WO2023064766A1

WO2023064766A1 - Lateral flow device and uses thereof

Info

Publication number: WO2023064766A1
Application number: PCT/US2022/077904
Authority: WO
Inventors: Tong-Ming Fu; John Wade SHIVER
Original assignee: Igm Biosciences, Inc.
Priority date: 2021-10-11
Filing date: 2022-10-11
Publication date: 2023-04-20

Abstract

This disclosure provides lateral flow devices comprising a sample loading section, a conjugate section, and an analysis section, wherein the conjugate section comprises a multimeric binding molecule that specifically binds to an analyte. Also provided are kits comprising such devices and methods of using and manufacturing such devices and kits.

Description

LATERAL FLOW DEVICE AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/254,500, filed 11 October 2021, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on 11 October 2022, is named 041WOl-Sequence-Listing.xml, and is 390,035 bytes in size.

BACKGROUND

[0003] Lateral flow devices have an extensive history of use in both clinical and home settings, particularly in medical diagnostics. These devices are simple to use and provide the ability to test for a variety of targets, such as proteins, hormones, drugs, and plasma components.

[0004] Typically, lateral flow devices use an immunoassay-based approach where the presence of a target is determined by the binding of the target to a target-specific IgG antibody. The presence of the target is then determined by measuring the presence of a label bound to the target-specific IgG antibody.

[0005] Despite advances in lateral flow devices, there remains a need for improved lateral flow devices having increased sensitivity for targets, and ability to bind to multiple binding sites on one or more targets.

[0006] Multimeric binding molecules, such as IgA and IgM antibodies, have emerged as promising drug candidates because of their improved specificity, improved avidity, and ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134, 9,938,347, 10,570,191, 10,604,559, 10,618,978, 10,787,520, and 10,899,835, and U.S. Patent Application Publication Nos. 2019-0185570, 2019-0330360, 2019-0330374, 2019- 0338041, and 2019-0338040, the contents of which are incorporated herein by reference in their entireties.

SUMMARY

[0007] This disclosure provides a lateral flow device for detecting an analyte in a sample. In certain embodiments, the device includes a lateral flow strip that includes in series: (1) a sample loading section; (2) a conjugate section including a diffusively-bound labeled multimeric binding molecule that specifically binds to the analyte; and (3) an analysis section including in series a test section including an immobilized test binding molecule that includes an antigen-binding domain that specifically binds to the analyte, and a control section including an immobilized control binding molecule that specifically binds to the labeled multimeric binding molecule. In certain embodiments, the labeled multimeric binding molecule includes a label and five or six bivalent binding units, where each binding unit includes two IgM heavy chain constant regions or multimerizing fragments or multimerizing variants thereof, each associated with an antigen-binding domain, and where the IgM heavy chain constant regions each comprise a Cp4 domain, and a p-tail piece (ptp) domain.

[0008] In certain embodiments, the labeled multimeric binding molecule is a hexamer and comprises six bivalent binding units. In certain embodiments, the labeled multimeric binding molecule is a pentamer and comprises five bivalent binding units and a J-chain or a functional fragment thereof or a functional variant thereof.

[0009] In certain embodiments, the J-chain or functional fragment thereof, or functional variant thereof includes the amino acid sequence of SEQ ID NOs: 3-47 or a functional fragment thereof, or a functional variant thereof. In certain embodiments, the J-chain or functional fragment thereof, or functional variant thereof is a modified J-chain further including a heterologous moiety. In certain embodiments, the heterologous moiety is fused or conjugated to the J-chain or functional fragment or functional variant thereof. In certain embodiments, the heterologous moiety includes the label. In certain embodiments, the heterologous moiety includes a histidine (His) tag.

[0010] In certain embodiments, the label includes a colloidal gold nanoparticle, a colloidal silver nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a quantum dot, a magnetic particle, an up converting phosphor, a color dye, a fluorescent dye, or an oligonucleotide. In certain embodiments, the label is attached to latex bead.

[0011] In certain embodiments, each heavy chain constant region or fragment or variant thereof is associated with a copy of a heavy chain variable region (VH).

[0012] In certain embodiments, each binding unit further includes two light chains each including a light chain constant region or fragment or variant thereof. In certain embodiments, the light chain constant regions or fragments or variants thereof are associated with a copy of a light chain variable region (VL).

