Anti-ETEC adhesin protein antibodies and methods of use

Abstract

The invention provides anti-enterotoxigenic E. coli (ETEC) adhesin protein antibodies and methods of using the same. One embodiment is an isolated antibody that binds ETEC coli surface antigen 3 (CS3) protein and ETEC coli surface antigen 6 (CS6) protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following complementary-determining regions (CDRs): (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21). Also disclosed are antibodies comprising said CS3 / CS6 binding VHH and a binding domain capable of binding to an ETEC adhesin protein selected from colonization factor antigen I adhesin subunit E (CfaE), coli surface antigen 1 (CS1), coli surface antigen 4 (CS4), coli surface antigen 14 (CS14), coli surface antigen 17 (CS17), coli surface antigen 19 (CS19), and coli surface antigen 2 (CS2).

Description

[0001] PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0002] ANTI-ETEC ADHESIN PROTEIN ANTIBODIES AND METHODS OF USE

[0003] STATEMENT AS TO FEDERALLY FUNDED RESEARCH

[0004] This invention was made with government support under W81 XWH-21 -2-0018, HT9425-24-1 - 0302, and W81 XWH-21 -9-0019 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 7, 2025, is named 50811 -013WO2_Sequence_Listing_8_7_25 and is 53,252 bytes in size.

[0007] BACKGROUND OF THE INVENTION

[0008] Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in infants in the developing world, as well as the major cause of traveler’s diarrhea. Transmission of ETEC occurs when contaminated food or water is ingested. ETEC infections are characterized by diarrhea, vomiting, stomach cramps, and in some cases mild fever. Symptoms usually occur 1 -3 days after infection and last for a few days. When adult travelers develop ETEC-related diarrhea, a short course of antibiotics can decrease the duration and volume of diarrhea. However, ETEC strains are becoming increasingly resistant to antibiotics, and there are currently no licensed vaccines for protecting travelers against ETEC- related diarrhea. Accordingly, there exists a need for improved treatments or prevention of ETEC-related disorders.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, the disclosure features an isolated antibody that binds enterotoxigenic E. coli (ETEC) coli surface antigen 3 (CS3) protein and ETEC coli surface antigen 6 (CS6) protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following complementary- determining regions (CDRs): (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ).

[0011] In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0012] In some embodiments, the CS3 / CS6 VHH binding domain comprises one or more of the following framework regions (FRs): (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 22); (b) an FR-H2 comprising the amino acid sequence of MGWFRQAPGKEREGVSC (SEQ ID NO: 23); (c) an FR-H3 comprising the amino acid sequence of YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 24); and (d) an FREW comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 25).

[0013] In a second aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 26.

[0014] In a third aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ).

[0015] In a fourth aspect, the disclosure features an isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding to CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ).

[0016] In a fifth aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

[0017] In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0018] In some embodiments, the CS3 / CS6 VHH binding domain comprises one or more of the following FRs: (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 4); (b) an FR-H2 comprising the amino acid sequence of MGWFRQAPGKEREFVAA (SEQ ID NO: 5); (c) an FR-H3 comprising the amino acid sequence of PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0019] IYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 6); and (d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 7).

[0020] In a sixth aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 8.

[0021] In a seventh aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

[0022] In an eighth aspect, the disclosure features an isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding to CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

[0023] In a ninth aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

[0024] In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0025] In some embodiments, the CS3 / CS6 VHH binding domain comprises one or more of the following FRs: (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 13); (b) an FR-H2 comprising the amino acid sequence of MAWFRQAPGKEREIVAA (SEQ ID NO: 14); (c) an FR-H3 comprising the amino acid sequence of DYTDSVKGRFTISRDNAKNTGFLQMNRLKPEDTAVYYC (SEQ ID NO: 15); and (d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 16). PATENT

[0026] Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0027] In a tenth aspect, the disclosure features an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 17.

[0028] In an eleventh aspect, the disclosure features an isolated antibody that binds an ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

[0029] In a twelfth aspect, the disclosure features an isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding to CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

[0030] In some embodiments of any of the above aspects, the antibody comprises an Fc region. In some embodiments, the Fc region is an immunoglobulin A (IgA) Fc region. In some embodiments, the IgA Fc region is an lgA1 Fc region. In some embodiments, the Ig A1 Fc region has at least 90% identity to SEQ ID NO: 37 or comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the IgA Fc region is an lgA2 Fc region. In some embodiments, the lgA2 Fc region has at least 90% identity to SEQ ID NO: 45 or comprises the amino acid sequence of SEQ ID NO: 45.

[0031] In some embodiments, the CS3 / CS6 VHH binding domain is connected to the Fc region by a linker. In some embodiments, the linker is a hinge region. In some embodiments, the hinge region comprises or consists of the sequence of SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 50, or SEQ ID NO: 51.

[0032] In other embodiments, the antibody does not comprise an Fc region.

[0033] In some embodiments of any of the above aspects, the antibody comprises two or more VHH binding domains (e.g., comprises two VHH binding domains). In some embodiments, the antibody comprises a binding domain capable of binding to an ETEC adhesin protein selected from colonization factor antigen I adhesin subunit E (CfaE), coli surface antigen 1 (CS1 ), coli surface antigen 4 (CS4), coli surface antigen 14 (CS14), coli surface antigen 17 (CS17), coli surface antigen 19 (CS19), and coli surface antigen 2 (CS2).

[0034] In some embodiments, the antibody comprises a CfaE VHH binding domain. In some embodiments, the CfaE VHH binding domain comprises the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0035] In some embodiments, the CfaE VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 the CfaE VHH binding domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the CfaE VHH binding domain consists of the amino acid sequence of SEQ ID NO: 35.

[0036] In another aspect, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ); and (ii) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0037] In another aspect, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3); and (ii) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0038] In another aspect, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and (ii) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0039] In some embodiments, the antibody comprises an Fc domain comprising a first Fc domain subunit and a second Fc domain subunit, wherein the first Fc domain subunit and the second Fc domain subunit are capable of stable association, and wherein: (i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain; and (ii) the second Fc domain subunit is linked to the CfaE VHH binding domain.

[0040] In some embodiments, (i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain in an N-terminal-to-C-terminal direction as follows: CS3 / CS6 VHH binding domain-Fc domain subunit; and (ii) the second Fc domain subunit is linked to the CfaE VHH binding domain in an N-terminal- to-C-terminal direction as follows: CfaE VHH binding domain-Fc domain subunit. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0041] In some embodiments, (i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain by a hinge region; and (ii) the second Fc domain subunit is linked to the CfaE VHH binding domain by a hinge region.

[0042] In some embodiments, each Fc domain subunit is further linked to a single CH1 domain. In some embodiments, each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit. In some embodiments, each VHH binding domain, CH1 domain, hinge region, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-hinge region-Fc domain subunit.

[0043] In some embodiments, the hinge region comprises the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRP (SEQ ID NO: 36), ASPVPPPPPCCHPRLSLHRP (SEQ ID NO: 44), ASPVPSTPPTPSPSTPPTPSPS (SEQ ID NO: 50), or ASPVPPPPPP (SEQ ID NO: 51 ).

[0044] In some embodiments, the antibody is a full-length antibody.

[0045] In some embodiments, the antibody comprises one or more modifications that promote the association of the first Fc subunit with the second Fc subunit.

[0046] In some embodiments, the antibody comprises the following N-terminal-to-C-terminal structure: (P3-L2)n3-P2-(Ll-Pl)n1 wherein Pi, P2, and P3 are each independently selected from (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ); (ii) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3); (iii) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR- H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and (iv) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30); wherein Li and L2are each independently a linker; and ni and n3are each independently 0 or 1 , wherein at least one of ni and n3are 1 .

[0047] In some embodiments, ni is 1 and n3is 0, and the antibody comprises the following N-terminal-to- C-terminal structure:

[0048] P2-LI-PI .

[0049] In some embodiments, Pi and P2each comprise different VHH binding domains. In other embodiments, Pi and P2each comprise identical VHH binding domains. PATENT Attorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77

[0050] In some embodiments, ni is 1 and ns is 1 , and the antibody comprises the following N-terminal-to-C- terminal structure:

[0051] P3-L2-P2-L1-P1 .

[0052] In some embodiments, Pi , P2, and P3 each comprise different VHH binding domains. In other embodiments, Pi , P2, and P3 each comprise identical VHH binding domains.

[0053] In some embodiments, the antibody comprises a single VHH binding domain. In some embodiments, the antibody consists of a single VHH binding domain.

[0054] In some embodiments, the antibody is a monoclonal antibody.

[0055] In some embodiments, the antibody is a humanized or chimeric antibody.

[0056] In some embodiments, the antibody is capable of inhibiting mannose-resistant hemagglutination of human group A erythrocytes.

[0057] In some embodiments, the antibody binds CS3 with a 50% effective concentration (EC50) of between about 1 ng / mL and about 1000 ng / mL; between about 1 ng / mL and about 10 ng / mL; between about 10 ng / mL and about 100 ng / mL; or between about 100 ng / mL and about 1000 ng / mL.

[0058] In some embodiments, the antibody binds CS6 with an EC50 of between about 1 ng / mL and about 1000 ng / mL; between about 1 ng / mL and about 10 ng / mL; between about 10 ng / mL and about 100 ng / mL; or between about 100 ng / mL and about 1000 ng / mL.

[0059] In some embodiments, the EC50 is measured by ELISA.

[0060] In some embodiments, the antibody is capable of inhibiting the binding of ETEC bacteria to intestinal cells.

[0061] In another aspect, the disclosure features one or more isolated nucleic acids encoding the antibody of any one of the above aspects.

[0062] In another aspect, the disclosure features one or more vectors comprising the one or more nucleic acids of the above aspect.

[0063] In another aspect, the disclosure features one or more host cells comprising the one or more vectors of the above aspect. In some embodiments, each of the one or more host cells is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell, e.g., a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 239 cell. In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments, the eukaryotic cell is a plant cell, e.g., a tobacco plant cell, a soybean plant cell, or a rice plant cell. In some embodiments, each of the one or more host cells is a prokaryotic cell, e.g., an E. coli cell.

[0064] In another aspect, the disclosure features a method of producing a VHH antibody, the method comprising culturing a host cell comprising the one or more isolated nucleic acids of the above aspect in a culture medium. In some embodiments, the method further comprises recovering the antibody from the host cell or the culture medium.

[0065] In another aspect, the disclosure features a composition comprising the antibody of any one of the above aspects.

[0066] In another aspect, the disclosure features a composition comprising (i) the antibody of the first or second aspect; and (ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence PATENT

[0067] Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0068] In another aspect, the disclosure features a composition comprising: (i) the antibody of the fifth or sixth aspect; and (ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0069] In another aspect, the disclosure features a composition comprising: (i) the antibody of the ninth or tenth aspect; and (ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0070] In another aspect, the disclosure features a composition comprising any of the one or more isolated nucleic acids provided herein.

[0071] In another aspect, the disclosure features a composition comprising any of the one or more vectors provided herein.

[0072] In another aspect, the disclosure features a pharmaceutical composition comprising any of the antibodies, nucleic acids, or vectors provided herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is formulated for treating a disorder associated with an ETEC infection in a subject. In some embodiments, the disorder associated with an ETEC infection is ETEC-related diarrhea.

[0073] In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for intramuscular administration.

[0074] In another aspect, the disclosure features a lipid nanoparticle comprising any one of the compositions or pharmaceutical compositions provided herein.

[0075] In another aspect, the disclosure features a method of treating a subject having a disorder associated with an ETEC infection comprising administering to the subject an effective amount of any one of the antibodies, compositions or pharmaceutical compositions, or lipid nanoparticles provided herein, thereby treating the subject.

[0076] In another aspect, the disclosure features a method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising administering to the subject an effective amount of any one of the antibodies, compositions or pharmaceutical compositions, or lipid nanoparticles provided herein, thereby treating the subject.

[0077] In another aspect, the disclosure features a method of treating a subject having a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of the first or second aspect and an effective amount of an isolated antibody that binds CfAE, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR- H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0078] In another aspect, the disclosure features a method of treating a subject having a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of the fifth or sixth aspect and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR- H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0079] In another aspect, the disclosure features a method of treating a subject having a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of the ninth or tenth aspect and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR- H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0080] In another aspect, the disclosure features a method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of the first or second aspect and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0081] In another aspect, the disclosure features a method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of the fifth or sixth aspect and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0082] In another aspect, the disclosure features a method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of the ninth or tenth aspect and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30). PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0083] In some embodiments, the antibody that binds ETEC CS3 protein and ETEC CS6 protein and the antibody that binds CfAE are formulated together. In other embodiments, the antibody that binds ETEC CS3 protein and ETEC CS6 protein and the antibody that binds CfAE are formulated separately.

[0084] In some embodiments, the antibody that binds ETEC CS3 protein and ETEC CS6 protein is administered to the subject at a dosage of about 0.1 mg / kg to about 100 mg / kg; is administered to the subject at a dosage of about 1 mg / kg to about 80 mg / kg; is administered to the subject at a dosage of about 1 mg / kg to about 40 mg / kg; is administered to the subject at a dosage of about 5 mg / kg to about 20 mg / kg; or is administered to the subject at a dosage of about 10 mg / kg.

[0085] In some embodiments, the subject is administered at least one dose of the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle. In some embodiments, the subject is administered at least two doses of the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle.

[0086] In some embodiments, the disorder associated with an ETEC infection is ETEC-related diarrhea.

[0087] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered orally to the subject.

[0088] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered subcutaneously to the subject.

[0089] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered intramuscularly to the subject.

[0090] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered approximately once a day to the subject.

[0091] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered approximately once a month to the subject.

[0092] In another aspect, the disclosure features a method of detecting an ETEC in a sample from a subject, the method comprising contacting the sample with any one of the antibodies provided herein under conditions permissive for binding of the antibody to an ETEC and detecting whether a complex is formed between the antibody and the ETEC. In some embodiments, the sample is a swab sample, a lavage sample, a blood sample, a plasma sample, a sputum sample, a urine sample, a stool sample, a sample from the mucosal lining of the small intestine, or a mucosal secretion sample. In some embodiments, the sample is a sample from the mucosal lining of the small intestine. In some embodiments, the sample is a stool sample.

[0093] In some embodiments, the subject is presumed to have an ETEC infection.

[0094] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0095] In another aspect, the disclosure features a kit comprising any of the antibodies provided herein and a package insert comprising instructions for using the antibody to treat a subject having or at risk of developing a disorder associated with an ETEC infection.

[0096] In another aspect, the disclosure features a kit comprising any of the antibodies provided herein and an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0097] ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0098] In another aspect, the disclosure features a kit for detecting ETEC, the kit comprising any one of the antibodies provided herein and a package insert comprising instructions for using the antibody to detect ETEC. In some embodiments, the antibody in the kit is conjugated to a label or a tag.

[0099] BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Fig. 1 is a schematic diagram showing a workflow for identifying VHH antibodies that bind an antigen of interest. Llamas are immunized with a purified protein of interest. RNA from the llamas’ immune cells is isolated and used to generate a phage-display library. The phage-display library is screened against immobilized antigens of interest. Selected VHH antibodies are sequenced, expressed in bacteria, and purified. The purified VHH antibodies are then further assessed in in vitro and in vivo assays.

[0101] Fig. 2 is a bar graph showing the level of binding of the indicated VHH antibodies (assessed as VHH-lgA fusion proteins) to enterotoxigenic E. coli (ETEC) coli surface antigen 3 (CS3) protein, ETEC coli surface antigen 6 (CS6) protein, or maltose-binding protein (MBP) (provided as a negative control), as measured using an enzyme-linked immunosorbent assay (ELISA). Y-axis shows optical density (OD) at 450 nm (OD).

[0102] Fig. 3A is a chart showing the level of binding of the indicated VHH antibodies (assessed as VHH-lgA fusion proteins) to ETEC CS3 at the indicated concentrations of the VHH antibody (in pg / mL), as quantified by OD at 450 nm in an ELISA assay.

[0103] Fig. 3B is a chart showing the level of binding of the indicated VHH antibodies (assessed as VHH-lgA fusion proteins) to ETEC CS6 at the indicated concentrations of the VHH antibody (in pg / mL), as quantified by OD at 450 nm in an ELISA assay.

