGLPGP QGPPGFQGLQ GTVGEPGVPG PRGLPGLPGV PGMPGPKGPP GPPGPSGAW PLALQNEPTP APEDNGCPPH WKNFTDKCYY FSVEKEI FED AKLFCEDKSS HLVFINTREE QQWIKKQMVG RESHWIGLTD SERENEWKWL DGTSPDYKNW KAGQPDNWGH

GHGPGEDCAG LIYAGQWNDF QCEDVNNFIC EKDRETVLSS AL

[00106] Conglutinin collectin (CL-43; Bos taurus; GenBank: AAH6148; SEQ ID NO: 17):

MLPLPLS ILL LLTQSQSFLG EEMDVYSEKT LTDPCTVWC APPADSLRGH DGRDGKEGPQ GEKGDPGPPG MPGPAGREGP SGRQGSMGPP GTPGPKGEPG PEGGVGAPGM PGSPGPTGLK GERGTPGPGG AIGPQGPSGA MGPPGLKGDR GDPGEKGAKG ETSVLEVDTL RQRMRNLEGE VQRLQNIVTQ YRKAVLFPDG QAVGEKI FKT AGAVKSYSDA EQLCREAKGQ LASPRSSAEN EAVTQLVRAK

NKHAYLSMND ISKEGKFTYP TGGSLDYSNW APGEPNNRAK DEGPENCLEI YSDGNWNDIE CREERLVICE F

[00107] Collectin of 46 kDa (CL-46; Bos taurus; GenBank: AAM34742; SEQ ID NO: 18):

MLLLPLSVLL LLTQPWRSLG AEMKIYSQKT LANGCTLWC RPPEGGLPGR DGQDGREGPQ GEKGDPGSPG PAGRAGRPGP AGPIGPKGDN GSAGEPGPKG DTGPPGPPGM PGPAGREGPS GKQGSMGPPG TPGPKGDTGP KGGMGAPGMQ GSPGPAGLKG ERGAPGELGA PGSAGVAGPA GAIGPQGPSG ARGPPGLKGD RGDPGERGAK GES GLAD VNA LKQRVTILEG QLQRLQNAFS RYKKAVLFPD GQAVGKKI FK TAGAVKSYSD AQQLCREAKG QLASPRSAAE NEAVAQLVRA

KNNDAFLSMN DISTEGKFTY PTGESLVYSN WASGEPNNNN AGQPENCVQI YREGKWNDVP CSEPLLVICE F

[00108] Collectin kidney 1 (CL-K1; also known as collectin-11; Homo sapiens; GenBank:

BAF43301.1; SEQ ID NO: 19):

MRGNLALVGV LISLAFLSLL PSGHPQPAGD DACSVQILVP GLKGDAGEKG DKGAPGRPGR VGPTGEKGDM GDKGQKGSVG RHGKIGPIGS KGEKGDSGDI GPPGPNGEPG LPCECSQLRK AIGEMDNQVS QLTSELKFIK NAVAGVRETE

SKIYLLVKEE KRYADAQLSC QGRGGTLSMP KDEAANGLMA AYLAQAGLAR

VFIGINDLEK EGAFVYSDHS PMRTFNKWRS GEPNNAYDEE DCVEMVASGG

WNDVACHTTM YFMCEFDKEN M

[00109] Conglutinin (Bos taurus; GenBank: BAA04983; SEQ ID NO:20):

MLLLPLSVLL LLTQPWRSLG AEMTTFSQKI LANACTLVMC SPLESGLPGH DGQDGRECPH GEKGDPGSPG PAGRAGRPGW VGPIGPKGDN GFVGEPGPKG DTGPRGPPGM PGPAGREGPS GKQGSMGPPG TPGPKGETGP KGGVGAPGIQ GFPGPSGLKG EKGAPGETGA PGRAGVTGPS GAIGPQGPSG ARGPPGLKGD RGDPGETGAK GESGLAEVNA LKQRVTILDG HLRRFQNAFS QYKKAVLFPD GQAVGEKI FK TAGAVKSYSD AEQLCREAKG QLASPRSSAE NEAVTQMVRA QEKNAYLSMN DISTEGRFTY PTGEILVYSN WADGEPNNSD EGQPENCVEI FPDGKWNDVP CSKQLLVICE F

[00110] 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. As used herein, the term “collectin-based molecule” refers to a molecule comprising a microbe-binding domain derived from a collectin, such as a lectin. The term “lectin” as used herein refers to any molecule including proteins, natural or genetically modified (e.g., recombinant), that interacts specifically with saccharides (e.g., carbohydrates). The term “lectin” as used herein 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. Examples of 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. Examples of 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. In some aspects, 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). In some aspects, the C-type lectin can include, but are not limited to, collectin, DC-SIGN, and fragments thereof. Without wishing to be bound by theory, 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. [00111] 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. 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. When 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.

[00112] 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.

MBL

[00113] In particular aspects and embodiments of the invention, 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.

[00114] The amino acid sequence of full-length human MBL (SEQ ID NO: 1; GenBank: AAH69338.1) is: MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSS PGINGFPGKD

GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKS

PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK

VKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTG

NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI [00115] 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

[00116] The amino acid sequence of a truncated MBL that retains microbe surface-binding (SEQ ID NO:3) is:

AASERKALQT EMARIKKWLT FSLGKQVGNK FFLTNGEIMT FEKVKALCVK FQASVATPRN AAENGAIQNL IKEEAFLGIT DEKTEGQFVD LTGNRLTYTN WNEGEPNNAG SDEDCVLLLK NGQWNDVPCS TSHLAVCEFP I

[00117] 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

VPCSTSHLAV CEFPI

[00118] The amino acid sequence of the neck and carbohydrate recognition domain of MBL (SEQ ID NO:5) is: PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK

VKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTG

NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI

[00119] 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.

[00120] Alternatively, 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. [00121] As to 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. In addition, 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. In some aspects, 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. Accordingly, in some aspects, the carbohydrate recognition domain of an engineered MBL protein can comprise SEQ ID NO:4. In some aspects, the carbohydrate recognition domain of an engineered MBL protein can comprise a fragment of SEQ ID NO:4 as defined above. Also, 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. In some aspects, 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.

[00122] The sequence variants of the naturally-occurring protein and the truncated forms thereof (e.g. SEQ ID NOs: l-5) 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

[00123] In other particular aspects and embodiments of the invention, 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. In some aspects, 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. As discussed above, 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.

2016/0311877, U.S. Patent Pub. 2019/0077850, U.S. Patent No. 9,593,160, U.S. Patent No. 10,435,457, U.S. Patent Pub. 2015/0173883 and International Application Publication No. WO 2011/090954, the entire disclosures of which are hereby incorporated by reference in their entirety.

[00124] 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. For example, 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. In use, the 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.