[0013] In certain embodiments, the IgM heavy chain constant regions or fragments or variants thereof each further include a Cyl domain, a Cy2 domain, a Cy3 domain, or any combination thereof. In certain embodiments, the IgM heavy chain constant regions or multimerizing variant or fragment thereof each further include a Cpl domain, a Cp2 domain, a Cp3 domain, or any combination thereof.

[0014] In certain embodiments, each IgM heavy chain constant region or multimerizing variant or fragment thereof is a human IgM constant region or multimerizing variant or fragment thereof, including the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or a multimerizing variant or fragment thereof. In certain embodiments, each IgM heavy chain constant region or multimerizing variant or fragment thereof is a variant IgM constant region, where the multimeric binding molecule has reduced CDC activity relative to a multimeric binding molecule including IgM heavy chain constant regions including the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the variant human IgM constant region includes an amino acid substitution corresponding to position P311 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 of SEQ ID NO: 1 or SEQ ID NO: 2, or amino acid substitutions corresponding to positions K315 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] In certain embodiments, the control and/or the test binding molecule is a monomeric antibody. In certain embodiments, the control and/or the test binding molecule is an IgG antibody. In certain embodiments, the control and/or the test binding molecule is a multimeric antibody.

[0016] In certain embodiments, the control binding molecule specifically binds to the label, the light chain, the J-chain, or the heavy chain of the labeled multimeric binding molecule, or the labeled multimeric binding molecule includes a tag and the control binding molecule specifically binds to the tag. In certain embodiments, the tag is a His tag attached to the J- chain.

[0017] In certain embodiments, the sample is tissue, saliva, tears, urine, blood, sputum, sweat, vaginal sample, nasal sample, skin sample, semen, feces, mucous, or spinal fluid. In certain embodiments, the sample is a liquid sample.

[0018] In certain embodiments, the analyte is a tumor antigen, a cardiac marker, an infectious pathogen antigen, a hormone, or a therapeutic drug. In certain embodiments, the tumor antigen is p53, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), or prostate specific antigen (PSA). In certain embodiments, the cardiac marker is troponin C, creatine kinase, or myoglobin.

[0019] In certain embodiments, the infectious pathogen is a virus, bacterium, fungi, or parasite. In certain embodiments, the virus is a coronavirus, an influenza virus, Dengue virus, Ebola virus, hepatitis virus, respiratory syncytial virus (RSV), human metapneumovirus, human immunodeficiency virus (HIV), herpes virus, paramyxovirus, or enterovirus. In certain embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or Middle East respiratory syndrome-related coronavirus (MERS-CoV). In certain embodiments, the influenza virus is influenza A virus (IAV), influenza B virus (IBV), or influenza C virus (ICV). In certain embodiments, the hepatitis virus is hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E. In certain embodiments, the herpes virus is herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), roseolovirus, or Kaposi’s sarcoma-associated herpesvirus (KSHV). In certain embodiments, the paramyxovirus is a parainfluenza virus (HPIV). In certain embodiments, the enterovirus is a poliovirus, coxsackie virus A virus, coxsackie virus B, echovirus, or EV71. In certain embodiments, the bacterium is Helicobacter pylori, Streptococcus pyogenes. Mycobacterium tuberculosis, Bacillus anthracis, Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Salmonella, Listeria, or Pseudomonas aeruginosa.

[0020] In certain embodiments, the analyte is a hormone, and the hormone is human chorionic gonadotropin (hCG), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), or follicle-stimulating hormone (FSH). [0021] In certain embodiments, the liquid sample was pretreated to remove an existing component and/or add an additional component.

[0022] In certain embodiments, the sample loading section includes a buffer, non-specific protein, and/or a surfactant. In certain embodiments, the buffer in the sample loading section includes borate buffer. In certain embodiments, the non-specific protein in the sample loading section includes albumin and/or casein. In certain embodiments, the surfactant in the sample loading section includes sodium dodecyl sulfate (SDS) and/or polysorbate.

[0023] In certain embodiments, the conjugate section includes a carbohydrate. In certain embodiments, the carbohydrate is sucrose.