[0104] Fig. 4A is a chart showing the level of binding of the indicated VHH antibodies (assessed as VHH-lgA fusion proteins) to ETEC CS3 at the indicated concentrations of the VHH antibody (in pg / mL), as quantified by OD at 450 nm in an ELISA assay.

[0105] Fig. 4B is a chart showing the level of binding of the indicated VHH antibodies (assessed as VHH-lgA fusion proteins) to ETEC CS6 at the indicated concentrations of the VHH antibody (in pg / mL), as quantified by OD at 450 nm in an ELISA assay.

[0106] Fig. 5 is a chart showing the level of colonization of the ETEC strain B7A in the intestines of mice that were treated with the ETEC strain and the indicated VHH antibodies (assessed as VHH-lgA fusion proteins) or a PBS control, as quantified by the number of colony-forming units (CFUs) isolated from the intestine.

[0107] Fig. 6A is a chart showing the percent inhibition of colonization by the ETEC strain B7A in mice that were treated with the indicated VHH antibodies (assessed as VHH-lgA fusion proteins), as determined by comparing the number of CFUs of the ETEC strain in the intestine to mice that were treated with a PBS control.

[0108] Fig. 6B is a chart showing the percent inhibition of colonization by the ETEC strain E24377A in mice that were treated with the indicated VHH antibodies (assessed as VHH-lgA fusion proteins), as PATENT Attorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77 determined by comparing the number of CFUs of the ETEC strain in the intestine to mice that were treated with a PBS control.

[0109] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0110] I. DEFINITIONS

[0111] The terms “anti-ETEC adhesin protein antibody,” “an antibody that binds to an ETEC adhesin protein,” and “an antibody that specifically binds to an ETEC adhesin protein,” or variants thereof, refer to an antibody that is capable of binding to an ETEC adhesin protein (e.g., one or more of CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, PCF071 , and CS2) with sufficient affinity such that the antibody is useful as a preventative, diagnostic, and / or therapeutic agent in targeting an ETEC adhesin protein. In one embodiment, the extent of binding of an anti-ETEC adhesin protein antibody to an unrelated, non-ETEC adhesin protein is less than about 10% of the binding of the antibody to an ETEC adhesin protein as measured, e.g., by a radioimmunoassay (RIA) or surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to an ETEC adhesin protein has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, e.g., from 10_8M to 10'13M, e.g., from 109M to 1013M).

[0112] The term “antibody” as used herein in the broadest sense encompasses various antibody structures, including but not limited to VHH antibodies, including single-domain VHH antibodies (e.g., nanobodies), monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity (e.g., binding to at least one ETEC adhesin protein). In some embodiments, the VHH antibody can comprise a VHH binding domain linked to an Fc domain subunit.

[0113] The term “VHH antibody” refers to an antibody fragment or full-length antibody that includes at least one VHH binding domain.

[0114] The term “VHH binding domain” refers to a heavy chain variable domain that does not require domain pairing with a light chain variable domain. In some embodiments, a VHH binding domain is capable of binding to an ETEC adhesin protein (e.g., one or more of CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, PCF071 , and CS2)). VHH binding domains can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FRs). Each VHH binding domain may be composed, for example, of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The CDR3 region of VHH binding domains is often longer than that of conventional VH domains and has the capacity to form finger-like extensions, allowing for the targeting of conserved clefts and pocket regions (typically binding sites) on hypervariable pathogen moieties. The variable regions of the VHH binding domains provided herein contain a binding domain that interacts with at least one ETEC adhesin protein.

[0115] The term “class 5 fimbrial adhesin protein” refers to the largest class of ETEC colonization factors in humans. Class 5 includes eight serologically discrete colonization factors of ETEC that mediate small intestinal adhesion, and are subdivided into three sub-classes, class 5a, class 5b, and class 5c. Class 5a PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 includes CfaE, CS4, and CS14; class 5b includes CS1 , CS17, CS19, and PCF071 ; and class 5c includes CS2.

[0116] The term “colonization factor antigen I adhesin subunit E” or “CfaE” refers to subunit E of colonization factor antigen I (CFA / I). CFA / I is a filamentous structure on the surface of ETEC that is involved with ETEC adhesion to the small intestine, which allows the bacteria to cause infection.

[0117] The term “coli surface antigen 1 ” or “CS1 ” refers to colonization factor antigen 1 (CFA / II) class 5b fimbrial adhesin protein.

[0118] The term “coli surface antigen 2” or “CS2” refers to colonization factor antigen 2 belonging to colonization factor antigen group II (CFA / II) and is a class 5c fimbrial adhesin protein.

[0119] The term “coli surface antigen 3” or “CS3” refers to colonization factor antigen 3 belonging to colonization factor antigen group II (CFA / II) and is a fibrillary adhesin protein. CS3 assembles into fine wiry helical fibrillae encoded by the cstA-H gene cluster.

[0120] The term “coli surface antigen 4” or “CS4” refers to colonization factor antigen 4 belonging to colonization factor antigen group IV (CFA / IV) and is a class 5a fimbrial adhesin protein.

[0121] The term “coli surface antigen 5” or “CS5” refers to colonization factor antigen 5 belonging to colonization factor antigen group IV (CFA / IV).

[0122] The term “coli surface antigen 6” or “CS6” refers to colonization factor antigen 6 belonging to colonization factor antigen group IV (CFA / IV). The CS6 operon encodes two structural subunit proteins, CssA and CssB, a chaperon, CssC, and an usher, CssD.

[0123] The term “coli surface antigen 14” or “CS14” refers to colonization factor antigen 14 belonging to colonization factor antigen group III (CFA / I 11) and is a class 5a fimbrial adhesin protein.

[0124] The term “coli surface antigen 17” or “CS17” refers to colonization factor antigen 17 belonging to colonization factor antigen group III (CFA / I 11) and is a class 5b fimbrial adhesin protein.

[0125] The term “coli surface antigen 19” or “CS19” refers to colonization factor antigen 19 and is a class 5b fimbrial adhesin protein.

[0126] The term “coli surface antigen 21 ” or “CS21 ” refers to colonization factor antigen 21 and is a class 5b fimbrial adhesin protein, also a type IV pilus (T4P), with >20 pm in length.

[0127] The term “PCF071 ” refers to putative colonization factor antigen 071 and is a class 5b fimbrial adhesin protein closely related to CS1 .

[0128] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure including a VHH binding domain and having heavy chains that contain Fc domain subunits forming or capable of forming an Fc domain as defined herein. The term “Fc domain subunit” refers to a polypeptide chain that includes at least a second and a third antibody constant domains (CH2 and CH3) or functional fragments thereof (e.g., fragments that are capable of (i) dimerizing with another Fc domain subunit to form an Fc domain, and (ii) binding to an Fc receptor). In certain instances, the Fc domain subunit can include a hinge region linked to the second and third antibody constant domains. The term “Fc domain” refers to a dimer of two Fc domain subunits that dimerize by an interaction between the two CH3 constant domains; in some instances, one or more disulfide bonds form between the hinge regions of the two dimerizing Fc domain subunits. The full-length antibody can further include a first constant domain (CH1 ) linked to the second and third antibody PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 constant domain. The Fc domain subunit or Fc domain can be an IgA subtype. In some instances, the Fc domain subunit or Fc domain is an Ig A1 Fc domain subunit or Fc domain. In some instances, the Fc domain subunit or Fc domain is an lgA2 Fc domain subunit or Fc domain. In some instances, the Fc domain subunit or Fc domain is a secretory IgA (slgA) or dimeric IgA (dlgA). The Fc domain subunit or Fc domain may be of any IgA subtype (e.g., dlgA1 , dlgA2, slgA1 , and slgA2). Alternatively, the Fc domain subunit or Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, or lgG2b) (e.g., IgG 1 ).

[0129] In some instances, the full-length antibody includes an Fc domain including a first Fc domain subunit and a second Fc domain subunit, wherein the first Fc domain subunit and the second Fc domain subunit are capable of stable association. In some embodiments, each Fc domain subunit is linked to a single VHH binding domain and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-Fc domain subunit. In some instances, each Fc domain subunit is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal- to-C-terminal direction as follows: VHH binding domain-hinge region-Fc domain subunit. In some instances, the antibody is an IgA class antibody (e.g., lgA1 or lgA2). In some instances, each Fc domain subunit is further linked to a single CH1 domain. In some embodiments, each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit. In some instances, each CH1 domain is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-hinge region-Fc domain subunit. In some instances, each CH1 domain is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-hinge region-CH1 domain-Fc domain subunit. In certain instances, the hinge region includes the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRP (SEQ ID NO: 36), ASPVPPPPPCCHPRLSLHRP (SEQ ID NO: 44), ASPVPSTPPTPSPSTPPTPSPS (SEQ ID NO: 50), or ASPVPPPPPP (SEQ ID NO: 51 ).

[0130] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogenous antibodies that displays binding affinity for an epitope on one or more ETEC adhesin proteins.

[0131] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that specifically binds to the antigen (e.g., an ETEC adhesin protein) to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’- SH, F(ab’)2; nanobodies (e.g., VHH nanobodies), diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. These antibody fragments are obtained using conventional techniques, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0132] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0133] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0134] The term “KD,” as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the invention bind to an ETEC adhesin protein (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, CS5, CS21 , or PCF071 ) with a dissociation equilibrium constant (KD) of less than about 106M, such as less than approximately 107M, 10-8M, 10-9M, or 10-10M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 3000 instrument using recombinant ETEC adhesin protein as the analyte and the antibody as the ligand.

[0135] A “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose a mammal to the disorder in question.

[0136] As used herein, the term “disorder associated with an enterotoxigenic Escherichia coli infection” or “disorder associated with an ETEC infection” refers to any disease, the onset, progression, or the persistence of the symptoms of which requires the participation of ETEC. An exemplary disorder associated with an ETEC infection is, for example, diarrhea.

[0137] The term “ICso,” as used herein, refers to the concentration of an antibody or an antigen-binding portion thereof, which induces a response, either in an in vivo or an in vitro assay, needed to inhibit 50% of the maximal response (i.e., halfway between the maximal response and the baseline), for example, of ETEC adhesion to Caco-2 cells as compared to an irrelevant antibody.

[0138] The term “IC100,” as used herein, refers to the maximal inhibitory concentration of an antibody or an antigen-binding portion thereof, either in an in vivo or an in vitro assay.

[0139] The terms “effective amount,” “effective dose,” and “effective dosage” as used herein are defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term “therapeutically effective dose” or “therapeutically effective amount” is defined as an amount sufficient to prevent, cure, or at least partially arrest, the disease (e.g., diarrhea) and its complications in a patient already suffering from the disease or at risk of developing the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient’s own immune system.

[0140] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0141] Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). Epitopes can also be defined by point mutations in the target protein (e.g., an ETEC adhesin protein (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, CS5, CS21 , or PCF071 )), which affect the binding of the antibody (e.g., monoclonal antibody).

[0142] The term “host cell,” as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0143] An “isolated antibody” or “isolated VHH antibody” is one which has been identified and separated and / or recovered from a component of its natural environment and / or is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds to an ETEC adhesin protein is substantially free of antibodies that bind antigens other than an ETEC adhesin protein). Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In preferred embodiments, the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie™ blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Similarly, isolated antibody includes the antibody in medium around recombinant cells. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0144] The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is doublestranded DNA.

[0145] The term “isolated nucleic acid,” as used herein in reference to nucleic acids molecules encoding antibodies or antibody portions (e.g., VHH antibodies or fragments thereof) that bind to an ETEC adhesin protein (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, CS5, CS21 , and PCF071 ), is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies that bind antigens other than an ETEC adhesin protein, which other sequences may naturally flank the nucleic acid in human genomic DNA.

[0146] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0147] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0148] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0149] As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of approximately less than 107M, such as approximately less than 10-8M, 10-9M or 10-10M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 3000 instrument, which can be performed, for example, using recombinant ETEC adhesin protein (e.g., recombinant CS3, CS6, or CfaE) as the analyte and the antibody as the ligand. In some embodiments, binding by the antibody to the predetermined antigen is with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., mannose-binding protein (MBP), BSA, or casein) other than the predetermined antigen or a closely related antigen. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0150] A “subject” or an “individual” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, deer, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

[0151] The terms “treat,” “treating,” and “treatment,” as used herein, refer to preventative or therapeutic measures described herein. The methods of “treatment” involve administration to a subject in need of such treatment a VHH antibody of the present invention or pharmaceutical composition including a VHH antibody of the present invention, for example, administration to a subject at risk of developing a disorder associated with ETEC infection or a subject having a disorder associated with ETEC infection, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. In some embodiments, for example, the anti-ETEC adhesin protein antibodies of the invention would be administered to a subject at risk of developing a disorder associated with ETEC infection (e.g., a subject residing or traveling to a geographical location in which pathogenic ETEC is found). Accordingly, desirable effects of treatment include, but are not limited to, preventing occurrence of disease or disorder, such as a disorder associated with ETEC infection (e.g., ETEC-related diarrhea). PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0152] Other desirable effects of treatment may include preventing recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and improved prognosis.

[0153] As used herein, “administering” is meant a method of giving a dosage of a compound (e.g., an anti-ETEC adhesin protein antibody of the invention or a nucleic acid encoding an anti-ETEC adhesin protein VHH antibody of the invention) or a composition (e.g., a pharmaceutical composition including an anti-ETEC adhesin protein VHH antibody of the invention) to a subject. The compositions utilized in the methods described herein can be administered or formulated for administration, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In some embodiments, the compound (e.g., VHH antibody) or composition (e.g., pharmaceutical composition comprising one or more VHH antibodies) is administered orally or formulated for oral administration. In some embodiments, the compound (e.g., VHH antibody) or composition (e.g., pharmaceutical composition comprising one or more VHH antibodies) is administered intramuscularly or formulated for intramuscular administration (e.g., injection).

[0154] As used herein, the term “vector” is meant to include, but is not limited to, a nucleic acid molecule (e.g., a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked), a virus (e.g., a lentivirus or an adenovirus, e.g., a recombinant adeno-associated virus (rAAV)), a lipid nanoparticle, a liposome (e.g., a liposome comprising a cationic lipid), cationic polymer (e.g., polysome), virosome, nanoparticle, or dendrimer. In a preferred embodiment, the vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0155] II. VHH ANTIBODIES THAT BIND ETEC CS3 AND CS6

[0156] The present disclosure provides antibodies (e.g., VHH antibodies) that bind to one or more enterotoxigenic Escherichia coli (ETEC) adhesin proteins. Antibodies of the invention are useful, for example, for treating a subject having, or at risk of developing, a disorder associated with an ETEC infection. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0157] In particular, the disclosure provides isolated antibodies that bind to enterotoxigenic E. coli (ETEC) coli surface antigen 3 (CS3) protein and ETEC coli surface antigen 6 (CS6) protein, wherein the antibodies comprise a CS3 / CS6 VHH binding domain (i.e., comprise a binding domain that is cross- reactive for CS3 and CS6). Such cross-reactive antibodies are capable of binding to CS3 in the presence of an ETEC strain that expresses CS3 (e.g., E24377A); binding to CS6 in the presence of an ETEC strain that expresses CS6 (e.g., B7A); and binding to both of CS3 and CS6 (in a mutually exclusive manner, such that a single antibody molecule binds CS3 or CS6) in scenarios in which both antigens are present).

[0158] A. Antibodies comprising the CDR regions of the VHH antibody D10

[0159] In one aspect, the disclosure provides an isolated antibody that binds enterotoxigenic E. coli (ETEC) coli surface antigen 3 (CS3) protein and ETEC coli surface antigen 6 (CS6) protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following complementary- determining regions (CDRs): (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ). The disclosure also provides an isolated antibody that binds ETEC CS3 and CS6 and comprises a combination of one or more of the CDRs of SEQ ID NOs: 19-21 and one or more variants thereof having

[0160] (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 19-21 and / or

[0161] (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 19-21 , such that the isolated antibody comprises the CDR of SEQ ID NO: 19 or a variant thereof; the CDR of SEQ ID NO: 20 or a variant thereof; and the CDR of SEQ ID NO: 21 or a variant thereof.

[0162] In some embodiments, the CS3 / CS6 VHH binding domain includes a sequence having at least 95% sequence identity (having e.g., 96%, 97%, 98%, or 99% identity) to the amino acid sequence of SEQ ID NO: 26 and / or having one, two, three, four, five, or six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 26.