[00125] Specific examples of 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. [00126] 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

[00127] AKT-FcMBL (SEQ ID NO:7):

AKTEPKSSDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGAPDGDS SLAASERKAL QTEMARIKKW LTFSLGKQVG NKFFLTNGEI MTFEKVKALC VKFQASVATP RNAAENGAIQ NLIKEEAFLG ITDEKTEGQF VDLTGNRLTY TNWNEGEPNN AGSDEDCVLL LKNGQWNDVP CSTSHLAVCE EPI

[00128] 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

[00129] FcMBL (SEQ ID NO:9):

AKTEPKSSDK THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYDSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGAPDGDS SLAASERKAL QTEMARIKKW" LTFSLGKQVG NKFFLTNGEI MTFEKVKALC VKFQASVATP RNAAENGAIQ NLIKEEAFLG ITDEKTEGQF VDLTGNRLTY TNWNEGEPNN AGSDEDCVLL LKNGQWNDVP CSTSHLAVCE EPI

[00130] In SEQ ID NOV, the residues with a single underscore correspond to the Fc portion, the residues with a double underscore correspond to the MBL neck, and those residues without underscore correspond to the MBL carbohydrate-binding domain.

[00131] Various genetically engineered versions of the MTM (e.g., FcMBL) are described in

International Application Pub. Nos. WO 2011/090954 and WO 2013/012924, as well as US Patents 9,150,631 and 9,593,160, the contents of each of which are incorporated herein by reference in their entireties. Lectins and other mannose binding molecules are also described in, for example, US 9,150,631 and US 9,632,085, and PCT application publication nos.

WO/2011/090954, WO2013012924 and WO/2013/130875, the contents of all of which are incorporated herein by reference in their entireties.

[00132] In aspects where the MTM is FcMBL, the Fc region or a fragment thereof can comprise at least one mutation, e.g., to modify the performance of the engineered MBL. For example, in some aspects, 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.

[00133] SEQ ID NO: 12:

EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVWD

VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN

GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL

TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS

RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GA

[00134] In some aspects and embodiments, 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.

[00135] The exemplary MBL sequences provided herein are not construed to be limiting. For example, while 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.

Other Collectins

[00136] In addition to the aspects and embodiments of the invention defined above that comprise the collectin MBL (whether naturally-occurring or engineered), 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. Thus, 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).

[00137] As with MBL, 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.

[00138] 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.

[00139] Alternatively, 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.

[00140] The sequence variants of the naturally-occurring protein and the truncated forms thereof (e.g. SEQ ID NOs: 13-20) 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. Ficolin-based MTMs

[00141] 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. 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.

[00142] 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.

[00143] There are currently three recognized members of the family: ficolin 1 (M-ficolin), ficolin 2 (L-ficolin), and ficolin 3 (H-ficolin). Each of these proteins is an MTM of the invention. The amino acid sequences of the human forms of the proteins are provided in the following paragraphs, with the fibrinogen-like domain underlined:

[00144] Ficolin 1 precursor - SEQ ID NO:21 - NP 001994.2 - Homo sapiens

MELSGATMAR GLAVLLVLFL HIKNLPAQAA DTCPEVKVVG LEGSDKLTIL RGCPGLPGAP

GPKGEAGVIG ERGERGLPGA PGKAGPVGPK GDRGEKGMRG EKGDAGQSQS CATGPRNCKD

LLDRGYFLSG WHTIYLPDCR PLTVLCDMDT DGGGWTVFQR RMDGSVDFYR DWAAYKQGFG

SQLGEFWLGN DNIHALTAQG SSELRVDLVD FEGNHQFAKY KSFKVADEAE KYKLVLGAFV

GGSAGNSLTG HNNNFFSTKD QDNDVSSSNC AEKFQGAWWY ADCHASNLNG LYLMGPHESY

ANGINWSAAK GYKYSYKVSE MKVRPA

[00145] Ficolin 2 isoform a precursor - SEQ ID NO:22 - NP 004099.2 - Homo sapiens

MELDRAVGVL GAATLLLSFL GMAWALQAAD TCPEVKMVGL EGSDKLTILR GCPGLPGAPG PKGEAGTNGK RGERGPPGPP GKAGPPGPNG APGEPQPCLT GPRTCKDLLD RGHFLSGWHT IYLPDCRPLT VLCDMDTDGG GWTVFQRRVD GSVDFYRDWA TYKQGFGSRL GEFWLGNDNI

HALTAQGTSE LRVDLVDFED NYQFAKYRSF KVADEAEKYN LVLGAFVEGS AGDSLTFHNN

QSFSTKDQDN DLNTGNCAVM FQGAWWYKNC HVSNLNGRYL RGTHGSFANG INWKSGKGYN

YSYKVSEMKV RPA

[00146] Ficolin 3 isoform 1 precursor - SEQ ID NO:23 - NP 003656.2 - Homo sapiens

MDLLWILPSL WLLLLGGPAC LKTQEHPSCP GPRELEASKV VLLPSCPGAP GSPGEKGAPG PQGPPGPPGK MGPKGEPGDP VNLLRCQEGP RNCRELLSQG ATLSGWYHLC LPEGRALPVF CDMDTEGGGW LVFQRRQDGS VDFFRSWSSY RAGFGNQESE FWLGNENLHQ LTLQGNWELR VELEDFNGNR TFAHYATFRL LGEVDHYQLA LGKFSEGTAG DSLSLHSGRP FTTYDADHDS SNSNCAVIVH GAWWYASCYR SNLNGRYAVS EAAAHKYGID WASGRGVGHP YRRVRMMLR

[00147] As with the collectins, 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.

[00148] 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.

[00149] Alternatively, 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.

[00150] The sequence variants of the naturally-occurring protein and the truncated forms thereof (e.g. SEQ ID NOs:21-23) 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.

[00151] 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. As used herein, the term “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.

[00152] 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. 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.

[00153] 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.

[00154] In certain aspects, the at least one additional domain is an immunoglobulin domain. For example, 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. [00155] In some aspects, 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. In other aspects, 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. In further aspects, 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.

[00156] In some aspects, 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.

[00157] In some aspects, 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. [00158] In some aspects, 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.

[00159] The exemplary sequences provided herein for the ficolins are not to be construed as limiting. For example, while the 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 receptor-based MTMs

[00160] Toll-like receptors (TLRs) 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. 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.

[00161] Members of the family have a common structure, characterized by three parts or domains arranged in the following N- to C-terminal arrangement: (i) an N-terminal ligandbinding domain, i.e. the microbe-binding domain, (ii) a single transmembrane helix (~ 20 amino acids), and (iii) a C-terminal cytoplasmic signaling domain.

[00162] 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. 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.

[00163] 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.

[00164] 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.