[0024] In certain embodiments, the lateral flow strip further includes an absorbent section after the analysis section. In certain embodiments, the absorbent section includes an absorbent pad including cellulose. In certain embodiments, the absorbent pad is 0.45 mm to 2.50 mm thick.

[0025] In certain embodiments, the analysis section and the absorbent section partially overlap. In certain embodiments, the sample loading section and the conjugate section and/or the conjugate section and the test section partially overlap.

[0026] In certain embodiments, the sample loading section includes a sample pad including cellulose, rayon, and/or glass fiber. In certain embodiments, the sample pad is 0.45 mm to 2.50 mm thick.

[0027] In certain embodiments, the conjugate section includes a conjugate pad including glass fiber, cellulose, rayon, and/or polyester. In certain embodiments, the conjugate pad is 0.45 mm to 2.50 mm thick.

[0028] In certain embodiments, the analysis section includes a porous membrane. In certain embodiments, the porous membrane includes nitrocellulose, nylon, polyvinylidene fluoride, or cellulose acetate. In certain embodiments, the porous membrane includes nitrocellulose. In certain embodiments, the porous membrane is 100 mm to 150 mm thick. In certain embodiments, the porous membrane has a pore size of 0.10 pm to 12 pm. In certain embodiments, the porous membrane has a capillary flow time of 60 seconds to 240 seconds to traverse 4 cm. In certain embodiments, the control and test binding molecules are immobilized on the surface of the porous membrane. [0029] In certain embodiments, the sample loading section, the conjugate section, and the test section include a single monolithic hydrophilic matrix. In certain embodiments, the single monolithic hydrophilic matrix comprises a network of fibers including a mixture of polymer and glass fiber or glass microfiber. In certain embodiments, the control and test binding molecules are immobilized on the surface of the monolithic hydrophilic matrix. In certain embodiments, the control and/or test binding molecules are indirectly immobilized on the surface of the monolithic hydrophilic matrix. In certain embodiments, the control and/or test binding molecules are multimeric binding molecules including a J-chain, and the control and/or test binding molecules are indirectly attached to the surface through an antibody that binds the J-chain or a moiety attached to the J-chain.

[0030] In certain embodiments, the lateral flow strip further includes a non-porous backing layer beneath the sample loading section, the conjugate section, and the analysis section. In certain embodiments, the non-porous backing layer includes polystyrene, polyvinyl chloride, and/or polyester.

[0031] In certain embodiments, the lateral flow device further includes an external casing. In certain embodiments, the external casing does not fully enclose the sample loading section. In certain embodiments, the external casing does not fully enclose the analysis section or is transparent above the test and control sections.

[0032] This disclosure also provides various methods of use and manufacturing of a lateral flow device described herein. In certain embodiments, the disclosure provides a method of detecting an analyte in a liquid sample including (1) applying a liquid sample to the sample loading section of a lateral flow device described herein, (2) allowing the liquid sample to flow through the lateral flow strip of the lateral flow device, and (3) detecting the presence or absence of the label in the test and control sections of the lateral flow device. In certain embodiments, the liquid sample comprises urine, saliva, sputum, whole blood, plasma, serum, or an extract or a dilution thereof. In certain embodiments, prior to step (1), the method includes removing a component from a sample, adjusting the pH of the sample, and/or diluting the sample, thereby generating the liquid sample.

[0033] This disclosure also provides a kit including a lateral flow device described herein. In certain embodiments, the disclosure provides a kit for detecting an analyte in a liquid sample including a lateral flow device described herein, and instructions for use. In certain embodiments, the kit further includes a sample buffer. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0034] FIG. 1 is a diagram illustrating one exemplary embodiment of a lateral flow device provided herein. The flow of a liquid sample containing an analyte through the device is also illustrated in successive panel views.

[0035] FIG. 2 is a diagram illustrating the binding properties of multimeric binding molecules to an analyte in a lateral flow device provided herein, compared to the binding properties of an IgG binding molecule to an analyte. In the right panel of FIG. 2, exemplary multimeric binding molecules binding to an analyte (scalloped circle) are depicted as IgM/IgM-like molecules that contain a label (filled circle). In the left panel of FIG. 2, IgG molecules binding to an analyte (scalloped circle) are depicted that contain a label (filled circle). The association rate (“Kon”) and dissociation rate (“Koff’) of binding and release of the IgG and IgM/IgM to the analyte is depicted in arrows, with arrows in bold having a greater relative association or dissociation rate.