[0163] In some instances, the CS3 / CS6 VHH binding domain includes the following framework regions (FRs): (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 22); (b) an FR-H2 comprising the amino acid sequence of MGWFRQAPGKEREGVSC (SEQ ID NO: 23); (c) an FR-H3 comprising the amino acid sequence of YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 24); and (d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 25). The disclosure also provides an isolated antibody that binds ETEC CS3 and CS6 and comprises a combination of one or more of the FRs of SEQ ID NOs: 22-25 and one or more variants thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 22-25 and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 22-25, such that the isolated antibody comprises the FR of SEQ ID NO: 22 or a variant thereof; the FR of SEQ ID NO: 23 or a variant thereof; the FR of SEQ ID NO: 24 or a variant thereof; and the FR of SEQ ID NO: 25 or a variant thereof. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0164] In some instances, the CS3 / CS6 VHH binding domain comprises the amino acid sequence of SEQ ID NO: 26. In some instances, the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 26, i.e. , the CS3 / CS6 VHH binding domain is the VHH antibody D10.

[0165] The disclosure also features isolated antibodies that binds to CS3, CS6, or both of CS3 and CS6 and compete for binding with any of the antibodies provided above. For example, in some embodiments, the disclosure provides an isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ). In some embodiments, the disclosure provides an isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding to CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ). In some embodiments, the disclosure provides an isolated antibody that binds ETEC CS6 protein and ETEC CS6 protein, wherein the antibody competes for binding to CS3 and CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ).

[0166] In another aspect, the disclosure provides a nucleotide sequence encoding any one of the isolated antibodies provided above. In some embodiments, the nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 90-92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to the nucleotide sequence of SEQ ID NO: 27. In some embodiments, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 27.

[0167] B. Antibodies comprising the CDR regions of the VHH antibody A09

[0168] In another aspect, the disclosure provides an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3). The disclosure also provides an isolated antibody that binds ETEC CS3 and CS6 and comprises a combination of one or more of the CDRs of SEQ ID NOs: 1 -3 and one or more variants thereof having (i) at least about 80% sequence identity (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1 -3 and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 1 -3, such that the isolated antibody comprises the CDR of SEQ ID NO: 1 or a variant thereof; the CDR of SEQ ID NO: 2 or a variant thereof; and the CDR of SEQ ID NO: 3 or a variant thereof. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0169] In some embodiments, the CS3 / CS6 VHH binding domain includes a sequence having at least 95% sequence identity (having e.g., 96%, 97%, 98%, or 99% identity) to the amino acid sequence of SEQ ID NO: 8 and / or having one, two, three, four, five, or six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 8.

[0170] In some instances, the CS3 / CS6 VHH binding domain includes the following FRs: (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 4); (b) an FR-H2 comprising the amino acid sequence of MGWFRQAPGKEREFVAA (SEQ ID NO: 5); (c) an FR-H3 comprising the amino acid sequence of IYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 6); and (d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 7). The disclosure also provides an isolated antibody that binds ETEC CS3 and CS6 and comprises a combination of one or more of the FRs of SEQ ID NOs: 4-7 and one or more variants thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 4-7 and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 4- 7, such that the isolated antibody comprises the FR of SEQ ID NO: 4 or a variant thereof; the FR of SEQ ID NO: 5 or a variant thereof; the FR of SEQ ID NO: 6 or a variant thereof; and the FR of SEQ ID NO: 7 or a variant thereof.

[0171] In some instances, the CS3 / CS6 VHH binding domain comprises the amino acid sequence of SEQ ID NO: 8. In some instances, the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 8, i.e. , the CS3 / CS6 VHH binding domain is the VHH antibody A09.

[0172] The disclosure also features isolated antibodies that binds to CS3, CS6, or both of CS3 and CS6 and compete for binding with any of the antibodies provided above. For example, in some embodiments, the disclosure provides an isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3). In some embodiments, the disclosure provides an isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding to CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3). In some embodiments, the disclosure provides an isolated antibody that binds ETEC CS6 protein and ETEC CS6 protein, wherein the antibody competes for binding to CS3 and CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

[0173] In another aspect, the disclosure provides a nucleotide sequence encoding any one of the isolated antibodies provided above. In some embodiments, the nucleotide sequence has at least 90%, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0174] 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 90-92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 9.

[0175] C. Antibodies comprising the CDR regions of the VHH antibody C04

[0176] In another aspect, the disclosure provides an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12). The disclosure also provides an isolated antibody that binds ETEC CS3 and CS6 and comprises a combination of one or more of the CDRs of SEQ ID NOs: 10-12 and one or more variants thereof having (i) at least about 80% sequence identity (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 10-12 and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 10-12, such that the isolated antibody comprises the CDR of SEQ ID NO: 10 or a variant thereof; the CDR of SEQ ID NO: 11 or a variant thereof; and the CDR of SEQ ID NO: 12 or a variant thereof.

[0177] In some embodiments, the CS3 / CS6 VHH binding domain includes a sequence having at least 95% sequence identity (having e.g., 96%, 97%, 98%, or 99% identity) to the amino acid sequence of SEQ ID NO: 17 and / or having one, two, three, four, five, or six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 17.

[0178] In some instances, the CS3 / CS6 VHH binding domain includes the following FRs: (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 13); (b) an FR-H2 comprising the amino acid sequence of MAWFRQAPGKEREIVAA (SEQ ID NO: 14); (c) an FR-H3 comprising the amino acid sequence of DYTDSVKGRFTISRDNAKNTGFLQMNRLKPEDTAVYYC (SEQ ID NO: 15); and (d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 16).

[0179] The disclosure also provides an isolated antibody that binds ETEC CS3 and CS6 and comprises a combination of one or more of the FRs of SEQ ID NOs: 13-16 and one or more variants thereof having

[0180] (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 13-16 and / or

[0181] (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 13-16, such that the isolated antibody comprises the FR of SEQ ID NO: 13 or a variant thereof; the FR of SEQ ID NO: 14 or a variant thereof; the FR of SEQ ID NO: 15 or a variant thereof; and the FR of SEQ ID NO: 16 or a variant thereof.

[0182] In some instances, the CS3 / CS6 VHH binding domain comprises the amino acid sequence of SEQ ID NO: 17. In some instances, the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 17, i.e., the CS3 / CS6 VHH binding domain is the VHH antibody C04.

[0183] The disclosure also features isolated antibodies that binds to CS3, CS6, or both of CS3 and CS6 and compete for binding with any of the antibodies provided above. For example, in some embodiments, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 the disclosure provides an isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12). In some embodiments, the disclosure provides an isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding to CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12). In some embodiments, the disclosure provides an isolated antibody that binds ETEC CS6 protein and ETEC CS6 protein, wherein the antibody competes for binding to CS3 and CS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

[0184] In another aspect, the disclosure provides a nucleotide sequence encoding any one of the isolated antibodies provided above. In some embodiments, the nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 90-92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 18.

[0185] D. Antibody features

[0186] Antibodies of the invention may, for example, be monoclonal, human, humanized, or chimeric. The antibodies can be full-length antibodies or antibody fragments thereof (e.g., an antibody fragment that binds an ETEC adhesin protein). The antibody fragment may be selected from the group consisting of Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments. An antibody of the invention may have a half-life of > 3 days (e.g., > 1 week, e.g., > 2 weeks, e.g., > 1 month, e.g., > 2 months, e.g., > 3 months, e.g., > 4 months, e.g., > 5 months, e.g., > 6 months).

[0187] / . Fc regions

[0188] In some embodiments, the antibody that binds ETEC CS3 and CS6 (e.g., an isolated antibody comprising a CS3 / CS6 VHH binding domain as provided in any one of Sections IIA-IIC, above) comprises a fragment crystallizable region (Fc region). Any suitable Fc region may be used.

[0189] Antibodies of the invention may be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD.

[0190] In some embodiments, the antibody that binds ETEC CS3 and CS6 is an immunoglobulin A (IgA) antibody (e.g., comprises an IgA Fc region). IgA is an isotype that plays a crucial role in the immune function of mucous membranes. The antibody that binds ETEC CS3 and CS6 may be any IgA subtype. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0191] Subclasses of IgA antibodies include secretory IgA (slgA) and dimeric IgA (dlgA). The antibody that binds ETEC CS3 and CS6 may be, e.g., dlgA1 , dlgA2, slgA1 , or slgA2.

[0192] In some embodiments, the IgA Fc region is an Ig A1 Fc region. In some embodiments, the lgA1 Fc region has at least 90% identity to SEQ ID NO: 37 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to SEQ ID NO: 37). In some embodiments, the lgA1 Fc region has the amino acid sequence of SEQ ID NO: 37. The antibody that binds ETEC CS3 and CS6 may further comprise a tail, wherein the components of the antibody are arranged in an N-terminal-to-C-terminal direction as follows: CS3 / CS6 VHH binding domain- Fc region-tail. In some aspects, the tail has at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96- 98%, or more than 98% identity) to SEQ ID NO: 38). Thus, in some aspects, the antibody that binds ETEC CS3 and CS6 comprises an Fc region and tail having at least 90% identity to SEQ ID NO: 39 (e.g., having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94- 96%, 96-98%, or more than 98% identity) to SEQ ID NO: 39).

[0193] In some embodiments, the IgA Fc region is an lgA2 Fc region. In some embodiments, the lgA2 Fc region has at least 90% identity to SEQ ID NO: 45 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to SEQ ID NO: 45). In some embodiments, the lgA2 Fc region has the amino acid sequence of SEQ ID NO: 45. The antibody that binds ETEC CS3 and CS6 may further comprise a tail, wherein the components of the antibody are arranged in an N-terminal-to-C-terminal direction as follows: CS3 / CS6 VHH binding domain- Fc region-tail. In some aspects, the tail has at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96- 98%, or more than 98% identity) to SEQ ID NO: 38). Thus, in some aspects, the antibody that binds ETEC CS3 and CS6 comprises an Fc region and tail having at least 90% identity to SEQ ID NO: 46 (e.g., having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94- 96%, 96-98%, or more than 98% identity) to SEQ ID NO: 46).

[0194] In some embodiments, the antibody that binds ETEC CS3 and CS6 is an immunoglobulin G (IgG) antibody (e.g., comprises an IgG Fc region). In aspects in which the antibody that binds ETEC CS3 and CS6 is an IgG antibody (e.g., comprises an IgG Fc region) the antibody may be any IgG subtype (e.g., IgG 1 , lgG2a, lgG2b, lgG3, or lgG4). In some embodiments, the antibody that binds ETEC CS3 and CS6 is an IgG 1 antibody.

[0195] In some embodiments, the antibody that binds ETEC CS3 and CS6 includes an Fc domain including a first Fc domain subunit and a second Fc domain subunit, and the first Fc domain subunit and the second Fc domain subunit are capable of stable association. In some embodiments, each Fc domain subunit is linked to a single VHH binding domain and positioned relative to each other in an N-terminal-to- C-terminal direction as follows: VHH binding domain-Fc domain subunit. In some embodiments, each Fc domain subunit is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-hinge region-Fc domain subunit. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0196] In certain embodiments, each Fc domain subunit is further linked to a single CH1 domain. In some embodiments, each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit. In some embodiments, each CH1 domain is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-hinge region-CH1 domain-Fc domain subunit. In some embodiments, each CH1 domain is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-hinge region-CH1 domain- Fc domain subunit.

[0197] In some embodiments, the CS3 / CS6 VHH binding domain is connected to the Fc region by a linker. In some embodiments, the linker is a hinge region.

[0198] In some embodiments, the hinge region includes the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRP (SEQ ID NO: 36), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 36, and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 36.

[0199] In some embodiments, the hinge region includes the amino acid sequence of ASPVPPPPPCCHPRLSLHRP (SEQ ID NO: 44), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 44, and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 44.

[0200] In some embodiments, the hinge region includes the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPS (SEQ ID NO: 50), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 50, and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 50.

[0201] In some embodiments, the hinge region includes the amino acid sequence of ASPVPPPPPP (SEQ ID NO: 51 ), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 51 , and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 51 . In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 51.

[0202] In certain embodiments, the antibody is a full-length antibody.

[0203] In other embodiments, the antibody does not include an Fc region. PATENT Attorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77

[0204] In some embodiments, the antibody that binds ETEC CS3 and CS6 is a VHH antibody fragment that binds an ETEC adhesin protein. In certain embodiments, the antibody that binds ETEC CS3 and CS6 consists of a single VHH binding domain.

[0205] Hi. Labeled antibodies

[0206] In certain embodiments, labeled antibodies that bind ETEC CS3 and CS6 are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes32P,14C,125l,3H, and1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, p-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like. iv. Other features

[0207] In a further aspect, an antibody that binds ETEC CS3 and CS6 according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1 -6 below.

[0208] 1. Antibody A ffinity

[0209] In certain embodiments, an antibody provided herein may have a dissociation constant (KD) of < 10 pM, < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, or < 0.01 nM.

[0210] In one embodiment, an antibody provided herein may bind an ETEC adhesin protein with a KD of between about 0.1 nM and about 200 nM (e.g., between about 5 nM and about 100 nM, between about 15 nM and about 100 nM, between about 25 nM and about 100 nM, between about 35 nM and about 100 nM, between about 45 nM and about 100 nM, between about 55 nM and about 100 nM, between about 65 nM and about 100 nM, between about 75 nM and about 100 nM, between about 85 nM and about 100 nM, between about 95 nM and about 100 nM, between about 95 nM and about 200 nM, between about 105 nM and about 200 nM, between about 1 15 nM and about 200 nM, between about 125 nM and about 200 nM, between about 135 nM and about 200 nM, between about 145 nM and about 200 nM, between about 155 nM and about 200 nM, between about 165 nM and about 200 nM, between about 175 nM and about 200 nM, between about 185 nM and about 200 nM, or between about 195 nM and about 200 nM). In some embodiments, the antibody may bind an ETEC adhesin protein with a KD between about 1 nM and about 50 nM (e.g., between about 5 nM and about 50 nM, between about 8 nM and about 40 nM, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 between about 11 nM and about 30 nM, or between about 14 nM and about 20 nM). In particular embodiments, an antibody provided herein may bind an ETEC adhesin protein with a KD of about 15 nM.

[0211] In some embodiments, the antibody may bind ETEC adhesin protein CS4 with a KD between about 15 and 200 nM (e.g., between about 20 and about 40 nM, between about 35 and about 80 nM, between about 75 and about 90 nM, between about 85 and about 120 nM, between about 115 and about 170 nM, or between about 165 and about 200 nM).

[0212] In some embodiments, the antibody may bind ETEC adhesin protein CS14 with a KD between about 15 and 200 nM (e.g., between about 20 and about 40 nM, between about 35 and about 80 nM, between about 75 and about 90 nM, between about 85 and about 120 nM, between about 115 and about 170 nM, or between about 165 and about 200 nM).

[0213] In some embodiments, the antibody may bind ETEC adhesin protein CS1 with a KD between about 15 and about 200 nM (e.g., between about 20 and about 40 nM, between about 35 and about 80 nM, between about 75 and about 90 nM, between about 85 and about 120 nM, between about 115 and about 170 nM, or between about 165 and about 200 nM).

[0214] In some embodiments, the antibody may bind ETEC adhesin protein CS17 with a KD between about 3 and about 200 nM (e.g., between about 3 and about 20 nM (e.g., about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, or about 20 nM), about 3 and about 150 nM, about 3 and about 100 nM, about 3 and about 50 nM, or about 3 and about 35 nM).

[0215] In some embodiments, the antibody may bind ETEC adhesin protein CS19 with a KD between about 80 and 200 nM (e.g., between about 85 and about 110 nM, between about 105 and about 150 nM, between about 145 and about 180 nM, or between about 175 and about 200 nM).

[0216] In some embodiments, the antibody may bind ETEC adhesin protein CS2 with a KD between about 3 and about 200 nM (e.g., between about 5 nM and about 100 nM, between about 15 nM and about 100 nM, between about 25 nM and about 100 nM, between about 35 nM and about 100 nM, between about 45 nM and about 100 nM, between about 55 nM and about 100 nM, between about 65 nM and about 100 nM, between about 75 nM and about 100 nM, between about 85 nM and about 100 nM, between about 95 nM and about 100 nM, between about 95 nM and about 200 nM, between about 105 nM and about 200 nM, between about 115 nM and about 200 nM, between about 125 nM and about 200 nM, between about 135 nM and about 200 nM, between about 145 nM and about 200 nM, between about 155 nM and about 200 nM, between about 165 nM and about 200 nM, between about 175 nM and about 200 nM, between about 185 nM and about 200 nM, or between about 195 nM and about 200 nM).