[00165] There are a number of recognized human members of the family, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10. Each of these proteins may be used as an MTM of the invention. The amino acid sequences of the human forms of the proteins are provided in the following paragraphs, with the extracellular domain that comprises the N-terminal ligand-binding domain underlined:

[00166] TLR1 (toll-like receptor 1 precursor) - NCBI Reference Sequence: NP 003254.2

(SEQ ID NO:24)

MTS I FHFAI I FMLILQIRIQ LSEESEFLVD RSKNGLIHVP KDLSQKTTIL NI SQNY ISEL WTSDILSLSK LRILI I SHNR IQYLDISVFK FNQELEYLDL SHNKLVKISC HPTVNLKHLD LSFNAFDALP ICKE FGNMSQ LKFLGLSTTH LEKSSVLPIA HLNISKVLLV LGETYGEKED PEGLQDFNTE SLHIVFPTNK E FHFILDVSV KTVANLELSN IKCVLEDNKC SY FLSILAKL QTNPKLSNLT LNNIETTWNS FIRILQLVWH TTVWYFSI SN VKLQGQLDFR DFDYSGTSLK ALS IHQVVSD VFGFPQSY IY E I FSNMNIKN FTVSGTRMVH MLCPSKI SPF LHLDFSNNLL TDTVFENCGH LTELETLILQ MNQLKELSKI AEMTTQMKSL QQLDI SQNSV SYDEKKGDCS WTKSLLSLNM SSNILTDT I F RCLPPRIKVL DLHSNKIKSI PKQVVKLEAL QELNVAFNSL TDLPGCGSFS SLSVLI IDHN SVSHPSADFF QSCQKMRS IK AGDNPFQCTC ELGE FVKNID QVSSEVLEGW PDSYKCDYPE SYRGTLLKDF HMSELSCNIT LLIVT IVATM LVLAVTVTSL CSYLDLPWYL RMVCQWTQTR RRARNIPLEE LQRNLQFHAF I SYSGHDSFW VKNELLPNLE KEGMQICLHE RNFVPGKS IV ENI ITCIEKS YKSI FVLSPN FVQSEWCHYE LY FAHHNLFH EGSNSLILIL LEPI PQYS IP SSYHKLKSLM ARRTYLEWPK EKSKRGLFWA NLRAAINIKL TEQAKK

[00167] TLR2 (toll-like receptor 2 precursor) - NCBI Reference Sequence: NP_001305722.1 (SEQ ID NO:25)

MPHTLWMVWV LGVI ISLSKE ESSNQASLSC DRNGICKGSS GSLNS IPSGL TEAVKSLDLS NNRITYI SNS DLQRCVNLQA LVLTSNGINT IEEDSFSSLG SLEHLDLSYN YLSNLSSSWF KPLSSLT FLN LLGNPYKTLG ETSLFSHLTK LQILRVGNMD T FTKIQRKDF AGLT FLEELE IDASDLQSYE PKSLKS IQNV SHLILHMKQH ILLLEI FVDV TSSVECLELR DTDLDT FHFS ELSTGETNSL IKKFTFRNVK ITDESLFQVM KLLNQI SGLL ELE FDDCTLN GVGNFRASDN DRVIDPGKVE TLTIRRLHIP RFYLFYDLST LYSLTERVKR ITVENSKVFL VPCLLSQHLK SLEYLDLSEN LMVEEYLKNS ACEDAWPSLQ TLILRQNHLA SLEKTGETLL TLKNLTNIDI SKNSFHSMPE TCQWPEKMKY LNLSSTRIHS VTGCIPKTLE ILDVSNNNLN LFSLNLPQLK ELY ISRNKLM TLPDASLLPM LLVLKISRNA ITTFSKEQLD S FHTLKTLEA GGNNFICSCE FLS FTQEQQA LAKVLIDWPA NYLCDSPSHV RGQQVQDVRL SVSECHRTAL VS GMCC AL FL LILLTGVLCH RFHGLWYMKM MWAWLQAKRK PRKAPSRNIC YDAFVSYSER DAYWVENLMV QELENFNPPF KLCLHKRDFI PGKWI IDNI I DS IEKSHKTV FVLSENFVKS EWCKYELDFS HFRLFDENND AAILILLEPI EKKAI PQRFC KLRKIMNTKT YLEWPMDEAQ REGFWVNLRA AIKS

[00168] TLR3 (toll-like receptor 3 precursor) - NCBI Reference Sequence: NP 003256.1 (SEQ ID NO:26)

MRQTLPCIY F WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH NQLRRLPAAN FTRYSQLTSL DVGFNTI SKL EPELCQKLPM LKVLNLQHNE LSQLSDKT FA FCTNLTELHL MSNS IQKIKN NPFVKQKNLI TLDLSHNGLS STKLGTQVQL ENLQELLLSN NKIQALKSEE LDI FANSSLK KLELSSNQIK EFSPGCFHAI GRLFGLFLNN VQLGPSLTEK LCLELANTS I RNLSLSNSQL STTSNTT FLG LKWTNLTMLD LSYNNLNVVG NDSFAWLPQL EYFFLEYNNI QHLFSHSLHG LFNVRYLNLK RS FTKQSI SL ASLPKIDDFS FQWLKCLEHL NMEDNDI PGI KSNMFTGLIN LKYLSLSNS F TSLRTLTNET FVSLAHSPLH ILNLTKNKIS KIESDAFSWL GHLEVLDLGL NEIGQELTGQ EWRGLENI FE IYLSYNKYLQ LTRNSFALVP SLQRLMLRRV ALKNVDSSPS PFQPLRNLT I LDLSNNNIAN INDDMLEGLE KLEILDLQHN NLARLWKHAN PGGPIY FLKG LSHLHILNLE SNGFDE IPVE VFKDLFELKI IDLGLNNLNT LPASVFNNQV SLKSLNLQKN LITSVEKKVF GPAFRNLTEL DMRFNPFDCT CESIAWFVNW INETHTNIPE LSSHYLCNTP PHYHGFPVRL FDTSSCKDSA PEEL F FMINT SILLI FI FIV LLIHFEGWRI SFYWNVSVHR VLGFKEIDRQ TEQFEYAAYI IHAYKDKDWV WEHFSSMEKE DQSLKFCLEE RDFEAGVFEL EAIVNSIKRS RKI I FVITHH LLKDPLCKRF KVHHAVQQAI EQNLDSI ILV FLEE IPDYKL NHALCLRRGM FKSHCILNWP VQKERIGAFR HKLQVALGSK NSVH

[00169] TLR4 (toll-like receptor 4 isoform D) - NCBI Reference Sequence: NP 612567.1 (SEQ ID NO:27)

MMSASRLAGT LI PAMAFLSC VRPESWEPCV EVVPNITYQC MELNFYKIPD NLPFSTKNLD LSFNPLRHLG SYSFFS FPEL QVLDLSRCE I QT IEDGAYQS LSHLSTLILT GNPIQSLALG AFSGLSSLQK LVAVETNLAS LENFPIGHLK TLKELNVAHN LIQSFKLPEY FSNLTNLEHL DLSSNKIQS I YCTDLRVLHQ MPLLNLSLDL SLNPMNFIQP GAFKE IRLHK LTLRNNFDSL NVMKTCIQGL AGLEVHRLVL GEFRNEGNLE KFDKSALEGL CNLTIEE FRL AYLDYYLDDI IDLFNCLTNV SS FSLVSVTI ERVKDFSYNF GWQHLELVNC KFGQFPTLKL KSLKRLTFTS NKGGNAFSEV DLPSLE FLDL SRNGLSFKGC CSQSDFGTTS LKYLDLS FNG VITMSSNFLG LEQLEHLDFQ HSNLKQMSEF SVFLSLRNLI YLDI SHTHTR VAFNGI FNGL SSLEVLKMAG NSFQENFLPD I FTELRNLTF LDLSQCQLEQ LSPTAFNSLS SLQVLNMSHN NFFSLDTFPY KCLNSLQVLD YSLNHIMTSK KQELQHFPSS LAFLNLTQND FACTCEHQS F LQWIKDQRQL LVEVERMECA TPSDKQGMPV LSLNITCQMN KT I IGVSVLS VLVVSVVAVL VYKFYFHLML LAGCIKYGRG ENIYDAFVIY SSQDEDWVRN ELVKNLEEGV PPFQLCLHYR DFIPGVAIAA NI IHEGFHKS RKVIVVVSQH FIQSRWCI FE YE IAQTWQFL SSRAGI I FIV LQKVEKTLLR QQVELYRLLS RNTYLEWEDS VLGRHI FWRR LRKALLDGKS WNPEGTVGTG CNWQEATS I [00170] TLR5 (toll-like receptor 5 precursor) - NCBI Reference Sequence: NP 003259.2 (SEQ ID NO:28)