[0036] FIG. 3A-B shows the results of an ELISA measuring binding of IgG and IgM versions of Abl4 (FIG. 3A) and Ab2 (FIG. 3B) monoclonal anti-SARS-CoV-2 antibodies to pseudotyped vesicular stomatitis virus (VSV) decorated with a SARS-CoV-2 spike protein.

[0037] FIG. 4A-B shows the results of an ELISA measuring binding of IgG and IgM versions of Ab 10 anti-SARS-CoV-2 monoclonal antibodies (FIG. 4 A) and chimeric IgG or IgM heavy chain constant regions fused to an angiotensin converting enzyme type 2 ectodomain (FIG. 4B) to pseudotyped VSV decorated with a SARS-CoV-2 spike protein.

[0038] FIG. 5A shows the results of an ELISA measuring binding of an anti-RSV (respiratory syncytial virus) monoclonal antibody (Abl5) to an RSV reporter virus. FIG. 5B shows the results of an ELISA measuring binding of an anti-PIV3 (parainfluenza virus, type 3) monoclonal antibody (Abl6) to a PIV3 reporter virus.

[0039] FIG. 6 schematically depicts the workflow of the ELISA assays depicted in FIGs. 3-5. FIG. 6A shows pseudovirus particles floating above or bound to IgGl molecules on the surface of the ELISA plate. FIG. 6B shows the next step in the workflow, in which labeled IgM antibodies are incubated with the surface-bound pseudovirus particles. FIG. 6C shows the last stage of the workflow, in which unbound pseudovirus particles and unbound IgM antibodies have been washed out, leaving on those pseudovirus particles that are bound to the IgG, and only those labeled IgM antibodies that are bound to the pseudovirus particles.

[0040] FIG. 7A shows an octet sensorgram of binding of the ACE2-h-IgM-J molecule (740 amino acid ACE2 ectodomain joined by the IgGl hinge domain to the IgM constant domain of a pentameric IgM antibody with a J chain) to SARS-CoV-2 Spike protein. FIG. 7B shows an octet sensorgram of binding of the ACE2-IgG molecule (740 amino acid ACE2 ectodomain joined to the IgGl CH2-CH3 domains) to SARS-CoV-2 Spike protein.

DETAILED DESCRIPTION

General Definitions

[0041] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a binding molecule,” is understood to represent one or more binding molecules. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0042] Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0043] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0044] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various embodiments or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0045] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0046] A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxy gen-containing or a nitrogen-containing side chain of an amino acid, e.g., a serine or an asparagine.

[0047] By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0048] As used herein, the term “a non-naturally occurring polypeptide” or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial body, to be “naturally-occurring.”

[0049] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms “fragment,” “variant,” “derivative” and “analog” as disclosed herein include any polypeptides which retain at least some of the properties of the corresponding native antibody or polypeptide, for example, specifically binding to an antigen. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of, e.g., a polypeptide include fragments as described above, and polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants can be non-naturally occurring. Non-naturally occurring variants can be produced using art- known mutagenesis techniques. Variant polypeptides can comprise conservative or nonconservative amino acid substitutions, deletions, or additions. Derivatives are polypeptides that have been altered to exhibit additional features not found on the original polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as “polypeptide analogs.” As used herein a “derivative” of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those peptides that contain one or more derivatives of the twenty standard amino acids. For example, 4-hy dr oxy proline can be substituted for proline; 5 -hydroxy lysine can be substituted for lysine; 3- methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0050] A “conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks etal., Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)). [0051] The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acid sequence” refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0052] By an “isolated” nucleic acid or polynucleotide is intended any form of the nucleic acid or polynucleotide that is separated from its native environment. For example, gel- purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be “isolated.” Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to have restriction sites for cloning is considered to be “isolated.” Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not one that would be found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0053] As used herein, the term “a non-naturally occurring polynucleotide” or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the nucleic acid or polynucleotide that are, or might be, determined or interpreted by a judge, or an administrative or judicial body, to be “naturally-occurring. ”

[0054] As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can include heterologous coding regions, either fused or unfused to another coding region. Heterologous coding regions include without limitation, those encoding specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0055] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

[0056] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron- A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g, promoters inducible by interferons or interleukins).