[0217] In some embodiments, the antibody may bind ETEC adhesin protein CS3 with a KD between about 15 and 200 nM (e.g., between about 20 and about 40 nM, between about 35 and about 80 nM, between about 75 and about 90 nM, between about 85 and about 120 nM, between about 115 and about 170 nM, or between about 165 and about 200 nM).

[0218] In some embodiments, the antibody may bind ETEC adhesin protein CS5 with a KD between about 1 and about 200 nM (e.g., between about 1 and about 20 nM (e.g., about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 PATENT Attorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, or about 20 nM), about 1 and about 150 nM, about 1 and about 100 nM, about 1 and about 50 nM, or about 1 and about 35 nM).

[0219] In some embodiments, the antibody may bind ETEC adhesin protein CS6 with a KD between about 1 and about 200 nM (e.g., between about 7 and about 30 nM (e.g., about 8 nM, about 9 nM, about 10 nM, about 1 1 nM, about 12 nM, about 12.5 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, or about 29 nM), about 1 and about 150 nM, about 1 and about 100 nM, about 1 and about 50 nM, or about 1 and about 35 nM).

[0220] In some embodiments, the antibody may bind ETEC adhesin protein CS21 with a KD between about 10 and about 200 nM (e.g., about 10 and about 150, about 10 and about 100, about 15 and about 50, or about 20 and about 30 nM (e.g., about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM, or about 29 nM)).

[0221] In some embodiments, the antibody binds ETEC CS3 with a 50% effective concentration (ECso) of between about 1 ng / mL and about 1000 ng / mL; between about 1 ng / mL and about 10 ng / mL; between about 10 ng / mL and about 100 ng / mL; or between about 100 ng / mL and about 1000 ng / mL.

[0222] In some embodiments, the antibody binds ETEC adhesin protein CS3 with an ECso of between about 500-1000 ng / mL (e.g., about 600-700 ng / mL) and includes a VHH binding domain including (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ).

[0223] In some embodiments, the antibody binds ETEC adhesin protein CS3 with an ECso of between about 1 -100 ng / mL (e.g., about 50-60 ng / mL) and includes a VHH binding domain including (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

[0224] In some embodiments, the antibody binds ETEC adhesin protein CS3 with an ECso of between about 1 -10 ng / mL (e.g., about 5-7 ng / mL) and includes a VHH binding domain including (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 1 1 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

[0225] In some embodiments, the antibody binds ETEC CS6 with an ECso of between about 1 ng / mL and about 1000 ng / mL; between about 1 ng / mL and about 10 ng / mL; between about 10 ng / mL and about 100 ng / mL; or between about 100 ng / mL and about 1000 ng / mL.

[0226] In some embodiments, the antibody binds ETEC adhesin protein CS6 with an ECso of between about 500-1000 ng / mL (e.g., about 600-700 ng / mL) and includes a VHH binding domain including (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ). PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0227] In some embodiments, the antibody binds ETEC adhesin protein CS6 with an EC50 of between about 1 -10 ng / mL (e.g., about 5-7 ng / mL) and includes a VHH binding domain including (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

[0228] In some embodiments, the antibody binds ETEC adhesin protein CS6 with an EC50 of between about 50-100 ng / mL (e.g., about 60-80 ng / mL) and includes a VHH binding domain including (a) a CDR- H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

[0229] According to another embodiment, KD is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE ®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with A / -ethyl- / V- (3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and A / -hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg / ml (~0.2 pM) before injection at a flow rate of 5 pl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 pl / min. Association rates (kon) and dissociation rates (kotf) are calculated using a simple one-to-one Langmuir binding model (BIACORE ® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k0tf / k0n. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106M‘1s‘1by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM- AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0230] In one embodiment, KD is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125l)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg / ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125l]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1 % polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 pl / well of scintillant (MICROSCINT-20 ™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

[0231] 2. Inhibition of Colonization by ETEC

[0232] In certain embodiments, an antibody provided herein is an antibody capable of inhibiting colonization of the small intestine of a mammal (e.g., a human) by ETEC. In some embodiments, the antibody is capable of inhibiting colonization of the small intestine by CS3-expressing ETEC (e.g., ETEC strain E24377A), CS6-expressing ETEC (e.g., ETEC strain B7A) and / or CfaE-expressing ETEC (e.g., ETEC strain H10407). In some instances, the antibody of the invention is capable of inhibiting colonization of the small intestine by ETEC by at least 20% (e.g., about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%). In some embodiments, the antibody of the invention is capable of inhibiting colonization of the small intestine by ETEC by between about 20% and 100% (e.g., between about 20% and about 40%, between about 35% and about 50%, between about 45% and about 60%, between about 55% and about 70%, between about 65% and about 80%, between about 75% and about 90%, or between about 85% and about 100%). In particular embodiments, the antibody is capable of inhibiting colonization of the small intestine by CS3-expressing ETEC, CS6-expressing ETEC, and / or CfaE-expressing ETEC when administered at a dose of 100 mg / kg.

[0233] In some embodiments, the antibody is capable of inhibiting mannose-resistant hemagglutination of human group A erythrocytes with a maximal inhibitory concentration (IC100) of between about 0.10 pg / mL and about 250 pg / mL (e.g., between about 0.5 pg / mL and about 30 pg / mL, about 25 pg / mL and about 50 pg / mL, about 45 pg / mL and about 100 pg / mL, about 95 pg / mL and about 150 pg / mL, about 145 pg / mL and about 200 pg / mL, or about 195 pg / mL and about 250 pg / mL). In some embodiments, the ICwo is between about 6 pg / mL and about 200 pg / mL. In some embodiments, the inhibiting is measured using a mannose-resistant hemagglutination (MRHA) assay.

[0234] In some embodiments, the antibody is capable of inhibiting the binding of ETEC bacteria to intestinal cells with a 50% inhibitory concentration (IC50) of between about 0.10 pM and about 10 pM (e.g., between about 0.50 pM and about 6 pM, about 3 pM and about 8 pM, or about 5 pM and about 10 pM). In some embodiments, the intestinal cells are Caco-2 human intestinal epithelial cells. In some embodiments, the inhibiting is measured using a Caco-2 adhesion assay at 37eC.

[0235] 3. Antibody Fragments

[0236] In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, which are PATENT Attorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77 known in the art. Also included are diabodies, which have two antigen-binding sites that may be bivalent or bispecific, as is known in the art. Triabodies and tetrabodies are also known. Single-domain antibodies are also antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. In particular embodiments, a single-domain antibody is a single-domain VHH antibody.

[0237] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0238] 4. Multimeric Antibodies

[0239] In certain embodiments, an antibody provided herein is a multimeric antibody (e.g., a multimeric VHH antibody). In one example, a multimeric antibody comprises two or more VHH antibodies (e.g., VHH antibodies described herein) connected by way of a linker (e.g., a peptide linker). In some instances, the VHH multimer includes the following N-terminal-to-C-terminal structure:

[0240] (P3-l-3)n3-P2-(Ll -Pl )n1 wherein Pi , P2, and P3 are each independently selected from any VHH binding domain described herein (e.g., a VHH binding domain of D10, A09, or CO4); Li and L3 are each independently a linker; and ni and ns are each independently 0 or 1 , wherein at least one of ni and ns are 1 .

[0241] In some instances, ni is 1 and ns is 0, and the antibody comprises the following N-terminal-to-C- terminal structure:

[0242] P2-L1 -P1 .

[0243] In some instances, the peptide linker includes between 2 and 200 amino acids (e.g., between 5 and 50 (e.g., between 5 and 20, 15 and 30, 25 and 40, or 35 and 50), between 45 and 100 (e.g., between 45 and 60, 55 and 70, 65 and 80, 75 and 90, or 85 and 100), 95 and 150 (e.g., between 95 and 1 10, 105 and 120, 1 15 and 130, 125 and 140, or 135 and 150), or 145 and 200 amino acids (e.g., between 145 and 160, 155 and 170, 165 and 180, 175 and 190, or 185 and 200)). In some instances, the peptide linker comprises glycine (Gly) and serine (Ser) amino acids. In some instances, the peptide linker includes the amino acid sequence of any one of (GS)x, (GGS)x, (GGGGS (SEQ ID NO: 52))x, (GGSG)x, and (SGGG)x, wherein x is an integer from 1 to 10. In certain embodiments the linker includes the amino acid sequence of (GGGGS (SEQ ID NO: 52))x, wherein x is an integer from 2-5. In some instances, P2 and Pi are different VHH binding domains. In some instances, P2 and Pi are identical VHH binding domains. In some embodiments, Pi and P2 each include identical VHH binding domains including the amino acid sequence of SEQ ID NO: 26. In some embodiments, Pi and P2 each include identical VHH binding domains including the amino acid sequence of SEQ ID NO: 8. In some embodiments, Pi and P2 each include identical VHH binding domains including the amino acid sequence of SEQ ID NO: 17.

[0244] In some instances, ni is 1 and ns is 1 , and the antibody comprises the following N-terminal-to-C- terminal structure:

[0245] P3-L3-P2-L1 -P1 . PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0246] In some instances, each peptide linker independently includes between 2 and 200 amino acids (e.g., between 5 and 50 (e.g., between 5 and 20, 15 and 30, 25 and 40, or 35 and 50), between 45 and 100 (e.g., between 45 and 60, 55 and 70, 65 and 80, 75 and 90, or 85 and 100), 95 and 150 (e.g., between 95 and 110, 105 and 120, 115 and 130, 125 and 140, or 135 and 150), or 145 and 200 amino acids (e.g., between 145 and 160, 155 and 170, 165 and 180, 175 and 190, or 185 and 200)). In some instances, the peptide linker comprises glycine (Gly) and serine (Ser) amino acids. In some instances, the peptide linker includes the amino acid sequence of any one of (GS)x, (GGS)x, (GGGGS (SEQ ID NO: 52))x, (GGSG)x, (SGGG)x, wherein x is an integer from 1 to 10. In certain embodiments the linker includes the amino acid sequence of (GGGGS (SEQ ID NO: 52))x, wherein x is an integer from 2-5. In some instances, P3, P2, and Pi are different VHH binding domains. In some instances, P3, P2, and Pi are identical VHH binding domains. In some embodiments, Pi, P2, and P3 each include identical VHH binding domains including the amino acid sequence of SEQ ID NO: 26. In some embodiments, Pi, P2, and P3 each include identical VHH binding domains including the amino acid sequence of SEQ ID NO: 8. In some embodiments, Pi, P2, and P3 each include identical VHH binding domains including the amino acid sequence of SEQ ID NO: 17.

[0247] Nanobodies may be multimerized to dimeric and trimeric forms with (GGGGS (SEQ ID NO: 52))xlinkers. Dimeric forms may be generated using a (GGGGS)e (SEQ ID NO: 53) linker to connect two monomeric VHHs in tandem N-terminus-to-C-terminus orientation. Trimers were generated using two (GGGGS)3 (SEQ ID NO: 54) linkers between monomeric VHH units. The multimers may be cloned into pET26b, adding a C-terminal 6xHis tag to the nanobody sequence. Nanobodies may be purified from the periplasmic fraction by Ni-NTA chromatography (Gold Biotechnology) and dialyzed against PBS to remove imidazole.

[0248] 5. Chimeric, Humanized, and Fc-containing Antibodies

[0249] In certain embodiments, an antibody provided herein is a chimeric antibody. In one example, a chimeric antibody comprises a non-human variable region (e.g., a VHH binding domain) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0250] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody (e.g., a VHH binding domain antibody), and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0251] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611 -22618 (1996)).

[0252] In some instances, an antibody provided herein includes an Fc domain including a first Fc domain subunit and a second Fc domain subunit capable of stable association, with each Fc domain subunit having a CH2 domain and a CH3 domain, or functional fragments thereof (e.g., fragments that are capable of (i) dimerizing with another variant Fc domain monomer to form a variant Fc domain, and (ii) binding to an Fc receptor). In certain instances, each Fc domain subunit can include a hinge region linked to the CH2 domain. In some instances, the hinge regions can include the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 50, or SEQ ID NO: 51 . In some instances, each Fc domain subunit is further linked to a single CH1 domain. In some embodiments, each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit. In some embodiments, each CH1 domain is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N- terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-hinge region-Fc domain subunit. In some embodiments, each CH1 domain is linked to a single VHH binding domain by a hinge region and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-hinge region-CH1 domain-Fc domain subunit. The Fc domain can be an IgA subtype, in particular a secretory IgA (slgA) or dimeric IgA (dlgA). In some instances, the Fc domain may be of any IgA subtype (e.g., dig A1 , dlgA2, slgA1 , and slgA2). The Fc domain can be an IgG subtype (e.g., IgG 1 , lgG2a, or lgG2b) (e.g., lgG1 ).

[0253] In some instances, in the antibodies provided herein VHH binding domains were ligated into a pcDNA 3.1 vector containing heavy constant lgA1 and lgA2 chains without CH1 domain. Each vector was transformed in NEB5 competent cells, and sequences were verified ahead of transient transfection. In other instances, VHH binding domains may be ligated into vectors containing heavy constant lgA1 and lgA2 chains including a CH1 domain. In some instances, each Fc domain subunit is linked to a single VHH binding domain and positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-Fc domain subunit. In some instances, when each Fc domain subunit is further linked to a single CH1 domain, and each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit.

[0254] 6. Antibody Variants

[0255] In certain embodiments, amino acid sequence variants of the antibodies of the invention (e.g., antibodies that bind ETEC CS3 and CS6) are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence PATENT

[0256] Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, for example, antigen-binding. In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0257] Table 1. Exemplary and Preferred Amino Acid Substitutions PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0258] Amino acids may be grouped according to common side-chain properties:

[0259] (1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;

[0260] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0261] (3) acidic: Asp, Glu;

[0262] (4) basic: His, Lys, Arg;

[0263] (5) residues that influence chain orientation: Gly, Pro;

[0264] (6) aromatic: Trp, Tyr, Phe.

[0265] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0266] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., an isolated VHH antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0267] Alterations (e.g., substitutions) may be made in CDRs, for example, to improve antibody affinity. Such alterations may be made in CDR “hotspots,” i.e. , residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and / or residues that contact antigen, with the resulting variant VHH binding domain being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is known in the art. In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0268] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variant VHH binding domain sequences provided above, each CDR either is unaltered, or contains no more than one, two, or three amino acid substitutions.

[0269] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081 -1085. In this method, a residue or group of target residues (e.g., charged residues PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 such as Arg, Asp, His, Lys, And Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigenantibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0270] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0271] In certain embodiments, alternations may be made to the Fc region, when present, of an antibody. These alterations can be made alone, or in addition to, alterations to the VHH binding domain or regions thereof (e.g., one or more CDRs or FRs). The alterations to the Fc region, when present, may result in enhanced antibody effector functions (e.g., complement-dependent cytotoxicity (CDC)), for example, by increasing C1 q avidity to opsonized cells. Exemplary mutations that enhance CDC include, for example, Fc mutations E345R, E430G, and S440Y. Accordingly, antibodies of the invention may contain one or more CDC-enhancing Fc mutations, which promote IgG hexamer formation and the subsequent recruitment and activation of C1 , the first component of complement (see, e.g., Diebolder et al. Science. 343: 1260-1263, 2014).

[0272] In certain embodiments, alterations of the amino acid sequences of the Fc region of the antibody may alter the half-life of the antibody in the host. Certain mutations that alter binding to the neonatal Fc receptor (FcRn) may extend half-life of antibodies in serum. For example, antibodies that have tyrosine in heavy chain position 252, threonine in position 254, and glutamic acid in position 256 of the heavy chain can have dramatically extended half-life in serum (see, e.g., U.S. Patent No. 7,083,784).

[0273] III. VHH ANTIBODIES THAT BIND ETEC CS3, CS6, AND AN ADDITIONAL ANTIGEN

[0274] Any of the antibodies provided herein that bind ETEC CS3 and CS6 (e.g., comprise a CS3 / CS6 VHH binding domain) may comprise one or more additional, non-identical binding domains (e.g., VHH binding domains). Thus, in some aspects, any of the antibodies provided herein may comprise two or more VHH binding domains (e.g., may comprise two binding domains.