MGDHLDLLLG VVLMAGPVFG I PSCS FDGRI AFYRFCNLTQ VPQVLNTTER LLLS FNYIRT VTASS FPFLE QLQLLELGSQ YTPLT IDKEA FRNLPNLRIL DLGSSKIYFL HPDAFQGLFH LFELRLY FCG LSDAVLKDGY FRNLKALTRL DLSKNQIRSL YLHPS FGKLN SLKS IDFSSN QI FLVCEHEL EPLQGKTLSF FSLAANSLYS RVSVDWGKCM NPFRNMVLE I LDVSGNGWTV DITGNFSNAI SKSQAFSLIL AHHIMGAGFG FHNIKDPDQN T FAGLARSSV RHLDLSHGFV FSLNSRVFET LKDLKVLNLA YNKINKIADE AFYGLDNLQV LNLSYNLLGE LYSSNFYGLP KVAYIDLQKN HIAI IQDQTF KFLEKLQTLD LRDNALTT IH FIPSI PDI FL SGNKLVTLPK INLTANLIHL SENRLENLDI LYFLLRVPHL QILILNQNRF SSCSGDQTPS ENPSLEQLFL GENMLQLAWE TELCWDVFEG LSHLQVLYLN HNYLNSLPPG VFSHLTALRG LSLNSNRLTV LSHNDLPANL EILDISRNQL LAPNPDVFVS LSVLDITHNK FICECELST F INWLNHTNVT IAGPPADIYC VYPDSFSGVS LFSLSTEGCD EEEVLKSLKF SLFIVCTVTL TLFLMT ILTV TKFRGFCFIC YKTAQRLVFK DHPQGTEPDM YKYDAYLCFS SKDFTWVQNA LLKHLDTQYS DQNRFNLCFE ERDFVPGENR IANIQDAIWN SRKIVCLVSR HFLRDGWCLE AFSYAQGRCL SDLNSALIMV VVGSLSQYQL MKHQS IRGFV QKQQYLRWPE DLQDVGWFLH KLSQQILKKE KEKKKDNNI P LQTVAT IS

[00171] TLR6 (toll-like receptor 6 precursor) - NCBI Reference Sequence: NP 006059.2 (SEQ ID NO:29)

MTKDKEPIVK SFHFVCLMI I IVGTRIQFSD GNEFAVDKSK RGLIHVPKDL PLKTKVLDMS QNY IAELQVS DMSFLSELTV LRLSHNRIQL LDLSVFKFNQ DLEYLDLSHN QLQKISCHPI VSFRHLDLS F NDFKALPICK E FGNLSQLNF LGLSAMKLQK LDLLPIAHLH LSYILLDLRN YYIKENETES LQILNAKTLH LVFHPTSLFA IQVNISVNTL GCLQLTNIKL NDDNCQVFIK FLSELTRGST LLNFTLNHIE TTWKCLVRVF QFLWPKPVEY LNIYNLT I IE SIREEDFTYS KTTLKALTIE HITNQVFLFS QTALYTVFSE MNIMMLTI SD TPFIHMLCPH APST FKFLNF TQNVFTDSI F EKCSTLVKLE TLILQKNGLK DLFKVGLMTK DMPSLEILDV SWNSLESGRH KENCTWVES I VVLNLSSNML TDSVFRCLPP RIKVLDLHSN KIKSVPKQVV KLEALQELNV AFNSLTDLPG CGSFSSLSVL I IDHNSVSHP SADFFQSCQK MRS IKAGDNP FQCTCELREF VKNIDQVSSE VLEGWPDSYK CDYPESYRGS PLKDFHMSEL SCNITLL I VT IGATMLVLAV TVTSLCIYLD LPWYLRMVCQ WTQTRRRARN IPLEELQRNL QFHAFISYSE HDSAWVKSEL VPYLEKEDIQ ICLHERNFVP GKS IVENI IN CIEKSYKS I F VLSPNFVQSE WCHYELYFAH HNLFHEGSNN LILILLEPIP QNS IPNKYHK LKALMTQRTY LQWPKEKSKR GLFWANIRAA FNMKLTLVTE NNDVKS

[00172] TLR7 (toll-like receptor 7 precursor) - NCBI Reference Sequence: NP 057646.1 (SEQ ID NO:30)

MVFPMWTLKR QILILFNI IL I SKLLGARWF PKTLPCDVTL DVPKNHVIVD CTDKHLTE IP

GGI PTNTTNL TLTINHIPDI SPASFHRLDH LVEIDFRCNC VPI PLGSKNN MCIKRLQIKP

RSFSGLTYLK SLYLDGNQLL E IPQGLPPSL QLLSLEANNI FSIRKENLTE LANIEILYLG

QNCYYRNPCY VSYS IEKDAF LNLTKLKVLS LKDNNVTAVP TVLPSTLTEL YLYNNMIAKI

QEDDFNNLNQ LQILDLSGNC PRCYNAPFPC APCKNNSPLQ I PVNAFDALT ELKVLRLHSN

SLQHVPPRWF KNINKLQELD LSQNFLAKE I GDAKFLHFLP SLIQLDLSFN FELQVYRASM

NLSQAFSSLK SLKILRIRGY VFKELKS FNL SPLHNLQNLE VLDLGTNFIK IANLSMFKQF

KRLKVIDLSV NKISPSGDSS EVGFCSNART SVESYEPQVL EQLHY FRYDK YARSCRFKNK

EAS FMSVNES CYKYGQTLDL SKNSI FFVKS SDFQHLSFLK CLNLSGNLI S QTLNGSEFQP

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 RDWLPGQPVL ENLSQSIQLS KKTVFVMTDK YAKTENFKIA FYLSHQRLMD EKVDVIILI F LEKPFQKSKF LQLRKRLCGS SVLEWPTNPQ AHPYFWQCLK NALATDNHVA YSQVFKETV

[00173] TLR8 (toll-like receptor 8 isoform 1) - UniProtKB/Swiss-Prot: Q9NR97.1 (SEQ ID

NO:31)