[0057] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0058] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0059] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse B-glucuronidase.

[0060] As used herein, the term “binding molecule” refers in its broadest sense to a molecule that specifically binds to a binding target, e.g., an epitope or an antigenic determinant. As described further herein, a binding molecule can comprise one of more “antigen-binding domains” described herein. A non-limiting example of a binding molecule is an antibody or antibody -like molecule that retains antigen-specific binding, or an antibody-like molecule or fragment thereof as described in detail herein that retains antigen-specific binding. In certain embodiments a “binding molecule” comprises an antibody or antibody-like molecule as described in detail herein.

[0061] As used herein, the terms “binding domain” or “antigen-binding domain” (can be used interchangeably) refer to a region or fragment of a binding molecule e.g., an antibody or antibody-like molecule, that is necessary and sufficient to specifically bind to a binding target, e.g., an epitope. For example, an “Fv,” e.g, a heavy chain variable region and a light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is considered to be a “binding domain.” Other antigen-binding domains include, without limitation, the heavy chain variable region (VHH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a scaffold, e.g., a fibronectin scaffold.

[0062] A “binding molecule,” e.g, an antibody or antibody -like molecule as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or even more “antigen-binding domains.” As used herein, a “binding unit-associated antigen-binding domain” refers to an antigen-binding domain that is part of an antibody heavy chain and/or an antibody light chain.

[0063] The terms “antibody” and “immunoglobulin” can be used interchangeably herein. An antibody as provided in this disclosure must specifically bind to an antigen, i.e., it includes at least the variable domain of a heavy chain (for camelid species) or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise stated, the term “antibody” encompasses anything ranging from a small antigenbinding fragment of an antibody to a full sized antibody, e.g. , an IgG antibody that includes two IgG heavy chains or fragments thereof and two complete light chains, an IgA antibody that includes two, four, or eight IgA heavy chains or multimerizing fragments thereof and two, four, or eight light chains and optionally includes a J-chain or functional fragment or variant thereof and/or a secretory component, or an IgM antibody that includes ten or twelve IgM heavy chains or multimerizing fragments thereof and ten or twelve light chains and optionally includes a J-chain or functional fragment or variant thereof.

[0064] The term “immunoglobulin” comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as, e.g., gamma, mu, alpha, delta, or epsilon, ( , p, a, 8, s) with some subclasses among them (e.g, yl-y4 or al-a2). It is the nature of this chain that determines the “isotype” of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. The immunoglobulin subclasses (subtypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.

[0065] Light chains are classified as either kappa or lambda (K, X). Each heavy chain class can be associated with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other via disulfide bonds, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non- covalent linkages when the immunoglobulins are expressed, e.g, by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of certain antibodies, e.g, IgG antibodies, includes two heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a “Y” structure, also referred to herein as an “H2L2” structure, or a “binding unit.” [0066] The term “binding unit” is used herein to refer to the portion of a binding molecule, e.g., an antibody or antibody-like molecule that corresponds to a standard “H2L2” immunoglobulin structure, e.g. , two heavy chains or fragments thereof and two light chains or fragments thereof. In certain embodiments a binding unit can correspond to two heavy chains, e.g, in a camelid antibody. In certain embodiments, e.g, where the binding molecule is a bivalent IgG antibody or antibody-like molecule the terms “binding molecule” and “binding unit” are equivalent. In other embodiments, e.g., where the binding molecule is multimeric, e.g., a dimeric or tetrameric IgA antibody or IgA-like antibody, a pentameric IgM antibody or IgM-like antibody, or a hexameric IgM antibody or IgM-like antibody, the binding molecule comprises two or more “binding units.” Two or four in the case of an IgA dimer or tetramer, or five or six in the case of an IgM pentamer or hexamer, respectively. A binding unit need not include full-length antibody heavy and light chains, but will typically be bivalent, i.e., will include two “antigen-binding domains,” as defined above. As used herein, certain binding molecules provided in this disclosure are “dimeric” or “tetrameric,” and include two or four bivalent binding units that include IgA heavy chain constant regions or multimerizing fragments thereof. Certain binding molecules provided in this disclosure are “pentameric” or “hexameric,” and include five or six bivalent binding units that include IgM heavy chain constant regions or multimerizing fragments thereof. A binding molecule, e.g., an antibody or antibody-like molecule comprising two or more, e.g., two, four, five, or six binding units, is referred to herein as “multimeric.”