[0275] For example, in some aspects, an antibody provided herein that comprises a CS3 / CS6 VHH binding domain further comprises a binding domain capable of binding to an ETEC adhesin protein selected from colonization factor antigen I adhesin subunit E (CfaE), coli surface antigen 1 (CS1 ), coli surface antigen 4 (CS4), coli surface antigen 14 (CS14), coli surface antigen 17 (CS17), coli surface antigen 19 (CS19), and coli surface antigen 2 (CS2). In some aspects, an antibody provided herein that comprises a CS3 / CS6 VHH binding domain further comprises a binding domain capable of binding to CS5, CS21 , or PCF071. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0276] A. Antibodies that bind ETEC CS3, CS6, and CfaE

[0277] In some aspects, an antibody provided herein that binds ETEC CS3 and CS6 and comprises a CS3 / CS6 VHH binding domain (e.g., an antibody provided in any one of Sections A-C, above) further comprises a colonization factor antigen I adhesin subunit E (CfaE) VHH binding domain.

[0278] In some embodiments, the CfaE VHH binding domain comprises the following CDRs: (a) a CDR- H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30). The disclosure also provides an isolated antibody comprising a CfaE VHH binding domain that comprises a combination of one or more of the CDRs of SEQ ID NOs: 28-30 and one or more variants thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 28-31 and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 28-31 , such that the isolated antibody comprises the CDR of SEQ ID NO: 28 or a variant thereof; the CDR of SEQ ID NO: 29 or a variant thereof; and the CDR of SEQ ID NO: 30 or a variant thereof.

[0279] In some embodiments, the CfaE VHH binding domain includes a sequence having at least 95% sequence identity (having e.g., 96%, 97%, 98%, or 99% identity) to the amino acid sequence of SEQ ID NO: 35 and / or having one, two, three, four, five, or six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 35.

[0280] In some instances, the CfaE VHH binding domain includes the following framework regions (FRs): (a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLTLSCAAS (SEQ ID NO: 31 ); (b) an FR-H2 comprising the amino acid sequence of MAWFRQAPGKERE (SEQ ID NO: 32); (c) an FR-H3 comprising the amino acid sequence of YADSVKGRFTISRDNAENTGYLQMNGLKPEDTAVYYCA (SEQ ID NO: 33); and (d) an FR-H4 comprising the amino acid sequence of WGRGTQVTVSSAA (SEQ ID NO: 34). The disclosure also provides an isolated antibody comprising a CfaE VHH binding domain that comprises a combination of one or more of the FRs of SEQ ID NOs: 31 -34 and one or more variants thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 31 -34 and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 31 -34, such that the isolated antibody comprises the FR of SEQ ID NO: 31 or a variant thereof; the FR of SEQ ID NO: 32 or a variant thereof; the FR of SEQ ID NO: 33 or a variant thereof; and the FR of SEQ ID NO: 34 or a variant thereof.

[0281] In some instances, the CfaE VHH binding domain comprises the amino acid sequence of SEQ ID NO: 35. In some instances, the CfaE VHH binding domain consists of the amino acid sequence of SEQ ID NO: 35, i.e., the CfaE VHH binding domain is the VHH antibody 4H4.

[0282] In some aspects, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ); and (ii) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30). In some aspects, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the amino acid sequence of SEQ ID NO: 26 and (ii) a CfaE VHH binding domain that comprises the amino acid sequence of SEQ ID NO: 35.

[0283] In some aspects, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3); and (ii) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30). In some aspects, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the amino acid sequence of SEQ ID NO: 8 and (ii) a CfaE VHH binding domain that comprises the amino acid sequence of SEQ ID NO: 35.

[0284] In some aspects, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and (ii) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30). In some aspects, the disclosure features an isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises (i) a CS3 / CS6 VHH binding domain that comprises the amino acid sequence of SEQ ID NO: 17 and (ii) a CfaE VHH binding domain that comprises the amino acid sequence of SEQ ID NO: 35.

[0285] / . Fc regions

[0286] In some aspects, an antibody provided herein comprising a CS3 / CS6 VHH binding domain and a CfaE VHH binding domain is a full-length antibody.

[0287] In some aspects, an antibody provided herein comprising a CS3 / CS6 VHH binding domain and a CfaE VHH binding domain comprises an Fc domain comprising a first Fc domain subunit and a second Fc domain subunit, wherein the first Fc domain subunit and the second Fc domain subunit are capable of stable association, and wherein: (i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 domain; and (ii) the second Fc domain subunit is linked to the CfaE VHH binding domain. In some embodiments, (i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain in an N- terminal-to-C-terminal direction as follows: CS3 / CS6 VHH binding domain-Fc domain subunit; and (ii) the second Fc domain subunit is linked to the CfaE VHH binding domain in an N-terminal-to-C-terminal direction as follows: CfaE VHH binding domain-Fc domain subunit. In some embodiments, the antibody comprises one or more modifications that promote the association of the first Fc subunit with the second Fc subunit (e.g., comprises knob-and-hole mutations).

[0288] In some embodiments, (i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain by a hinge region; and (ii) the second Fc domain subunit is linked to the CfaE VHH binding domain by a hinge region.

[0289] In some embodiments, each Fc domain subunit is further linked to a single CH1 domain. In some embodiments, each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit. In some embodiments, each VHH binding domain, CH1 domain, hinge region, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-hinge region-Fc domain subunit.

[0290] In some embodiments, the Fc region is an IgA Fc region.

[0291] In some embodiments, the IgA Fc region is an Ig A1 Fc region. In some embodiments, the lgA1 Fc region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 37 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96- 98%, or more than 98% identity) to SEQ ID NO: 37). In some embodiments, the lgA1 Fc region comprises the amino acid sequence of SEQ ID NO: 37. The antibody may further comprise a tail, wherein the components of the antibody are arranged in an N-terminal-to-C-terminal direction as follows: VHH binding domain- Fc region-tail. In some aspects, the tail has at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to SEQ ID NO: 38). Thus, in some aspects, the antibody comprises an Fc region and tail having at least 90% identity to SEQ ID NO: 39 (e.g., having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to SEQ ID NO: 39).

[0292] In some embodiments, the IgA Fc region is an lgA2 Fc region. In some embodiments, the lgA2 Fc region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 45 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96- 98%, or more than 98% identity) to SEQ ID NO: 45). In some embodiments, the lgA2 Fc region comprises the amino acid sequence of SEQ ID NO: 45. The antibody may further comprise a tail, wherein the components of the antibody are arranged in an N-terminal-to-C-terminal direction as follows: VHH binding domain- Fc region-tail. In some aspects, the tail has at least 90% identity to SEQ ID NO: 38 (e.g., has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to SEQ ID NO: 38). Thus, in some aspects, the antibody comprises an Fc region and tail having at least 90% identity to SEQ ID NO: 46 (e.g., having at least 91%, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0293] 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (e.g., 91 -92%, 92-94%, 94-96%, 96-98%, or more than 98% identity) to SEQ ID NO: 46).

[0294] In some embodiments, the Fc region is an IgG Fc region. The IgG Fc region may be any IgG subtype (e.g., lgG1 , lgG2a, lgG2b, lgG3, or lgG4).

[0295] In some embodiments, the hinge region includes the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRP (SEQ ID NO: 36), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 36, and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the hinge region consists of the amino acid sequence of SEQ ID NO: 36.

[0296] In some embodiments, the hinge region includes the amino acid sequence of ASPVPPPPPCCHPRLSLHRP (SEQ ID NO: 44), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 44, and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the hinge region consists of the amino acid sequence of SEQ ID NO: 44.

[0297] In some embodiments, the hinge region includes the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPS (SEQ ID NO: 50), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 50, and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the hinge region consists of the amino acid sequence of SEQ ID NO: 50.

[0298] In some embodiments, the hinge region includes the amino acid sequence of ASPVPPPPPP (SEQ ID NO: 51 ), or a variant thereof having (i) at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 51 , and / or (ii) one, two, or three amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 51 . In some embodiments, the hinge region consists of the amino acid sequence of SEQ ID NO: 51 . ii. Multimeric architectures

[0299] In some aspects, an antibody provided herein comprising a CS3 / CS6 VHH binding domain and a CfaE VHH binding domain comprises the following N-terminal-to-C-terminal structure:

[0300] (P3-L2)n3-P2-(Ll-Pl)n1 wherein Pi, P2, and P3 are each independently selected from (i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ); (ii) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0301] AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3); (iii) a CS3 / CS6 VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR- H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and (iv) a CfaE VHH binding domain that comprises the following three CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30); wherein Li and L2 are each independently a linker; and ni and ns are each independently 0 or 1 , wherein at least one of ni and ns are 1 .

[0302] In some embodiments, ni is 1 and ns is 0, and the antibody comprises the following N-terminal-to- C-terminal structure:

[0303] P2-L1-P1.

[0304] In some embodiments, Pi and P2 each comprise different VHH binding domains. In some embodiments, Pi and P2 each comprise identical VHH binding domains.

[0305] In some embodiments, ni is 1 and ns is 1 , and the antibody comprises the following N-terminal-to- C-terminal structure:

[0306] P3-L2-P2-L1-P1 .

[0307] In some embodiments, Pi, P2, and P3 each comprise different VHH binding domains. In some embodiments, Pi, P2, and P3 each comprise identical VHH binding domains.

[0308] In some aspects of any of the above embodiments, the peptide linker includes between 2 and 200 amino acids (e.g., between 5 and 50 (e.g., between 5 and 20, 15 and 30, 25 and 40, or 35 and 50), between 45 and 100 (e.g., between 45 and 60, 55 and 70, 65 and 80, 75 and 90, or 85 and 100), 95 and 150 (e.g., between 95 and 110, 105 and 120, 115 and 130, 125 and 140, or 135 and 150), or 145 and 200 amino acids (e.g., between 145 and 160, 155 and 170, 165 and 180, 175 and 190, or 185 and 200)). In some instances, the peptide linker comprises glycine (Gly) and serine (Ser) amino acids. In some instances, the peptide linker includes the amino acid sequence of any one of (GS)x, (GGS)x, (GGGGS (SEQ ID NO: 52))x, (GGSG)x, and (SGGG)x, wherein x is an integer from 1 to 10. In certain embodiments the linker includes the amino acid sequence of (GGGGS (SEQ ID NO: 52))x, wherein x is an integer from 2-5.

[0309] IV. PRODUCTION AND CHARACTERIZATION OF ANTI-ETEC ADHESIN PROTEIN VHH ANTIBODIES

[0310] A. Production

[0311] 1. Immunizations

[0312] A mammal from the Camelidae family (e.g., llamas, camels, or alpacas) can be subcutaneously immunized with N-terminal fragments representing ETEC antigens. A phage-displayed VHH library can be constructed from PBMC total RNA, retrieved from the mammal, and selection of target-binding VHHs can be performed by phage-display selections as described in Hultberg, A., et al (PLoS One 6(4): e17665, 2011 ), which is incorporated by reference herein in its entirety. The initial screening can be PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 performed with desired antigens (e.g., an ETEC adhesin protein (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, or PCF071 )). Additionally, another round of screening can be performed with a different ETEC adhesin protein antigen.

[0313] Identified clones of interest can be completely sequenced and clones including unique sequences can be selected for further characterization.

[0314] 2. Yeast Library screening

[0315] Yeast library screening can be performed as described in McMahon et al. Nat. Struct. Mol. Biol. 25: 289-296, 2018, which is incorporated by reference herein in its entirety. For a first round of magnetic- activated cell sorting (MACS), 1 x1010S. cerevisiae cells expressing a surface displayed library of synthetic nanobodies (see McMahon et al. Nat. Struct. Mol. Biol. 25: 289-296, 2018) can be centrifuged, resuspended in binding buffer (20 mM HEPES pH 7.5, 150 mM NaCI, 2.8 mM CaCl2, 0.05% MNG, 0.005% CHS, 0.1% BSA, 0.2% maltose), and then incubated with anti-fluorescein isothiocyanate (FITC) microbeads (Miltenyi Biotec) and FITC labeled MBP for 40 min at 4 °C. The selected yeast can then be passed through an LD column (Miltenyi Biotec) to remove any yeast expressing nanobodies which interacted with the microbeads or MBP. Remaining yeast that flow through the column can be centrifuged, resuspended in binding buffer, and incubated with 1 mM of FITC-labeled N-terminal ETEC adhesin (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, or PCF071 ) protein for 1 h at 4 °C. Yeast can then be centrifuged, resuspended in binding buffer with anti-FITC microbeads, and incubated for 15 min at 4 °C before passing into an LS column (Miltenyi Biotec) and collecting the eluate enriched for ETEC adhesin-binding nanobodies. The eluted yeast can be expanded and used in a subsequent round of MACS to further enrich for ETEC adhesin-binding nanobodies. A second round can be performed similarly to the first, but beginning with 4x108yeast and substituting FITC-labeled ETEC adhesin protein with AlexaFluor647-labeledETEC adhesin protein, and anti- FITC microbeads with anti- AlexaFluor647 microbeads. High-affinity binding yeast can be isolated with FACS. In the first round of FACS, yeast binding to 300 nM of AlexaFluor488 labeled ETEC adhesin protein can be collected. The yeast can be grown and subjected to a second round of FACS in the presence of human monoclonal antibody capable of binding to an ETEC adhesin protein (e.g., a human monoclonal antibody of Guintini et al. Infect. Immun. 86(8): e00355-18, 2018) that was shown to bind in the proximity of the receptor binding domain of CfaE). Clones that are outcompeted from binding to the antigen can be collected. Individual clones from the two rounds of FACS can be grown, stained in a 96-well plate, assessed via flow cytometry for binding specificity to an ETEC adhesin protein (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, or PCF071 ), and subsequently sequenced. Clones with unique sequences can be isolated and chosen for further characterization, that may include further panning with FITC-labeled ETEC adhesin proteins (e.g., class 5 adhesins CS1 (class5b) and CS2 (class5c)).

[0316] 3. Nanobody purification

[0317] Nanobody sequences can be ligated into a suitable vector (e.g., a pET26b vector), and a tag (e.g., a C-terminal 6xHis tag) can be added to the nanobody sequence to aid in purification. Sequence- verified clones can be transformed into T7 Express lysY BL21 E. coli. Bacteria can be grown in Terrific PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0318] Broth containing 1 mM MgCl2 and 0.01% glucose to an OD600 = 0.7 before induction with 1 mM IPTG. Cells can be harvested after an overnight incubation at 27 °C. Following osmotic shock, nanobodies can be purified from the periplasmic fraction by Ni-NTA chromatography (Gold Biotechnology) and dialyzed against PBS to remove imidazole.

[0319] 4. Recombinant Generation of VHH antibodies to an ETEC adhesin protein Anti-ETEC adhesin protein VHH antibodies of the invention (e.g., antibodies including anti-ETEC adhesin protein VHH binding domains D10, A09, C04, 4H4, or variants thereof) may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, one or more isolated nucleic acids encoding any one of the antibodies provided herein (e.g., antibodies comprising a CS3 / CS6 VHH binding domain or antibodies comprising a CS3 / CS6 VHH binding domain and a CfaE binding domain) are provided. Such nucleic acids may encode an amino acid sequence comprising the VHH of the antibody (e.g., the light and / or heavy chains of the antibody). The nucleic acids may be, e.g., mRNA or DNA. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, one or more host cells comprising such nucleic acids is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VHH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell). In one embodiment, a method of making an anti-ETEC adhesin protein antibody is provided, wherein the method comprises culturing one or more host cells comprising one or more nucleic acids encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the one or more host cells (or host cell culture medium).

[0320] In one embodiment, a method of making an antibody comprising a CS3 / CS6 VHH binding domain and a CfaE binding domain is provided, wherein the method comprises culturing a single host cell line comprising one or more nucleic acids encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell line (or host cell culture medium).

[0321] For recombinant production of an anti-ETEC adhesin protein antibody, one or more nucleic acids encoding an antibody, e.g., as described above, can be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0322] Suitable host cells for cloning or expression of anti-ETEC adhesin protein antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, or when an Fc region is not present. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0323] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern.

[0324] Suitable host cells for the expression of anti-ETEC adhesin protein VHH antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. Furthermore, high expression of IgA-Fc fusions can be easily produced in plant seeds (e.g., Arabidopsis, tobacco, rice, and soybean) and are specifically contemplated for the production of anti-ETEC adhesin protein VHH antibodies provided herein.

[0325] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells, and myeloma cell lines such as Y0, NSO, and Sp2 / 0.