MENMFLQSSM LTCI FLLISG SCELCAEENF SRSYPCDEKK QNDSVIAECS NRRLQEVPQT VGKYVTELDL SDNFITHITN ESFQGLQNLT KINLNHNPNV QHQNGNPGIQ SNGLNITDGA FLNLKNLREL LLEDNQLPQI PSGLPESLTE LSLIQNNIYN ITKEGISRLI NLKNLYLAWN CYFNKVCEKT NIEDGVFETL TNLELLSLSF NSLSHVPPKL PSSLRKLFLS NTQIKYISEE DFKGLINLTL LDLSGNCPRC FNAPFPCVPC DGGASINIDR FAFQNLTQLR YLNLSSTSLR KINAAWFKNM PHLKVLDLEF NYLVGEIASG AFLTMLPRLE ILDLSFNYIK GSYPQHINIS RNFSKLLSLR ALHLRGYVFQ ELREDDFQPL MQLPNLSTIN LGINFIKQID FKLFQNFSNL EIIYLSENRI SPLVKDTRQS YANSSSFQRH IRKRRSTDFE FDPHSNFYHF TRPLIKPQCA AYGKALDLSL NSIFFIGPNQ FENLPDIACL NLSANSNAQV LSGTEFSAIP HVKYLDLTNN RLDFDNASAL TELSDLEVLD LSYNSHYFRI AGVTHHLEFI QNFTNLKVLN LSHNNIYTLT DKYNLESKSL VELVFSGNRL DILWNDDDNR YISI FKGLKN LTRLDLSLNR LKHIPNEAFL NLPASLTELH INDNMLKFFN WTLLQQFPRL ELLDLRGNKL LFLTDSLSDF TSSLRTLLLS HNRISHLPSG FLSEVSSLKH LDLSSNLLKT INKSALETKT TTKLSMLELH GNPFECTCDI GDFRRWMDEH LNVKIPRLVD VICASPGDQR GKSIVSLELT TCVSDVT A V I LFFFTFFITT MVMLAALAHH LFYWDVWFIY NVCLAKVKGY RSLSTSQTFY DAYISYDTKD ASVTDWVINE LRYHLEESRD KNVLLCLEER DWDPGLAIID NLMQSINQSK KTVFVLTKKY AKSWNFKTAF YLALQRLMDE NMDVII FILL EPVLQHSQYL RLRQRICKSS ILQWPDNPKA EGLFWQTLRN VVLTENDSRY NNMYVDSIKQ Y

[00174] TLR9 (toll-like receptor 9 precursor) - NCBI Reference Sequence: NP 059138.1

(SEQ ID NO:32)

MGFCRSALHP LSLLVQAIML AMTLALGTLP AFLPCELQPH GLVNCNWLFL KSVPHFSMAA PRGNVTSLSL SSNRIHHLHD SDFAHLPSLR HLNLKWNCPP VGLSPMHFPC HMTIEPSTFL AVPTLEELNL SYNNIMTVPA LPKSLISLSL SHTNILMLDS ASLAGLHALR FLFMDGNCYY KNPCRQALEV APGALLGLGN LTHLSLKYNN LTVVPRNLPS SLEYLLLSYN RIVKLAPEDL ANLTALRVLD VGGNCRRCDH APNPCMECPR HFPQLHPDTF SHLSRLEGLV LKDSSLSWLN ASWFRGLGNL RVLDLSENFL YKCITKTKAF QGLTQLRKLN LSFNYQKRVS FAHLSLAPSF GSLVALKELD MHGI FFRSLD ETTLRPLARL PMLQTLRLQM NFINQAQLGI FRAFPGLRYV DLSDNRISGA SELTATMGEA DGGEKVWLQP GDLAPAPVDT PSSEDFRPNC STLNFTLDLS RNNLVTVQPE MFAQLSHLQC LRLSHNCISQ AVNGSQFLPL TGLQVLDLSH NKLDLYHEHS FTELPRLEAL DLSYNSQPFG MQGVGHNFSF VAHLRTLRHL SLAHNNIHSQ VSQQLCSTSL RALDFSGNAL GHMWAEGDLY LHFFQGLSGL IWLDLSQNRL HTLLPQTLRN LPKSLQVLRL RDNYLAFFKW WSLHFLPKLE VLDLAGNQLK ALTNGSLPAG TRLRRLDVSC NSISFVAPGF FSKAKELREL NLSANALKTV DHSWFGPLAS ALQILDVSAN PLHCACGAAF MDFLLEVQAA VPGLPSRVKC GSPGQLQGLS I FAQDLRLCL DEALSWDCFA LSLLAVALGL GVPMLHHLCG WDLWYCFHLC LAWLPWRGRQ SGRDEDALPY DAFVVFDKTQ SAVADWVYNE LRGQLEECRG RWALRLCLEE RDWLPGKTLF ENLWASVYGS RKTLFVLAHT DRVSGLLRAS FLLAQQRLLE DRKDVVVLVI LSPDGRRSRY VRLRQRLCRQ SVLLWPHQPS GQRSFWAQLG MALTRDNHHF YNRNFCQGPT AE

[00175] TLR10 (toll-like receptor 10 isoform a) - NCBI Reference Sequence:

NP_001017388.1 (SEQ ID NO:33)

MRLIRNIYI F CS IVMTAEGD APELPEEREL MTNCSNMSLR KVPADLTPAT TTLDLSYNLL

FQLQSSDFHS VSKLRVLILC HNRIQQLDLK TFEFNKELRY LDLSNNRLKS VTWYLLAGLR

YLDLS FNDFD TMPICEEAGN MSHLE ILGLS GAKIQKSDFQ KIAHLHLNTV FLGFRTLPHY

EEGSLPILNT TKLHIVLPMD TNFWVLLRDG IKTSKILEMT NIDGKSQFVS YEMQRNLSLE

NAKTSVLLLN KVDLLWDDLF LILQFVWHTS VEHFQIRNVT FGGKAYLDHN SFDYSNTVMR

T IKLEHVHFR VFYIQQDKIY LLLTKMDIEN LT ISNAQMPH MLFPNYPTKF QYLNFANNIL

TDELFKRTIQ LPHLKTLILN GNKLETLSLV SCFANNTPLE HLDLSQNLLQ HKNDENCSWP

ETVVNMNLSY NKLSDSVFRC LPKSIQILDL NNNQIQTVPK ETIHLMALRE LNIAFNFLTD

LPGCSHFSRL SVLNIEMNFI LSPSLDFVQS CQEVKTLNAG RNPFRCTCEL KNFIQLETYS

EVMMVGWSDS YTCEYPLNLR GTRLKDVHLH ELSCNTALLI VTIVVIMLVL GLAVAFCCLH

FDLPWYLRML GQCTQTWHRV RKTTQEQLKR NVRFHAFI SY SEHDSLWVKN ELIPNLEKED GSILICLYES YFDPGKSI SE NIVSFIEKSY KS I FVLSPNF VQNEWCHYE F YFAHHNLFHE NSDHI ILILL EPIPFYCI PT RYHKLKALLE KKAYLEWPKD RRKCGLFWAN LRAAINVNVL ATREMYELQT FTELNEESRG STI SLMRTDC L

[00176] As with the collectins, 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.

[00177] 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.