[0067] The term “J-chain” as used herein refers to the J-chain associated with pentameric IgM or dimeric or tetrameric IgA antibodies of any animal species, any functional fragment thereof, derivative thereof, and/or variant thereof, including the mature human J- chain, the amino acid sequence of which is presented as SEQ ID NO: 3. Various J-chain variants and modified J-chain derivatives are disclosed herein. As persons of ordinary skill in the art will recognize, “a functional fragment” or a “functional variant” includes those fragments and variants that can associate with IgM heavy chain constant regions to form a pentameric IgM antibody (or alternatively can associate with IgA heavy chain constant regions to form a dimeric tetrameric IgA antibody).

[0068] The term “modified J-chain” is used herein to refer to a derivative of a J-chain polypeptide comprising a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain introduced into the J-chain polypeptide. The introduction can be achieved by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety or by attachment through a peptide or chemical linker. The term “modified human J-chain” encompasses, without limitation, a human J-chain comprising the amino acid sequence of SEQ ID NO: 3 or functional fragment thereof, or functional variant thereof, modified by the introduction of a heterologous moiety, e.g, a heterologous polypeptide, e.g, an extraneous binding domain. In certain embodiments the heterologous moiety does not interfere with efficient polymerization of IgM into a pentamer or IgA into a dimer or tetramer, and binding of such polymers to a target. Exemplary modified J-chains can be found, e.g, in U.S. Patent Nos. 9,951,134, 10,975,147, 10,400,038, and 10,618,978, and in U.S. Patent Application Publication No. US-2019-0185570, each of which is incorporated herein by reference in its entirety.

[0069] As used herein, the terms “IgM-derived binding molecule,” “IgM-like antibody,” “IgM-like binding unit,” or “IgM-like heavy chain constant region” refer to a variant antibody-derived binding molecule, antibody, binding unit, or heavy chain constant region that still retains the structural portions of an IgM heavy chain necessary to confer the ability to form multimers, i.e., hexamers, or in association with J-chain, form pentamers. An IgM- like antibody or IgM-derived binding molecule typically includes at least the Cp4 and tailpiece (tp) domains of the IgM constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. An IgM-like antibody or IgM-derived binding molecule can likewise be an antibody fragment in which one or more constant regions are deleted, as long as the IgM-like antibody is capable of forming hexamers and/or pentamers. Thus, an IgM-like antibody or IgM-derived binding molecule can be, e.g., a hybrid IgM/IgG antibody or can be a “multimerizing fragment” of an IgM antibody.

[0070] As used herein, the terms “IgA-derived binding molecule,” “IgA-like antibody,” “IgA-like binding unit,” or “IgA-like heavy chain constant region” refer to a variant antibody-derived binding molecule, antibody, binding unit, or heavy chain constant region that still retains the structural portions of an IgA heavy chain necessary to confer the ability to form multimers, i.e., dimers, in association with J-chain. An IgA-like antibody or IgA- derived binding molecule typically includes at least the Ca3 and tailpiece (tp) domains of the IgA constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. An IgA- like antibody or IgA-derived binding molecule can likewise be an antibody fragment in which one or more constant regions are deleted, as long as the IgA-like antibody is capable of forming dimers in association with a J-chain. Thus, an IgA-like antibody or IgA-derived binding molecule can be, e.g., a hybrid IgA/IgG antibody or can be a “multimerizing fragment” of an IgA antibody.

[0071] The terms “valency,” “monovalent,” “bivalent,” “multivalent” and grammatical equivalents, refer to the number of antigen-binding domains in given binding molecule, e.g., an antibody orantibody-like molecule or in a given binding unit. As such, the terms “bivalent,” “tetravalent,” and “hexavalent” in reference to a given binding molecule, e.g, an IgM antibody, IgM-like antibody or multimerizing fragment thereof, denote the presence of two antigen-binding domains, four antigen-binding domains, and six antigenbinding domains, respectively. A typical IgM antibody or IgM-like antibody or IgM - derived binding molecule where each binding unit is bivalent can have 10 or 12 valencies. A bivalent or multivalent binding molecule, e.g, antibody or antibody-like molecule, can be monospecific, i.e., all of the antigen-binding domains are the same, or can be bispecific or multispecific, e.g., where two or more antigen-binding domains are different, e.g., bind to different epitopes on the same antigen, or bind to entirely different antigens.