[0326] B. Characterization of anti-ETEC adhesin protein VHH antibodies

[0327] Sequence information for VHH antibodies of the invention can be ascertained using sequencing techniques which are well known in the art.

[0328] Similarly, affinity of the antibodies for ETEC adhesin proteins can also be assessed using standard techniques. For example, BIACORE 3000 can be used to determine the affinity of VHH antibodies to ETEC adhesin proteins. VHH antibodies are captured on the surface of a BIACORE® chip (GE healthcare), for example, via amine coupling (Sensor Chip CM5). The captured VHH antibodies can be exposed to various concentrations of ETEC adhesin proteins (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, or PCF071 ) in solution, and the KOn and Kotf for an affinity (KD) can be calculated, for example, by BIAevaluation software.

[0329] VHH antibodies of the invention can also be characterized for binding to ETEC adhesin proteins using a variety of known techniques, such as ELISA, Western blot, etc. Generally, the antibodies are initially characterized by ELISA. Briefly, microtiter plates can be coated with purified ETEC adhesin protein(s) (e.g., CS3, CS6, or CfaE) in PBS, and then blocked with irrelevant proteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from ETEC adhesin protein immunized mammals from the Camelidae family (e.g., llamas, camels, and alpacas), or purified VHHs are added to each well and incubated for 1 -2 hours at 37 °C. The plates are washed with PBS / Tween 20 and then incubated with a hydroxy peroxidase-conjugated rabbit anti-camelid IgG Fc (1 :10,000) for 1 h for staining. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0330] After staining, the plates can be developed using TMB Peroxidase substrate (SeraCare) and analyzed at OD of 450.

[0331] In some instances, an ELISA assay as described above can be used to screen for antibodies and, thus, hybridomas that produce antibodies that show positive reactivity with the ETEC adhesin protein immunogen (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, or PCF071 ). Hybridomas that bind, preferably with high affinity, to an ETEC adhesin protein (e.g., CS3, CS6, CfaE, CS4, CS14, CS1 , CS17, CS19, CS2, or PCF071 ) can then be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cell (by ELISA), can then be chosen for making a cell bank, and for antibody purification.

[0332] In some instances, the antibodies can be evaluated by a mannose-resistant hemagglutination (MRHA) assay of human group A erythrocyte inhibition. The MRHA assay is considered a surrogate method for assessment of ETEC adhesion to the intestinal mucosa (Hagberg et al., Infect. Immun. 31 :564-570, 1981 ). In summary, the MRHA assay is performed as follows. First, ETEC cultures are taken from frozen cell banks and diluted in saline solution, reaching an ODeoonm of 1 for the assay. Human erythrocytes type A+ are washed in saline solution and resuspended in the same solution. Serial antibody dilutions are prepared in a 96-well plate. The diluted ETEC and a solution of D-mannose are added to each well, then incubated at room temperature for 10 minutes. After incubation, the blood solution is added to the plates and mixed well, then allowed to sit stagnant at 4 °C for two hours. Hemagglutination is then observed without the aid of magnification. The absence of a pellet of red blood cells at the bottom of the well is indicative of positive hemagglutination.

[0333] In other instances, the antibodies are evaluated for their ability to inhibit binding of ETEC to intestinal cells by a Caco-2 cell adhesion assay. Briefly, Caco-2 cells are seeded and grown in 24-well tissue plates containing Dulbecco’s modified Eagle’s medium (DMEM), and frozen bacterial banks are streaked on CFA agar plates and grown overnight at 37 °C. Bacteria are then resuspended in PBS and diluted until reaching an ODeoonm of 0.1 . Serial antibody dilutions are also prepared in a deep well plate. The antibody dilutions and bacteria are combined and allowed to shake at 300 rpm for one hour at room temperature. The antibody / bacteria mixture is then added to Caco-2 cells and incubated statically for 3 hours at 37 °C. After incubation, cells are washed with PBS to remove non-adherent ETEC cells, then dislodged with trypsin, collected via centrifugation, and resuspended in PBS. Dilutions are plated on CFA agar plates and colonies are counted the next day.

[0334] In other instances, competition assays may be used to identify an antibody that competes with an anti-ETEC adhesin protein antibody of the invention for binding to an ETEC adhesin protein. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by an anti-ETEC adhesin protein antibody of the invention. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0335] In an exemplary competition assay, immobilized ETEC adhesin protein (e.g., CS3, CS6, or CfaE) can be incubated in a solution comprising a first labeled antibody that binds to an ETEC adhesin protein (e.g., CS3, CS6, or CfaE) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the ETEC adhesin protein (e.g., CS3, CS6, or CfaE). As a control, PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 immobilized ETEC adhesin protein (e.g., CS3, CS6, or CfaE) can be incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to an ETEC adhesin protein (e.g., CS3, CS6, or CfaE), excess unbound antibody can be removed, and the amount of label associated with immobilized ETEC adhesin protein (e.g., CS3, CS6, or CfaE) can be measured. If the amount of label associated with immobilized ETEC adhesin protein (e.g., CS3, CS6, or CfaE) is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to an ETEC adhesin protein (e.g., CS3, CS6, or CfaE).

[0336] V. PHARMACEUTICAL COMPOSITIONS

[0337] In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or more (e.g., 1 , 2, 3, or 4 or more) of the anti-ETEC adhesin protein VHH antibodies, or antibody fragments thereof, of the present invention. The pharmaceutical compositions may be formulated together with a pharmaceutically acceptable carrier, excipient, or diluent. In some instances, the pharmaceutical compositions include two or more of the anti-ETEC VHH antibodies of the invention. In some instances, each of the antibodies of the composition binds to distinct ETEC adhesin proteins. Preferably, each of the antibodies of the composition binds to a distinct, pre-selected epitopes of ETEC adhesin proteins.

[0338] A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

[0339] To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, lipid nanoparticles, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.

[0340] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0341] The anti-ETEC adhesin protein antibodies of the invention may be orally administered as a pharmaceutical composition, for example, with an inert diluent or with an assimilable edible carrier, or it PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the anti-ETEC adhesin protein antibodies of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. Oral formulations including antibodies are described in Jaison et al., Nutrition Journal. 14:22 (2015), which is incorporated herein by reference in its entirety. For example, the anti-ETEC adhesin protein antibodies may be formulated in a lyophilized composition, mixed into a liquid or a powder formulation. The anti- ETEC adhesin protein antibodies may also be formulated in enteric-coated capsules containing the antibodies. The composition formulated for oral administration may contain at least 0.01% (w / v) of the antibody. For example, the composition may contain about 0.1% to 70% (w / v) of the antibody, e.g., about 0.1% to 65% (w / v), about 0.1% to 65% (w / v), about 0.1% to 55% (w / v), about 0.1% to 50% (w / v), about 0.1% to 45% (w / v), about 0.1% to 40% (w / v), about 0.1% to 35%(w / v), about 0.1% to 30% (w / v), about 0.1% to 25% (w / v), about 0.1% to 20% (w / v), about 0.1% to 15% (w / v), about 0.1% to 10% (w / v), about 0.1% to 5% (w / v), about 0.1% to 2% (w / v), about 2% to 70% (w / v), or about 2% to 60% (w / v) of the antibody.

[0342] Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. , combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one or more additional therapeutic agents as necessary for the particular indication (e.g., ETEC-related diarrhea) being treated.

[0343] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

[0344] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, films, or microcapsules.

[0345] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-f iltered solution thereof. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, such as TWEEN® 80. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0346] Alternatively, genes encoding the anti-ETEC adhesin protein antibodies of the invention may be delivered directly into the subject for expression rather than administering purified antibodies for prevention or therapy. For example, viral vectors, such as recombinant viruses, can be used to deliver the heavy and light chain genes. In one example, rAAV virus particles can be used to deliver anti-ETEC adhesin protein antibodies (Balazs et al. Nature. 481 : 81 , 2012). Antibody genes could also be effectively delivered by electroporation of muscle cells with plasmid DNA containing heavy chain genes (e.g., VHH) (Muthumani et al. Hum Vaccin Immunother. 10: 2253, 2013). Lentivirus vectors or other nucleic acids (e.g., RNA) capable of delivering transgenes could also be used to deliver antibody genes to establish serum antibody levels capable of prevention.

[0347] Also within the scope of the present invention are kits including human anti-ETEC adhesin protein VHH antibodies of the invention and, optionally, a package insert comprising instructions for use (e.g., instructions for using the antibody to treat a subject having or at risk of developing a disorder associated with an ETEC infection). The kits can further contain one or more additional reagents, such as a second, different anti-ETEC adhesin protein VHH antibody having a complementary activity that binds to an epitope on an ETEC adhesin protein (e.g., CfaE) that is distinct from the epitope to which the first anti- ETEC adhesin protein VHH antibody binds.

[0348] In some aspects, the disclosure provides a composition comprising:

[0349] (i) an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19); (b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and (c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ); and

[0350] (ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0351] In some aspects, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising:

[0352] (i) an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 ); (b) a CDR-H2 comprising the amino acid sequence PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 of ISGSGRST (SEQ ID NO: 2); and (c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3); and

[0353] (ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0354] In some aspects, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising:

[0355] (i) an isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10); (b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and (c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and

[0356] (ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

[0357] In some aspects, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising one or more isolated nucleic acids (e.g., mRNA or DNA molecules) encoding any one of the antibodies provided herein. In some aspects, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising a single isolated nucleic acid (e.g., mRNA or DNA molecule) encoding any one of the antibodies provided herein. In some aspects, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising one or more vectors comprising one or more isolated nucleic acids encoding any one of the antibodies provided herein.

[0358] In some embodiments, any one of the compositions provided herein may be formulated for oral administration.

[0359] In some embodiments, any one of the compositions provided herein may be formulated for intramuscular administration.

[0360] In some aspects, the disclosure provides a composition (e.g., a pharmaceutical composition) comprising a lipid nanoparticle comprising any one of the antibodies, nucleic acids, or vectors provided herein or a composition (e.g., pharmaceutical composition) comprising the same.

[0361] VI. THERAPEUTIC METHODS AND RELATED COMPOSITIONS FOR USE

[0362] Any of the anti-ETEC adhesin protein antibodies of the invention (e.g., antibodies including anti- ETEC adhesin protein VHH binding domains D10, A09, or C04), compositions containing the antibodies, and / or lipid nanoparticles comprising the antibodies or nucleic acids encoding the same can be used in a variety of in vitro and in vivo therapeutic applications. PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0363] In one aspect, the invention features a method of treating a subject having a disorder associated with an ETEC infection (e.g., ETEC-related diarrhea) comprising administering a therapeutically effective amount of one or more (e.g., 1 , 2, 3, or 4 or more) anti-ETEC adhesin protein antibodies of the invention (e.g., isolated antibodies that bind ETEC CS3 protein and ETEC CS6 protein and comprise a CS3 / CS6 VHH binding domain), a pharmaceutical composition(s) including the one or more antibodies, or a lipid nanoparticle comprising the one or more antibodies or nucleic acids encoding the same.

[0364] In another aspect, an anti-ETEC adhesin protein antibody of the invention may be used in a method of treating a subject at risk of developing a disorder associated with an ETEC infection (e.g., treating a subject at risk of developing a disorder associated with an ETEC infection with an anti-ETEC adhesin protein antibody of the invention in order to prevent the subject from developing a disorder associated with an ETEC infection, such as ETEC-related diarrhea). In one embodiment, the method comprises administering to a subject at risk of developing a disorder associated with an ETEC infection a therapeutically effective amount of one or more (e.g., 1 , 2, 3, or 4 or more) anti-ETEC adhesin protein antibodies of the invention or a pharmaceutical composition(s) including the one or more anti-ETEC adhesin protein antibodies. In some instances, a subject can be considered at risk of an ETEC infection if the subject is in a geographic region in which ETEC is commonly found (e.g., in Asia, the Middle East, Africa, and Central and South America). In other instances, subject can be considered at risk of an ETEC infection if the subject had travelled, or will travel, to a geographic region in which ETEC is commonly found.

[0365] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered in a single dose. In other embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered, e.g., approximately once a day, every other day, every week, every two weeks, every four weeks, every month, every two months, or every six months.

[0366] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered approximately once a day to the subject. In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is orally administered approximately once a day to the subject.

[0367] In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered approximately once a month to the subject. In some embodiments, the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is intramuscularly administered approximately once a month to the subject.

[0368] Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeutic agent. Such combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-ETEC adhesin protein antibody and administration of an additional therapeutic agent occur PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.

[0369] An antibody of the invention, such as antibodies including anti-ETEC adhesin protein VHH binding domains D10, A09, or C04 (and / or any additional therapeutic agent) can be administered by any suitable means, including oral, parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Preferably, the antibodies are administered orally, intramuscularly, or subcutaneously. In certain instances, antibody genes (e.g., genes encoding any one or more of the anti-ETEC adhesin protein antibodies of the invention could be administered as a gene therapy to produce the one or more anti-ETEC adhesin protein antibodies in the subject using either DNA vectors or viral vectors (e.g., rAAV vectors) comprising nucleic acids encoding the antibodies. In some aspects, nucleic acids for use in such a gene therapy are administered in a lipid nanoparticle, e.g., are administered intramuscularly in a lipid nanoparticle. Dosing can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0370] Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with, one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / cli nically determined to be appropriate.

[0371] For the prevention or treatment of disease, such as ETEC-related diarrhea, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be prevented / treated, the duration of effective antibody concentration required, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. In some embodiments, a dosing schedule can include delivery, for example oral delivery, 1 -3 days before a subject is at risk of developing a disorder associated with an ETEC infection (e.g., -3 days, -2 days, and / or -1 day), on the day a subject is at risk of developing a disorder associated with an ETEC infection (e.g., 0 day), and / or 1 -3 days after a subject was at risk of developing a disorder associated with an ETEC infection (e.g., +1 day, +2 days, and / or +3 days). In some embodiments, a dosing schedule can PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 include delivery, for example oral delivery, on the day before a subject is at risk of developing a disorder associated with an ETEC infection (e.g., -1 days), the day a subject is at risk of developing a disorder associated with an ETEC infection (e.g., 0 day), and / or on the day after a subject is at risk of developing a disorder associated with an ETEC infection (e.g., +1 day).

[0372] As a general proposition, the therapeutically effective amount of the anti-ETEC adhesin protein antibody administered to human will be in the range of about 0.01 to about 100 mg / kg of patient body weight whether by one or more administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.1 to about 10 mg / kg, or about 1 to about 10 mg / kg administered one (single administration) or more times (multiple administrations, e.g., daily administrations). In one example, the antibody used is about 10 mg / kg, preferably administered orally. In one embodiment, an anti-ETEC adhesin protein antibody described herein is administered to a human at a flat dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21 -day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.01 mg / kg to about 10 mg / kg. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses of the anti-ETEC adhesin protein antibody). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

[0373] The anti-ETEC adhesin protein antibodies of the invention may be orally administered as a pharmaceutical composition, for example, with an inert diluent or with an assimilable edible carrier; may be enclosed in hard or soft shell gelatin capsules; may be compressed into tablets; or may be incorporated directly with the food of the diet. For oral therapeutic administration, the anti-ETEC adhesin protein antibodies of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. Oral formulations including antibodies are described in Jaison et al., Nutrition Journal. 14:22 (2015), which is incorporated herein by reference in its entirety. For example, the anti-ETEC adhesin protein antibodies may be formulated in a lyophilized composition, mixed into a liquid or a powder formulation. The anti- ETEC adhesin protein antibodies may also be formulated in enteric-coated capsules containing the antibodies. The composition formulated for oral administration may contain at least 0.01% (w / v) of the antibody. For example, the composition may contain about 0.1% to 70% (w / v) of the antibody, e.g., about

[0374] 0.1% to 65% (w / v), about 0.1% to 65% (w / v), about 0.1% to 55% (w / v), about 0.1% to 50% (w / v), about

[0375] 0.1% to 45% (w / v), about 0.1% to 40% (w / v), about 0.1% to 35%(w / v), about 0.1% to 30% (w / v), about

[0376] 0.1% to 25% (w / v), about 0.1% to 20% (w / v), about 0.1% to 15% (w / v), about 0.1% to 10% (w / v), about PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77

[0377] 0.1% to 5% (w / v), about 0.1% to 2% (w / v), about 2% to 70% (w / v), or about 2% to 60% (w / v) of the antibody.