[00178] Alternatively, 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.

[00179] The sequence variants of the naturally-occurring protein and the truncated forms thereof (e.g. SEQ ID NOs:24-33) 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.

[00180] 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. As used herein, the term “TLR-based molecule” refers to a molecule comprising a microbe-binding domain (i.e. an N-terminal ligand-binding domain) derived from a TLR. The term “TLR” as used herein 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. The term “TLR” as used herein 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.

[00181] 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. a 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. 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.

[00182] 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.

[00183] In certain aspects of this embodiment, the at least one additional domain is an immunoglobulin domain. For example, 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. [00184] In some aspects, 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, or the N-terminal ligand-binding domain of TLR10 of SEQ ID NO:33. In each of these examples, MTMs and engineered MTMs may further comprise an immunoglobulin domain of SEQ ID NO: 12.

[00185] In some aspects, 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.

[00186] In certain aspects of this embodiment, 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.

[00187] In some aspects, 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.

[00188] The exemplary sequences provided herein for the TLRs are not to be construed as limiting. For example, while 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.

[00189] In some further aspects, 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, 15/668,794, 15/750,788, 15/839,352, 16/059,799, 16/302,023, 16/553,635; and US patent numbers 9,150,631, 9,593,160, 9,632,085, 9,791,440, and 10,435,457; the contents of each of which are incorporated by reference herein in their entireties.

Chimeric antigen receptors

[00190] As indicated above, 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. The 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.

[00191] 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).

[00192] As suggested above, 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.

[00193] When the CAR has two or more primary target-binding domains, these primary domains may bind to the same or different MAMP. Similarly, when the CAR has two or more secondary target-binding domains, these secondary domains may bind to the same or different target molecule. Thus, the CARs of the invention may be bi-specific, tri-specific, quadraspecific, quinta-specific, sexta-specific, septa-specific, octo-specific, etc. [00194] 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. Alternatively, 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.

[00195] 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. For example, 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. Alternatively, or in addition, 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. Alternatively, or in addition, the secondary domain could bind a non-mutable target of a pathogen, thus preventing the pathogen from escaping immune surveillance. For example, and with respect to a virus known to rapidly mutate such as the SARS-CoV-2 virus, 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. As a result, while the composition of the spike proteins may change over time, 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. Thus, such an CAR would offer broad-spectrum protection and avoid the need to generate new antibody cocktails for each new emergent infectious disease.

[00196] 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. As a non-limiting example, the secondary binding domain may be a portion of an antibody, such as a single-chain variable fragment (scFv).

[00197] 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_xSer (where x is 1-5 Gly), Gly_xSerGly_xSer (where x is 1-5 Gly), Gly4Ser, and Gly4Ser repeats.

[00198] 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.

[00199] 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. In some aspects, a hinge domain is inserted between the target-binding domain closest to the surface of the cell and the transmembrane domain (see Figure 2).

[00200] 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. When a hinge domain is included in the CAR, an exemplary hinge and transmembrane domain includes, but is not limited to, the hinge and transmembrane regions of the human CD8a chain.

[00201] 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. When the CAR-expressing cell is a T cell and a T cell activation domain is used, 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.

[00202] When present, 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.

[00203] 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.

CAR-expressing Cells

[00204] As indicated above, the present invention is also directed to cells expressing the CARs of the invention.

[00205] It will be apparent that 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.

Production of CAR-expressing Cells

[00206] Selected cells may be engineered to express CARs by means readily known to the skilled artisan. Generally, 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. [00207] In one aspect, 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

[00208] The invention also includes compositions comprising one or more of types of MTM- based CARs defined herein. Different “types” of MTM-based CARs vary based on the identity of the primary and/or secondary target binding domains. For example, and as indicated above, 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 identity of the MAMP bound by the PRR portion of the microbebinding domain; the affinity or avidity of binding to the MAMP; etc.), or (iii) both structural and functional terms. Thus, in one instance, when a composition is defined as comprising two or more types of MTM-based CARs, 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. 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.

[00209] The 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. Thus, for example, 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.

[00210] The 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).

Compositions Comprising MTM-based CAR-expressing Cells

[00211] The invention also includes compositions comprising one or more of types of MTM- based CAR-expressing cells as defined herein.

[00212] The 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).

Carriers and diluents

[00213] Depending on the manner in which the MTM-based CARs and the MTM-based CAR-expressing cells are used, the 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. Examples of suitable carriers and diluents include water, buffered water, saline, buffered saline, dextrose, glycerol, ethanol, and combinations thereof, propylene glycol, polysorbate 80 (Tween-80™), 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. In one embodiment of the invention, the composition comprises as a carrier or diluent, an aqueous solution comprising sodium acetate having a pH of about 3.2.

[00214] The 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.

[00215] When the 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). Examples of 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.

[00216] Some exemplary 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, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Cefmetazole, Cefuroxime, Loracarbef), Cephalosporin III Generation (e.g., Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone), Cephalosporin IV Generation (e.g., Cefepime), Macrolides and Lincosamides (e.g., Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin), Quinolones and Fluoroquinolones (e.g., Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Perfloxacin), Carbapenems (e.g., Imipenem-Cilastatin, Meropenem), Monobactams (e.g., Aztreonam), Aminoglycosides (e.g., Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin), Glycopeptides (e.g., Teicoplanin, Vancomycin), Tetracyclines (e.g., Demeclocycline, Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline, Chlortetracycline), Sulfonamides (e.g., Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfamethizole), Rifampin (e.g., Rifabutin, Rifampin, Rifapentine), Oxazolidinones (e.g., Linezolid, Streptogramins, Quinupristin Dalfopristin), Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazole, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Ethionamide, Pyrazinamide, Para-aminosalicylic acid, Erythromycin ethylsuccinate, and the like.

[00217] 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, Allylamines, amorolfin, butenafine, naftifine, terbinafine, Echinocandins, Anidulafungin, Caspofungin, Micafungin, Aurones, Benzoic acid, Ciclopirox, Flucytosine, 5-fluorocytosin, Griseofulvin, Haloprogin, Tolnaftate, Undecylenic acid, Triacetin, Crystal violet, Castellani's paint, Orotomide, Miltefosine, Potassium iodide, Coal tar, Copper(II) sulfate, Selenium disulfide, Sodium thiosulfate, Piroctone olamine, lodoquinol, clioquinol, Acrisorcin, Zinc pyrithione, and Sulfur. Additional antifungals known in the art can also be used.

[00218] 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, Imunovir, Indinavir, Inosine, Integrase inhibitor, Interferon, Interferon type I, Interferon type II, Interferon type III, Lamivudine, Ledipasvir, Lopinavir, Lopiravir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide, Norvir, Nucleoside analogues, Ombitasvir, Oseltamivir (Tamiflu), Paritaprevir, Peglyated Interferon-alpha, Peginterferon alfa-2a, Penciclovir, Peramivir, Pibrentasvir, Pleconaril, Podophyllotoxin, Protease inhibitor, Pyramidine, Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rilpivirine, Rimantadine, Ritonavir, Saquinavir, Simeprevir, Sofosbuvir, Stavudine, Synergistic enhancer (antiretroviral), Telaprevir, Telbivudine, Tenofovir, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Velpatasvir, Vicriviroc, Vidarabine, Viramidine, Voxilaprevir, Zalcitabine, Zanamivir (Relenza), Zidovudine. Additional antivirals known in the art can also be used.