[0072] The term “epitope” includes any molecular determinant capable of specific binding to an antigen-binding domain of an antibody or antibody-like molecule. In certain embodiments, an epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, can have three-dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of a target that is bound by an antigen-binding domain of an antibody.

[0073] The term “target” is used in the broadest sense to include substances that can be bound by a binding molecule, e.g., an antibody or antibody -like molecule. A target can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule. Moreover, a “target” can, for example, be a cell, an organ, or an organism that comprises an epitope that can be bound by a binding molecule, e.g., an antibody or antibody-like molecule.

[0074] Both the light and heavy chains of an antibody or antibody-like molecule are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally, but refer to particular structures of the molecule. The variable regions of both the light (VL) and heavy (VH) chains determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain ( .g., CHI, CH2, CH3, or CH4) confer biological properties such as the ability to multimerize, secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen-binding regions or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 (or CH4 in the case of IgM) and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0075] A “full length IgM antibody heavy chain” is a polypeptide that includes, in N- terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM1 or Cpl), an antibody heavy chain constant domain 2 (CM2 or Cp2), an antibody heavy chain constant domain 3 (CM3 or Cp3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a tailpiece.

[0076] As indicated above, variable region(s) form the antigen-binding domain of the antibody or antibody-like molecule, allowing it to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or an antigen-binding subset of the complementarity determining regions (CDRs), of a binding molecule, e.g., an antibody or antibody-like molecule combine to form the antigen-binding domain. More specifically, an antigen-binding domain can be defined by three CDRs on each of the VH and VL chains. Certain antibodies or antibody-like molecules form larger structures. For example, IgA heavy chains can form a molecule that includes two or four H2L2 binding units and a J-chain covalently connected via disulfide bonds, which can be further associated with a secretory component, and IgM heavy chains can form a pentameric or hexameric molecule that includes five or six H2L2 binding units and optionally a J-chain covalently connected via disulfide bonds.

[0077] The six “complementarity determining regions” or “CDRs” present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, referred to as “framework” regions, show less inter- molecular variability. The framework regions largely adopt a |3-sheet conformation and the CDRs form loops that connect, and in some cases form part of, the |3-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the target antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways (see, e.g, “Sequences of Proteins of Immunological Interest,” Kabat, E., etal., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 796:901-917 (1987), which are incorporated herein by reference in their entireties).

[0078] In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described, for example, by Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 796:901-917 (1987), which are incorporated herein by reference. The Kabat and Chothia definitions include overlapping or subsets of amino acids when compared against each other. Nevertheless, application of either definition (or other definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated. The appropriate amino acids which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact amino acid numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids comprise a particular CDR given the variable region amino acid sequence of the antibody. Table 1: CDR Definitions*

*Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below).

[0079] Antibody variable domains can also be analyzed, e.g., using the IMGT information system (imgt_dot_cines_dot_fr/) (IMGT®/V -Quest) to identify variable region segments, including CDRs. See, e.g., Brochet et al., Nucl. Acids Res., 36:W503-508, 2008).

[0080] Kabat et al. also defined a numbering system for variable region and constant region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure.

[0081] The Kabat numbering system for the human IgM constant domain can be found in Kabat, et al. “Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, [3-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, Post-gamma Globulin, a-2 Macroglobulins, and Other Related Proteins,” U.S. Dept, of Health and Human Services (1991). IgM constant regions can be numbered sequentially (i.e., amino acid #1 starting with the first amino acid of the constant region, or by using the Kabat numbering scheme. A comparison of the numbering of two alleles of the human IgM constant region sequentially (presented herein as SEQ ID NO: 1 (allele IGHM*03) and SEQ ID NO: 2 (allele IGHM*04)) and by the Kabat system is set out below. The underlined amino acid residues are not accounted for in the Kabat system (“X,” double underlined below, can be serine (S) (SEQ ID NO: 1) or glycine (G) (SEQ ID NO: 2)): Sequential (SEQ ID NO: 1 or SEQ ID NO: 2)/KABAT numbering key for IgM heavy chain