[0378] Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. , combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one or more additional therapeutic agents as necessary for the particular indication (e.g., ETEC-related diarrhea) being treated. In some embodiments, an isolated antibody of the invention that comprises a CS3 / CS6 VHH binding domain is administered in combination with an antibody comprising a CfaE VHH binding domain, e.g., is administered in combination with an antibody comprising a CfaE VHH binding domain comprising the following CDRs: (a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28); (b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and (c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30). In some embodiments, the antibody that comprises the CS3 / CS6 VHH binding domain and the antibody that binds CfAE are formulated together. In other embodiments, the antibody that comprises the CS3 / CS6 VHH binding domain and the antibody that binds CfAE are formulated separately.

[0379] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response and duration for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of compositions of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. If desired, the effective daily dose of therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[0380] Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851 , 5,312,335, 5,064,413, 4,941 ,880, 4,790,824, or 4,596,556. Examples of well-known implants PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0381] In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery. Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; p 120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0382] In some instances, the antibody-based therapy may be combined with an additional therapy for more efficacious treatment (e.g., additive or synergistic treatment) of the subject. Accordingly, subjects treated with antibodies of the invention can be additionally administered (prior to, simultaneously with, or following administration of a human antibody of the invention) with another therapeutic agent which enhances or augments the therapeutic effect of the human antibodies.

[0383] VII. METHODS AND COMPOSITIONS FOR DETECTION OR DIAGNOSIS

[0384] In certain embodiments, any of the anti-ETEC adhesin protein antibodies of the invention are useful for in vitro or in vivo detection of the presence of an ETEC adhesin protein (e.g., CS3 or CS6) in a biological sample (e.g., a swab sample, a lavage sample, a blood sample, a plasma sample, a sputum sample, a urine sample, a stool sample, a sample from the mucosal lining of the small intestine, or a mucosal secretion sample) from a subject (e.g., a mammalian subject, e.g., a human subject). The term “detecting” as used herein encompasses quantitative or qualitative detection. In some embodiments, the PATENT Attorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77 sample is from a subject presumed to have an ETEC infection (e.g., a subject showing symptoms of an ETEC adhesin protein (e.g., ETEC-related diarrhea)).

[0385] In some aspects, an anti-ETEC adhesin protein antibody for use in a method of diagnosis (e.g., diagnosis of a disorder associated with an ETEC infection) or detection (e.g., detection of an ETEC infection) is provided. For example, in one aspect, a method of detecting an ETEC (e.g., detecting the presence of an ETEC adhesin protein (e.g., CS3 or CS6)) in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-ETEC adhesin protein antibody as described herein under conditions permissive for binding of the anti-ETEC adhesin protein antibody to the ETEC adhesin protein (e.g., CS3 or CS6), and detecting whether a complex is formed between the anti-ETEC adhesin protein antibody and ETEC adhesin protein (e.g., CS3 or CS6). Such a method of detection may be an in vitro or in vivo method.

[0386] In another aspect, an anti-ETEC adhesin protein VHH antibody for use in a method of diagnosis (e.g., diagnosis of a disorder associated with an ETEC infection) is provided. In certain embodiments, the method includes contacting the biological sample with an anti-ETEC adhesin protein antibody as described herein under conditions permissive for binding of the anti-ETEC adhesin protein antibody to the ETEC adhesin protein (e.g., CS3 or CS6), and detecting whether a complex is formed between the anti- ETEC adhesin protein antibody and ETEC. Such a diagnostic method may be an in vitro or in vivo method.

[0387] In certain embodiments, labeled anti-ETEC adhesin protein antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes32P,14C,125l,3H, and1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, p-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0388] In one aspect, the disclosure provides a kit for detecting ETEC, the kit comprising any one of the antibodies provided herein (e.g., wherein the antibody is conjugated to a label or a tag) and a package insert comprising instructions for using the antibody to detect ETEC.

[0389] VIII. Articles of Manufacture

[0390] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. In some embodiments, the invention provides a kit comprising an antibody of the invention and a package insert with instructions for using the antibody for treating a subject having or at risk of developing a disorder associated with an ETEC infection (e.g., ETEC-related diarrhea). In some embodiments, the invention provides a kit for detecting ETEC including an antibody of the invention and a package insert with instructions for using the antibody to detect ETEC. In some embodiments, the antibody is conjugated to a label or a tag. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0391] Other embodiments of the present invention are described in the following Examples. The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0392] IX. EXAMPLES

[0393] The following are examples of the methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the description provided herein.

[0394] Example 1. Screening for VHH antibodies that bind to ETEC CS3 and CS6

[0395] Infectious diarrhea historically has been a substantial cause of morbidity for children in the developing world, travelers, and deployed U.S. warfighters. Enterotoxigenic Escherichia coli (ETEC) is one of the most prevalent pathogens responsible for diarrheal disease. Traditionally, infected adults have been given short courses of antibiotics to which the ETEC strain is sensitive and which may shorten the duration and volume of diarrhea. However, ETEC strains are becoming increasingly resistant to antibiotics. Attachment and colonization of ETEC through filamentous colonization factors (CF) are critical for bacteria to effectively produce toxin and represent a potential strategic target for preventing ETEC infection. Development of vaccine against bacterial attachment and colonization factors has long PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 been considered as an effective approach against ETEC diarrhea. However, a main challenge is the heterogeneity of ETEC strains with a high degree of clonal diversity within an epidemic area. An alternative approach to active vaccination is pre-exposure prophylaxis by antibodies. Despite a great deal of effort over the past 20 years, a cross-protective antibody has not yet been discovered. Isolated antibodies are specific for one or at most two colonization factors, while there are over 25 colonization factors associated with pathogenic ETEC.

[0396] Therefore, VHH or nanobody discovery is being explored to identify cross-protective antibodies. Camelids (namely camels, llamas, and alpacas) have an immune repertoire of three different IgG subclasses - IgG 1 , lgG2, and lgG3. Of these, IgG 1 is a conventional IgG with two heavy chains and two light chains, whereas lgG2 and lgG3 are heavy chain only antibodies (HcAbs), each comprising two heavy chains that are linked by disulfide bonds. As well as lacking light chains, HcAbs also differ from conventional IgG antibodies in terms of the heavy chain structure. Specifically, while conventional IgG heavy chains have three constant domains (CH1 , CH2, and CH3) and a variable domain (VH), HcAbs lack a CH1 domain and are thus smaller. The term nanobody (used interchangeably with VHH) describes a single heavy chain variable domain of a HcAb. Nanobodies have a molecular weight of approximately 15 kDa, which is about 10-fold smaller than that of a conventional IgG antibody, and bind their antigens monovalently but with equivalent strength to their larger (homobivalent) counterparts. More importantly, especially in the context of ETEC, nanobodies have a longer complementarity-determining region 3 (CDR3) which allows access to more conserved, protective epitopes within ETEC colonization factors.

[0397] To generate VHH antibodies (nanobodies) that bind to enterotoxigenic E. coli (ETEC) coli surface antigen 3 (CS3) protein and / or coli surface antigen 6 (CS6), two llamas were immunized and boosted with CS3 or CS3 / CS6 proteins over a period of five weeks. A phage-displayed VHH library was constructed. 84 clones were sequenced, and 36 unique VHH sequences were recovered.

[0398] Example 2. Characterization of VHH antibodies that bind to CS3 and / or CS6

[0399] Thirteen of the unique VHH antibody clones identified in Example 1 were further characterized. Each of these clones was expressed as a VHH-lgA1 fusion protein. Each VHH antibody was assessed for binding to CS3 and CS6 proteins using an enzyme-linked immunosorbent assay (ELISA) (Fig. 2; Figs. 3A and 3B); binding to maltose-binding protein (MBP) was assessed as a negative control.

[0400] Table 2 shows the level of binding of each of the VHH antibodies to CS3, as measured by optical density in an ELISA assay (protein concentration: 2 pg / mL, OD measured at 450 nM). Antibodies are ranked by level of binding, in descending order.

[0401] Table 2. Selected VHH antibodies ranked by level of binding to CS3 PATENT

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[0404] Table 3 shows the level of binding of each of the VHH antibodies to CS6, as measured by optical density in an ELISA assay (protein concentration: 2 pg / mL, OD measured at 450 nM). Antibodies are ranked by binding affinity, in descending order.

[0405] Table 3. Selected VHH antibodies ranked by level of binding to CS6

[0406] Each of the VHH antibodies bound CS3 (Fig. 2, Fig. 3A, and Table 2). The antibodies A09, C04, D09, and D10 demonstrated cross-reactivity (ability to bind to both CS3 and CS6) (Fig. 2; Figs. 3A and 3B; Table 2; and Table 3).

[0407] Half-maximal effective concentration (ECso) values for CS3 and CS6 of each of the cross-reactive clones (A09, C04, D09, and D10) (as VHH-lgA fusion proteins) were determined using ELISA assays (Figs. 4A and 4B; Table 4). Binding trends were consistent with the first round of screening described above.

[0408] Table 4. EC5o values for A09, C04, D09, and D10

[0409] The complementarity-determining region (CDR), framework region (FR), and full VHH sequences of A09, C04, and D10 are provided in Table 5. IgAf and lgA2 Fc region sequences are provided in Table 6.

[0410] Table 5. Antibody Sequences PATENT

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[0415] Table 6. Further Antibody Sequences PATENT

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[0420] Example 3. VHH antibodies that bind to CS3 and CS6 and inhibit ETEC colonization of the intestine

[0421] The cross-reactive clones A09, C04, and D10 were assessed (as VHH-lgA fusion proteins) for their ability to inhibit ETEC colonization in a mouse model. These experiments were performed to determine whether the VHH-lgA fusion proteins were able to bind to the ETEC strains expressing CS3 or CS6, preventing the bacteria from colonizing the intestine.

[0422] Mice were administered a VHH-lgA fusion protein (A09, C04, or D10) and either (i) the ETEC strain E24377A (which expresses CS3) or (ii) the ETEC strain B7A (which expresses CS6). All three VHH-lgA fusion proteins reduced colonization by both the B7A and E24377A ETEC strains, as measured by total number of colony-forming units (CFUs) detected in the intestine (Fig. 5), fold reduction of CFUs (calculated relative to a PBS control) in the intestine (Table 7), and percent inhibition of colonization as calculated relative to the control (Figs. 6A and 6B; Table 8). All three VHH-lgA fusion proteins had activity against both ETEC strains. The fusion protein comprising the C04 VHH exhibited the highest potency against both the B7A and E24377A ETEC strains.

[0423] Table 7. Fold reduction of bacterial colonization by VHH-lgA antibodies

[0424] Table 8. Percent inhibition of bacterial colonization by VHH-lgA antibodies PATENT

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[0427] Example 3. Treatment of subjects at risk of developing a disorder associated with an ETEC infection

[0428] Subjects (e.g., human subjects) who are at risk of developing a disorder associated with an ETEC infection (e.g., subjects residing or traveling to a geographical location in which pathogenic ETEC is found) are treated with one or more of the VHH antibodies D10, A09, and C04 to reduce the likelihood of development of the disorder associated with an ETEC infection.

[0429] Treatments include:

[0430] • The VHH antibody D10 administered as a single agent;

[0431] • The VHH antibody A09 administered as a single agent;

[0432] • The VHH antibody C04 administered as a single agent;

[0433] • The VHH antibodies D10 and A09; D10 and C04; or A09 and C04 administered as a combination therapy; and

[0434] • The VHH antibodies D10, A09, and C04 administered as a combination therapy.

[0435] Subjects (e.g., human subjects) who are at risk of developing a disorder associated with an ETEC infection are also treated with (i) one or more of the VHH antibodies D10, A09, and C04; and (ii) the VHH antibody 4H4 (an anti-ETEC antibody having the VHH sequence of SEQ ID NO: 35) to reduce the likelihood of development of the disorder associated with an ETEC infection.

[0436] Treatments include:

[0437] • The VHH antibodies D10 and 4H4 administered as a combination therapy;

[0438] • The VHH antibodies A09 and 4H4 administered as a combination therapy;

[0439] • The VHH antibodies C04 and 4H4 administered as a combination therapy;

[0440] • The VHH antibodies D10, A09, and 4H4; D10, C04 and 4H4; or A09, C04, and 4H4 administered as a combination therapy; and

[0441] • The VHH antibodies D10, A09, C04, and 4H4 administered as a combination therapy.

[0442] Any of the VHH antibodies (D10, A09, C04, and 4H4) may be an IgA fusion protein.

[0443] Administration includes (I) administration of the antibody or antibodies (e.g., by oral administration), and (II) administration of one or more nucleic acids encoding the antibody or antibodies, e.g., formulated in a lipid nanoparticle (e.g., by intramuscular injection).

[0444] In treatments comprising administration of two or more antibodies, the antibodies (or nucleic acids encoding the same) may be co-administered or separately administered. Co-administration includes administration of a molecule comprising two or more different binding domains (e.g., comprising binding domains comprising the VHH antibodies D10 and 4H4; A09 and 4H4; or C04 and 4H4).

[0445] Other Embodiments

[0446] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed PATENT

[0447] Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77 as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77What is claimed is:1 . An isolated antibody that binds enterotoxigenic E. coli (ETEC) coli surface antigen 3 (CS3) protein and ETEC coli surface antigen 6 (CS6) protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following complementary-determining regions (CDRs):(a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19);(b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and(c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO: 21 ).

2. The antibody of claim 1 , wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26.

3. The antibody of claim 2, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 26.

4. The antibody of claim 3, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 26.

5. The antibody of claim 4, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 26.

6. The antibody of claim 5, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 26.

7. The antibody of any one of claims 1 -6, wherein the CS3 / CS6 VHH binding domain comprises one or more of the following framework regions (FRs):(a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 22);(b) an FR-H2 comprising the amino acid sequence of MGWFRQAPGKEREGVSC (SEQ ID NO: 23);(c) an FR-H3 comprising the amino acid sequence ofYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 24); and(d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 25).

8. An isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the amino acid sequence of SEQ ID NO: 26.

9. The antibody of claim 8, wherein the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 26.PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-7710. An isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding toCS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19);(b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and(c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO:21 ).11 . An isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding toCS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19);(b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and(c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO:21 ).

12. An isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 );(b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and(c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

13. The antibody of claim 12, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8.

14. The antibody of claim 13, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 8.

15. The antibody of claim 14, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 8.

16. The antibody of claim 15, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8.

17. The antibody of claim 16, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.

18. The antibody of any one of claims 12-17, wherein the CS3 / CS6 VHH binding domain comprises one or more of the following FRs:(a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ IDNO: 4);(b) an FR-H2 comprising the amino acid sequence of MGWFRQAPGKEREFVAA (SEQ ID NO: 5);PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77(c) an FR-H3 comprising the amino acid sequence ofIYADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 6); and(d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 7).

19. An isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the amino acid sequence of SEQ ID NO: 8.

20. The antibody of claim 19, wherein the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 8.21 . An isolated antibody that binds ETEC CS3 protein, wherein the antibody competes for binding toCS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 );(b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and(c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

22. An isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding toCS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 );(b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and(c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO: 3).

23. An isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10);(b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and(c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

24. The antibody of claim 23, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17.

25. The antibody of claim 24, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 17.

26. The antibody of claim 25, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 17.

27. The antibody of claim 26, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 17.PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-7728. The antibody of claim 27, wherein the CS3 / CS6 VHH binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17.

29. The antibody of any one of claims 23-28, wherein the CS3 / CS6 VHH binding domain comprises one or more of the following FRs:(a) an FR-H1 comprising the amino acid sequence of EVQLVESGGGLVQAGGSLRLSCAAS (SEQ IDNO: 13);(b) an FR-H2 comprising the amino acid sequence of MAWFRQAPGKEREIVAA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofDYTDSVKGRFTISRDNAKNTGFLQMNRLKPEDTAVYYC (SEQ ID NO: 15); and(d) an FR-H4 comprising the amino acid sequence of WGQGTQVTVSS (SEQ ID NO: 16).

30. An isolated antibody that binds ETEC CS3 protein and ETEC CS6 protein, wherein the antibody comprises a CS3 / CS6 VHH binding domain comprising the amino acid sequence of SEQ ID NO: 17.31 . The antibody of claim 30, wherein the CS3 / CS6 VHH binding domain consists of the amino acid sequence of SEQ ID NO: 17.