[00219] The 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.

[00220] The 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. Examples of suitable carriers and diluents include saline, buffered saline, dextrose, water-for- inj ection, glycerol, ethanol, and combinations thereof, propylene glycol, polysorbate 80 (Tween- 80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. 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. The formulations comprising MTM-based CARs and MTM-based CAR-expressing cells will typically have been prepared using MTM-based CAR proteins from cultures prepared in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin). [00221] In some aspects, 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.

[00222] 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. In some aspects, the delivery device comprises a nebulizer where the nebulizer can comprise a small volume nebulizer, a large volume nebulizer, or an ultrasound nebulizer. In some aspects, the delivery device comprises a metered-dose inhaler. In these aspects, the inhaler can include spacers or holding chambers. In some aspects, the delivery device comprises a dry-powder inhaler.

[00223] 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. Aerosol administration enables a direct, topical application of the composition to the target site, such as the lung, which may be particularly advantageous in preventing a respiratory-transmitted infection such as SARS-CoV-2. [00224] In some aspects, 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.

[00225] In some aspects, 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.

[00226] In some aspects, 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.

[00227] 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. In some aspects, if a subject is exposed to a known pathogen, a particular antimicrobial, based on the pathogen, can be selected and added to the composition.

[00228] In some aspects, MTM-based CAR or MTM-based CAR-expressing cellular compositions (e.g., engineered MTMs as described therein), methods, systems, and assays 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, 14/766,575, 14/831,480, 14/904,583, 15/105,298, 15/415,352, 15/483,216, 15/668,794, 15/750,788, 15/839,352, 16/059,799, 16/302,023, 16/553,635; and US patent numbers 9,150,631, 9,593,160, 9,632,085, 9,791,440, and 10,435,457; the contents of each of which are incorporated by reference herein in their entireties.

Uses

[00229] As suggested above, 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.

[00230] MTM-based CARs and MTM-based CAR-expressing cells can be used in diagnostic and/or therapeutic health care applications. As one non-limiting example, 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.

[00231] As a second non-limiting example, the MTM-based CARs and MTM-based CAR- expressing cells can be used in methods of treating a subject infected by a microbial pathogen. For example, 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.

[00232] As a third non-limiting example, the MTM-based CARs and MTM-based CAR- expressing cells can be used in methods of detecting a microbe in a sample. For example, 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.

[00233] As a fourth non-limiting example, 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. For example, 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.

[00234] 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.

[00235] In some aspects, the microbe is a human pathogen, in other words a microbe that causes at least one disease in a human.

[00236] In some aspects, the microbe is a Gram -positive bacterial species, a Gram -negative bacterial species, a mycobacterium, a fungus, a parasite, protozoa, or a virus. In some aspects, the Gram-positive bacterial species comprises bacteria from the class Bacilli. In some aspects, the Gram-negative bacterial species comprises bacteria from the class Gammaproteobacteria. In some aspects, the mycobacterium comprises bacteria from the class Actinobacteria. In some aspects, the fungus comprises fungus from the class Saccharomycetes.

[00237] In some aspects, the microbe is Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Candida albicans, ox Escherichia coli. In some aspects, 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.

[00238] In some aspects, 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 hepaticus, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacterium avium, Mycobacterium celatum, Mycobacterium celonae, Mycobacterium fortuitum, Mycobacterium genavense, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium ulcerans, Mycobacterium xenopi, Corynebacterium diptheriae, Rhodococcus equi, Rickettsia aeschlimannii, Rickettsia africae, Rickettsia conorii, Arcanobacterium haemolyticum, Bacillus anthracia, Bacillus cereus, Lysteria monocytogenes, Yersinia pestis, Yersinia enterocolitica, Shigella dysenteriae, Neisseria meningitides, Neisseria gonorrhoeae, Streptococcus bovis, Streptococcus hemolyticus, Streptococcus mutans, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Staphylococcus saprophyticus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Treponema pallidum, Human rhinovirus, Human coronavirus such as SARS-CoV-2, Dengue virus, Filoviruses (e.g., Marburg and Ebola viruses), Hantavirus, Rift Valley virus, Hepatitis B, C, and E, Human Immunodeficiency Virus (e.g., HIV-1, HIV-2), HHV-8, Human papillomavirus, Herpes virus (e.g., HV-I and HV-II), Human T-cell lymphotrophic viruses (e.g., HTLV-I and HTLV-II), Bovine leukemia virus, Influenza virus, Guanarito virus, Lassa virus, Measles virus, Rubella virus, Mumps virus, Chickenpox (Varicella virus), Monkey pox, Epstein Bahr virus, Norwalk (and Norwalk-like) viruses, Rotavirus, Parvovirus Bl 9, Hantaan virus, Sin Nombre virus, Venezuelan equine encephalitis, Sabia virus, West Nile virus, Yellow Fever virus, causative agents of transmissible spongiform encephalopathies, Creutzfeldt-Jakob disease agent, variant Creutzfeldt-Jakob disease agent, Candida, Cryptcooccus, Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax, Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes, Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoon hellem, or Encephalitozoon cuniculi, among other viruses, bacteria, archaea, protozoa, and fungi. In yet other aspects, the microbe is a bioterror agent (e.g., B. anthracis, and smallpox).

[00239] As used herein, 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. Preferably, 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.

[00240] 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.

[00241] The 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.

[00242] In each of the embodiments of the present invention 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.

[00243] 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

[00244] 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. [00245] Other 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., ¹⁵²Eu), a radioactive marker (e.g., radioisotopes for diagnostic purposes, including ³H, ¹³¹I, ³⁵S, ¹⁴C, and ¹²⁵I), a chemiluminescent marker (e.g., luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester), and a protein tag (e.g., biotin, phycobiliprotein, c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS).

Side Groups

[00246] The MTMs of the invention can be engineered to display side groups that augment, enhance or otherwise alter selected characteristics of the MTMs. For example, 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

[00247] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

Equivalents

[00248] Various modifications of the invention and many further aspects thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various aspects and equivalents thereof.

Claims

WHAT IS CLAIMED IS:

1. A microbe-targeting molecule (MTM)-based chimeric antigen receptor (CAR) comprising (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, wherein the one or more primary target-binding domains comprise a microbebinding domain, wherein the microbe-binding domain comprises a collectin-based MTM comprising at least one collectin microbe-binding domain and optionally at least one additional domain, wherein the collectin microbe-binding domain comprises the carbohydrate recognition domain (CRD) of a collectin selected from the group consisting 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), and wherein the at least one additional domain is one or more domains selected from the group consisting 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 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)-(xxiv).

2. The MTM-based CAR of claim 1, wherein 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.

3. The MTM-based CAR of claim 1 or 2, wherein the CRD of MBL comprises 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.

4. The MTM-based CAR of any one of claims 1-3, wherein the optional at least one additional domain is an immunoglobulin domain.