32. An isolated antibody that binds an ETEC CS3 protein, wherein the antibody competes for binding to CS3 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10);(b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and(c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

33. An isolated antibody that binds ETEC CS6 protein, wherein the antibody competes for binding toCS6 with an antibody comprising a CS3 / CS6 VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10);(b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and(c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12).

34. The antibody of any one of claims 1 -33, wherein the antibody comprises an Fc region.

35. The antibody of claim 34, wherein the Fc region is an immunoglobulin A (IgA) Fc region.

36. The antibody of claim 35, wherein the IgA Fc region is an Ig A1 Fc region.

37. The antibody of claim 36, wherein the Ig A1 Fc region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 37.

38. The antibody of claim 37, wherein the IgA Fc region comprises the amino acid sequence of SEQID NO: 37.PATENTAttorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-7739. The antibody of claim 35, wherein the IgA Fc region is an lgA2 Fc region.

40. The antibody of claim 39, wherein the lgA2 Fc region comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 45.41 . The antibody of claim 40, wherein the lgA2 Fc region comprises the amino acid sequence of SEQ ID NO: 45.

42. The antibody of any one of claims 34-41 , wherein the CS3 / CS6 VHH binding domain is connected to the Fc region by a linker.

43. The antibody of claim 42, wherein the linker is a hinge region.

44. The antibody of claim 43, wherein the hinge region comprises the sequence of SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 50, or SEQ ID NO: 51 .

45. The antibody of claim 44, wherein the hinge region consists of the sequence of SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 50, or SEQ ID NO: 51 .

46. The antibody of any one of claims 1 -33, wherein the antibody does not comprise an Fc region.

47. The antibody of any one of claims 1 -46, wherein the antibody comprises two or more VHH binding domains.

48. The antibody of claim 47, wherein the antibody comprises two VHH binding domains.

49. The antibody of claim 47 or 48, wherein the antibody comprises a binding domain capable of binding to an ETEC adhesin protein selected from colonization factor antigen I adhesin subunit E (CfaE), coli surface antigen 1 (CS1 ), coli surface antigen 4 (CS4), coli surface antigen 14 (CS14), coli surface antigen 17 (CS17), coli surface antigen 19 (CS19), and coli surface antigen 2 (CS2).

50. The antibody of claim 49, wherein the antibody comprises a CfaE VHH binding domain.51 . The antibody of claim 50, wherein the CfaE VHH binding domain comprises the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-7752. The antibody of claim 51 , wherein the CfaE VHH binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 35.

53. The antibody of claim 52, wherein the CfaE VHH binding domain comprises the amino acid sequence of SEQ ID NO: 35.

54. The antibody of claim 53, wherein the CfaE VHH binding domain consists of the amino acid sequence of SEQ ID NO: 35.

55. An isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises:(i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19);(b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and(c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO:21 ); and(ii) a CfaE VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

56. An isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises:(i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 );(b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and(c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO:3); and(ii) a CfaE VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

57. An isolated antibody that binds to each of ETEC CS3, CS6, and CfaE proteins, wherein the antibody comprises:(i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10);(b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 11 ); and(c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and(ii) a CfaE VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

58. The antibody of any one of claims 50-57, wherein the antibody comprises an Fc domain comprising a first Fc domain subunit and a second Fc domain subunit, wherein the first Fc domain subunit and the second Fc domain subunit are capable of stable association, and wherein:(i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain; and(ii) the second Fc domain subunit is linked to the CfaE VHH binding domain.

59. The antibody of claim 58, wherein:(i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain in an N-terminal-to-C- terminal direction as follows: CS3 / CS6 VHH binding domain-Fc domain subunit; and(ii) the second Fc domain subunit is linked to the CfaE VHH binding domain in an N-terminal-to-C- terminal direction as follows: CfaE VHH binding domain-Fc domain subunit.

60. The antibody of claim 59, wherein:(i) the first Fc domain subunit is linked to the CS3 / CS6 VHH binding domain by a hinge region; and(ii) the second Fc domain subunit is linked to the CfaE VHH binding domain by a hinge region.61 . The antibody of claim 60, wherein each Fc domain subunit is further linked to a single CH1 domain.

62. The antibody of claim 61 , wherein each VHH binding domain, CH1 domain, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-Fc domain subunit.

63. The antibody of claim 62, wherein each VHH binding domain, CH1 domain, hinge region, and Fc domain subunit is positioned relative to each other in an N-terminal-to-C-terminal direction as follows: VHH binding domain-CH1 domain-hinge region-Fc domain subunit.

64. The antibody of any one of claims 60-63, wherein the hinge region comprises the amino acid sequence of ASPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRP (SEQ ID NO: 36), ASPVPPPPPCCHPRLSLHRP (SEQ ID NO: 44), ASPVPSTPPTPSPSTPPTPSPS (SEQ ID NO: 50), or ASPVPPPPPP (SEQ ID NO: 51 ).

65. The antibody of any one of claims 1 -45 and 47-64, wherein the antibody is a full-length antibody.

66. The antibody of any one of claims 58-65, wherein the antibody comprises one or more modifications that promote the association of the first Fc subunit with the second Fc subunit.PATENTAttorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-7767. The antibody of any one of claims 50-57, comprising the following N-terminal-to-C-terminal structure:(P3-L2)n3-P2-(Ll-Pl)n1 wherein Pi , P2, and P3 are each independently selected from:(i) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GFTFEDYA (SEQ ID NO: 19);(b) a CDR-H2 comprising the amino acid sequence of INMSDGST (SEQ ID NO: 20); and(c) a CDR-H3 comprising the amino acid sequence of AANLTGPSGGGLPGYNYDY (SEQ ID NO:21 );(ii) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 1 );(b) a CDR-H2 comprising the amino acid sequence of ISGSGRST (SEQ ID NO: 2); and(c) a CDR-H3 comprising the amino acid sequence of AARFQGSSGIYVYTEAYRD (SEQ ID NO:3);(Hi) a CS3 / CS6 VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of GRAFSNYN (SEQ ID NO: 10);(b) a CDR-H2 comprising the amino acid sequence of ISWSGSIT (SEQ ID NO: 1 1 ); and(c) a CDR-H3 comprising the amino acid sequence of AAYSITLRTNDYRY (SEQ ID NO: 12); and(iv) a CfaE VHH binding domain that comprises the following three CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30); wherein Li and L2are each independently a linker; and ni and n3are each independently 0 or 1 , wherein at least one of ni and n3are 1 .

68. The antibody of claim 67, wherein m is 1 and n3is 0, and the antibody comprises the following N- terminal-to-C-terminal structure:P2-LI-PI .

69. The antibody of claim 68, wherein Pi and P2each comprise different VHH binding domains.

70. The antibody of claim 68, wherein Pi and P2each comprise identical VHH binding domains.71 . The antibody of claim 67, wherein m is 1 and n3is 1 , and the antibody comprises the following N- terminal-to-C-terminal structure:P3-L2-P2-LI-PI .

72. The antibody of claim 71 , wherein Pi , P2, and P3each comprise different VHH binding domains.PATENTAttorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-7773. The antibody of claim 71 , wherein Pi , P2, and P3 each comprise identical VHH binding domains.

74. The antibody of any one of claims 1 -33, wherein the antibody comprises a single VHH binding domain.

75. The antibody of claim 74, wherein the antibody consists of a single VHH binding domain.

76. The antibody of any one of claims 1 -75, wherein the antibody is a monoclonal antibody.

77. The antibody of any one of claims 1 -76, wherein the antibody is a humanized or chimeric antibody.

78. The antibody of any one of claims 1 -77, wherein the antibody is capable of inhibiting mannoseresistant hemagglutination of human group A erythrocytes.

79. The antibody of any one of claims 1 -78, wherein the antibody binds CS3 with a 50% effective concentration (ECso) of between about 1 ng / mL and about 1000 ng / mL.

80. The antibody of claim 79, wherein the antibody binds CS3 with an ECso of between about 1 ng / mL and about 10 ng / mL.81 . The antibody of claim 79, wherein the antibody binds CS3 with an ECso of between about 10 ng / mL and about 100 ng / mL.

82. The antibody of claim 79, wherein the antibody binds CS3 with an ECso of between about 100 ng / mL and about 1000 ng / mL.

83. The antibody of any one of claims 1 -82, wherein the antibody binds CS6 with an ECso of between about 1 ng / mL and about 1000 ng / mL.

84. The antibody of claim 83, wherein the antibody binds CS6 with an ECso of between about 1 ng / mL and about 10 ng / mL.

85. The antibody of claim 83, wherein the antibody binds CS6 with an ECso of between about 10 ng / mL and about 100 ng / mL.

86. The antibody of claim 83, wherein the antibody binds CS6 with an ECso of between about 100 ng / mL and about 1000 ng / mL.

87. The antibody of any one of claims 79-86, wherein the ECso is measured by ELISA.PATENTAttorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-7788. The antibody of any one of claims 1 -87, wherein the antibody is capable of inhibiting the binding of ETEC bacteria to intestinal cells.

89. One or more isolated nucleic acids encoding the antibody of any one of claims 1 -88.

90. One of more vectors comprising the one or more nucleic acids of claim 89.91 . One or more host cells comprising the one or more vectors of claim 90.

92. The one or more host cells of claim 91 , wherein each of the one or more host cells is a eukaryotic cell.

93. The one or more host cells of claim 92, wherein the eukaryotic cell is a mammalian cell.

94. The one or more host cells of claim 93, wherein the mammalian cell is a Chinese hamster ovary(CHO) cell or a human embryonic kidney (HEK) 239 cell.

95. The one or more host cells of claim 92, wherein the eukaryotic cell is a yeast cell.

96. The one or more host cells of claim 92, wherein the eukaryotic cell is a plant cell.

97. The one or more host cells of claim 96, wherein the plant cell is a tobacco plant cell, a soybean plant cell, or a rice plant cell.

98. The one or more host cells of claim 91 , wherein each of the one or more host cells is a prokaryotic cell.

99. The one or more host cells of claim 98, wherein the prokaryotic cell is an E. coli cell.

100. A method of producing a VHH antibody, the method comprising culturing a host cell comprising the one or more isolated nucleic acids of claim 89 in a culture medium.101 . The method of claim 100, wherein the method further comprises recovering the antibody from the host cell or the culture medium.

102. A composition comprising the antibody of any one of claims 1 -88.

103. A composition comprising:(i) the antibody of any one of claims 1 -9; andPATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77(ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

104. A composition comprising:(i) the antibody of any one of claims 12-20; and(ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

105. A composition comprising:(i) the antibody of any one of claims 23-31 ; and(ii) an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

106. A composition comprising the one or more isolated nucleic acids of claim 89.

107. A composition comprising the one or more vectors of claim 90.

108. A pharmaceutical composition comprising:(a) the antibody of any one of claims 1 -88;(b) the one or more nucleic acids of claim 89; or(c) the one or more vectors of claim 90.

109. The pharmaceutical composition of claim 108, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

110. The pharmaceutical composition of claim 108 or 109, wherein the pharmaceutical composition is formulated for treating a disorder associated with an ETEC infection in a subject.

111. The pharmaceutical composition of claim 110, wherein the disorder associated with an ETEC infection is ETEC-related diarrhea.PATENT Attorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77112. The pharmaceutical composition of any one of claims 108-111 , wherein the pharmaceutical composition is formulated for oral administration.

113. The pharmaceutical composition of any one of claims 108-111 , wherein the pharmaceutical composition is formulated for intramuscular administration.

114. A lipid nanoparticle comprising the composition or pharmaceutical composition of any one of claims 102-113.

115. A method of treating a subject having a disorder associated with an ETEC infection comprising administering to the subject an effective amount of the antibody of any one of claims 1 -88, the composition or pharmaceutical composition of any one of claims 102-113, or the lipid nanoparticle of claim 114, thereby treating the subject.

116. A method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising administering to the subject an effective amount of the antibody of any one of claims 1 -88, the composition or pharmaceutical composition of any one of claims 102-113, or the lipid nanoparticle of claim 114, thereby treating the subject.

117. A method of treating a subject having a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of any one of claims 1 -9 and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

118. A method of treating a subject having a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of any one of claims 12-20 and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

119. A method of treating a subject having a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of any one of claims 23-31 and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

120. A method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of any one of claims 1 -9 and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).121 . A method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of any one of claims 12-20 and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

122. A method of treating a subject at risk of developing a disorder associated with an ETEC infection comprising co-administering to the subject an effective amount of the antibody of any one of claims 23-31 and an effective amount of an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

123. The method of any one of claims 117-122, wherein (a) the antibody that binds ETEC CS3 protein and ETEC CS6 protein and (b) the antibody that binds CfAE are formulated together.

124. The method of any one of claims 117-122, wherein (a) the antibody that binds ETEC CS3 protein and ETEC CS6 protein (b) and the antibody that binds CfAE are formulated separately.

125. The method of any one of claims 115-124, wherein the antibody that binds ETEC CS3 protein and ETEC CS6 protein is administered to the subject at a dosage of about 0.1 mg / kg to about 100 mg / kg.

126. The method of claim 125, wherein the antibody that binds ETEC CS3 protein and ETEC CS6 protein is administered to the subject at a dosage of about 1 mg / kg to about 80 mg / kg.PATENTAttorney Docket No.: 50811 -013WO2 UMMS Docket No.: UMMS 24-77127. The method of claim 126, wherein the antibody that binds ETEC CS3 protein and ETEC CS6 protein is administered to the subject at a dosage of about 1 mg / kg to about 40 mg / kg.

128. The method of claim 127, wherein the antibody that binds ETEC CS3 protein and ETEC CS6 protein is administered to the subject at a dosage of about 5 mg / kg to about 20 mg / kg.

129. The method of claim 128, wherein the antibody that binds ETEC CS3 protein and ETEC CS6 protein is administered to the subject at a dosage of about 10 mg / kg.

130. The method of any one of claims 115-129, wherein the subject is administered at least one dose of the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle.131 . The method of claim 130, wherein the subject is administered at least two doses of the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle.

132. The method of any one of claims 115-131 , wherein the disorder associated with an ETEC infection is ETEC-related diarrhea.

133. The method of any one of claims 115-132, wherein the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered orally to the subject.

134. The method of any one of claims 115-132, wherein the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered subcutaneously to the subject.

135. The method of any one of claims 115-132, wherein the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered intramuscularly to the subject.

136. The method of any one of claims 115-135, wherein the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered approximately once a day to the subject.

137. The method of any one of claims 115-135, wherein the antibody, the composition or pharmaceutical composition, or the lipid nanoparticle is administered approximately once a month to the subject.

138. A method of detecting an ETEC in a sample from a subject, the method comprising contacting the sample with the antibody of any one of claims 1 -88 under conditions permissive for binding of the antibody to an ETEC and detecting whether a complex is formed between the antibody and the ETEC.PATENTAttorney Docket No.: 5081 1 -013WO2 UMMS Docket No.: UMMS 24-77139. The method of claim 138, wherein the sample is a swab sample, a lavage sample, a blood sample, a plasma sample, a sputum sample, a urine sample, a stool sample, a sample from the mucosal lining of the small intestine, or a mucosal secretion sample.

140. The method of claim 139, wherein the sample is a sample from the mucosal lining of the small intestine.141 . The method of claim 139, wherein the sample is a stool sample.

142. The method of any one of claims 138-141 , wherein the subject is presumed to have an ETEC infection.

143. The method of any one of claims 138-142, wherein the subject is a mammal.

144. The method of claim 143, wherein the mammal is a human.

145. A kit comprising the antibody of any one of claims 1 -88 and a package insert comprising instructions for using the antibody to treat a subject having or at risk of developing a disorder associated with an ETEC infection.

146. A kit comprising the antibody of any one of claims 1 -48, 74, and 75, and an isolated antibody that binds CfAE, wherein the antibody comprises a CfaE VHH binding domain comprising the following CDRs:(a) a CDR-H1 comprising the amino acid sequence of ERTFSYYV (SEQ ID NO: 28);(b) a CDR-H2 comprising the amino acid sequence of FVAGISMGGDSTK (SEQ ID NO: 29); and(c) a CDR-H3 comprising the amino acid sequence of ADRDIRGNFRS (SEQ ID NO: 30).

147. A kit for detecting ETEC, the kit comprising the antibody of any one of claims 1 -88 and a package insert comprising instructions for using the antibody to detect ETEC.

148. The kit of claim 147, wherein the antibody is conjugated to a label or a tag.

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