5. The MTM-based CAR of claim 1 or 4, wherein the immunoglobulin domain comprises 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.

6. The MTM-based CAR of any one of claims 1-5, wherein the optional one or more secondary target binding domain is not present.

7. The MTM-based CAR of any one of claims 1-5, wherein the optional one or more secondary target binding domain is present.

8. The MTM-based CAR of claim 1, wherein the MTM is one or more of SEQ ID NOs: 6-9, or a sequence variant thereof having at least 85% sequence identity to any one of SEQ ID NOs:6-9.

9. A polynucleotide sequence encoding a MTM-based CAR of any one of claims 1- 8, or a complementary strand thereof.

10. A cloning or expression vector comprising a polynucleotide sequence of claim 9.

11. A cell comprising a polynucleotide sequence of claim 9.

12. A cell comprising a cloning or expression vector of claim 10.

13. A method of producing a MTM-based CAR comprising culturing a cell of claim

11 under conditions promoting expression of the MTM-based CAR, and recovering the MTM- based CAR from the cell or cell culture.

14. A method of producing a MTM-based CAR comprising culturing a cell of claim

12 under conditions promoting expression of the MTM-based CAR, and recovering the MTM- based CAR from the cell or cell culture.

15. A cell expressing a MTM-based CAR of any one of claims 1-8.

16. A MTM-based CAR comprising (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, wherein the one or more primary target-binding domains comprise a microbe-binding domain, wherein the microbe-binding domain comprises a ficolin- based microbe-targeting molecule (MTM) comprising at least one ficolin microbe-binding domain and optionally at least one additional domain, wherein the ficolin microbe-binding domain comprises the fibrinogen-like domain of a ficolin selected from the group consisting 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), and wherein the at least one additional domain is one or more domains selected from the group consisting 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).

17. The MTM-based CAR of claim 16, wherein the ficolin microbe-binding domain comprises 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.

18. The MTM-based CAR of claim 16 or 17, wherein the optional at least one additional domain is an immunoglobulin domain.

19. The MTM-based CAR of claim 16 or 18, wherein the immunoglobulin domain comprises 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.

20. The MTM-based CAR of any one of claims 16-19, wherein the optional one or more secondary target binding domain is not present.

21. The MTM-based CAR of any one of claims 16-19, wherein the optional one or more secondary target binding domain is present.

22. A polynucleotide sequence encoding a MTM-based CAR of any one of claims 16- 21, or a complementary strand thereof.

23. A cloning or expression vector comprising a polynucleotide sequence of claim 22.

24. A cell comprising a polynucleotide sequence of claim 22.

25. A cell comprising a cloning or expression vector of claim 23.

26. A method of producing a MTM-based CAR comprising culturing a cell of claim

24 under conditions promoting expression of the MTM-based CAR, and recovering the MTM- based CAR from the cell or cell culture.

27. A method of producing a MTM-based CAR comprising culturing a cell of claim

25 under conditions promoting expression of the MTM-based CAR, and recovering the MTM- based CAR from the cell or cell culture.

28. A cell expressing a MTM-based CAR of any one of claims 16-21.

29. A MTM-based CAR comprising (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, wherein the one or more primary target-binding domains comprise a microbe-binding domain, wherein the microbe-binding domain comprises a toll-like receptor (TLR)-based microbe-targeting molecule (MTM) comprising at least one TLR microbebinding domain and optionally at least one additional domain, wherein the TLR microbe-binding domain comprises the N-terminal ligand-binding domain of a TLR selected from the group consisting of

(i) TLR1,

(ii) TLR2,

(iii) TLR3,

(iv) TLR4,

(v) TLR5,

(vi) TLR6,

(vii) TLR7,

(vii) TLR8,

(viii) TLR9,

(ix) TLR10, and

(xi) a TLR transmembrane helix,

(xii) a TLR C-terminal cytoplasmic signaling domain,

(xiii) a ficolin short N-terminal domain,

(xiv) a ficolin collagen-like domain,

(xv) a collectin cysteine-rich domain,

(xvi) a collectin collagen-like domain,

(xvii) a collectin coiled-coil neck 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

30. The MTM-based CAR of claim 29, wherein the TLR microbe-binding domain comprises 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.

31. The MTM-based CAR of claim 29 or 30, wherein the optional at least one additional domain is an immunoglobulin domain.

32. The MTM-based CAR of claim 29 or 31, wherein the immunoglobulin domain comprises 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.

33. The MTM-based CAR of any one of claims 29-32, wherein the optional one or more secondary target binding domain is not present.

34. The MTM-based CAR of any one of claims 29-32, wherein the optional one or more secondary target binding domain is present.

35. A polynucleotide sequence encoding a MTM-based CAR of any one of claims 29- 34, or a complementary strand thereof.

36. A cloning or expression vector comprising a polynucleotide sequence of claim 35.

37. A cell comprising a polynucleotide sequence of claim 35.

38. A cell comprising a cloning or expression vector of claim 36.

39. A method of producing a MTM-based CAR comprising culturing a cell of claim

37 under conditions promoting expression of the MTM-based CAR, and recovering the MTM- based CAR from the cell or cell culture.

40. A method of producing a MTM-based CAR comprising culturing a cell of claim

38 under conditions promoting expression of the MTM-based CAR, and recovering the MTM- based CAR from the cell or cell culture.

41. A cell expressing a MTM-based CAR of any one of claims 29-34.

42. A composition comprising a population of cells expressing one or more MTM- based CARs, wherein the one or more MTM-based CARs are selected from any of the MTM- based CARs of claims 1-8, 16-21 and 29-34.

43. The composition of claim 42, further comprising one or more antimicrobial agents.

44. A method of treating a microbial infection in a subject comprising administering to a subject in need thereof a therapeutically-effective amount of a cell of any one of claims 15, 28 and 41, thereby treating a microbial infection in a subject.

45. A method of detecting a microbial infection in a subject comprising administering to a subject in need thereof a population of cells of any one of claims 15, 28 and 41 and detecting binding by the cells to a microbe, thereby detecting a microbial infection in a subject.

46. The method of claim 44 or 45, wherein the microbial infection is caused by a bacteria, virus, fungi, parasite, protozoa, archaea, or protist.

47. The MTM-based CAR of any one of claims 1-8, 16-21 and 29-34, wherein the MTM-based CAR is bi-specific or tri-specific.

48. The MTM-based CAR of any one of claims 1-8, 16-21 and 29-34, wherein the MTM-based CAR comprises 1, 2 or 3 primary binding domains and 1, 2 or 3 secondary binding domains.

49. The MTM-based CAR of any one of claims 1-8, 16-21 and 29-34, wherein targetbinding domains are linked by short peptides of 1-50 amino acids in length.

50. The MTM-based CAR of claim 49, wherein the short peptide is Gly_xSer or Gly_xSerGly_xSer, where each x is individually 1-5 Gly.

51. A MTM-based CAR of any one of claims 1-8, 16-21 and 29-34 comprising one primary target-binding domain and two secondary target binding domains, wherein the primary domain binds a viral MAMP, and wherein the secondary domains bind viral coat or spike proteins.