1 /127 GSASAPTLFP LVSCENSPSD TSSVAVGCLA QDFLPDSITF SWKYKNNSDI 51 /176 SSTRGFPSVL RGGKYAATSQ VLLPSKDVMQ GTDEHWCKV QHPNGNKEKN 101 /226 VPLPVIAELP PKVSVFVPPR DGFFGNPRKS KLICQATGFS PRQIQVSWLR 151 /274 EGKQVGSGVT TDQVQAEAKE SGPTTYKVTS TLTIKESDWL XQSMFTCRVD 201 /324 HRGLTFQQNA SSMCVPDQDT AIRVFAIPPS FASIFLTKST KLTCLVTDLT 251 /374 TYDSVTISWT RQNGEAVKTH TNISESHPNA TFSAVGEASI CEDDWNSGER 301 /424 FTCTVTHTDL PSPLKQTISR PKGVALHRPD VYLLPPAREQ LNLRESATIT 351 /474 CLVTGFSPAD VFVQWMQRGQ PLSPEKYVTS APMPEPQAPG RYFAHSILTV 401 /524 SEEEWNTGET YTCWAHEAL PNRVTERTVD KSTGKPTLYN VSLVMSDTAG 451 /574 TCY

[0082] Binding molecules, e.g., antibodies, antibody-like molecules, antigen-binding fragments, variants, or derivatives thereof, and/or multimerizing fragments thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab’ and F(ab’)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are described, e.g., in US patent 5,892,019.

[0083] By “specifically binds,” it is generally meant that a binding molecule, e.g., an antibody or antibody-like molecule binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule, e.g. , an antibody or antibodylike molecule is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain epitope. For example, binding molecule “A” can be deemed to have a higher specificity for a given epitope than binding molecule “B,” or binding molecule “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”

[0084] A binding molecule, e.g, an antibody or fragment, variant, or derivative thereof disclosed herein can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5 X 10'² sec , 10'² sec⁴, 5 X IO'³ sec⁴, IO'³ sec⁴, 5 X IO sec⁴, IO sec⁴, 5 X IO'⁵ sec⁴, IO'⁵ sec⁴, 5 X 10'⁶ sec⁴, 10'⁶ sec⁴, 5 X 10'⁷ sec⁴, or 10'⁷ sec⁴.

[0085] A binding molecule, e.g, an antibody or antibody-like molecule disclosed herein can be said to bind a target antigen with an on rate (k(on)) of greater than or equal to 10³ M sec⁴, 5 X IO³ M sec⁴, 10⁴ M sec⁴, 5 X 10⁴ M sec⁴, IO⁵ M sec⁴, 5 X IO⁵ M sec’ 10⁶ M sec⁴, 5 X 10⁶ M sec⁴ or 10⁷ M sec⁴.

[0086] A binding molecule, e.g., an antibody or antibody-like molecule is said to competitively inhibit binding of a reference antibody or antibody-like molecule to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen-binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays or OCTET assays. A binding molecule can be said to competitively inhibit binding of the reference antibody or antibody -like molecule to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0087] As used herein, the term “affinity” refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, e.g., of an antibody or antibody-like molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refers to the overall stability of the complex between a population of antigenbinding domains and an antigen. See, e.g., Harlow at pages 29-34. Avidity is related to both the affinity of individual antigen-binding domains in the population with specific epitopes, and the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. Likewise, the interaction between a multimeric antibody with four, eight, ten, or twelve valencies and a population of specific epitopes would be one of high avidity. An interaction between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.

[0088] Binding molecules, e.g., antibodies or fragments, variants, or derivatives thereof as disclosed herein can also be described or specified in terms of their cross-reactivity. As used herein, the term “cross-reactivity” refers to the ability of a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, a binding molecule is cross reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.

[0089] A binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof can also be described or specified in terms of their binding affinity to an antigen. For example, a binding molecule can bind to an antigen with a dissociation constant or KD no greater than 5 x IO’² M, IO’² M, 5 x 10’³ M, 10’³ M, 5 x IO’⁴ M, IO’⁴ M, 5 x 10’⁵ M, 10’⁵ M, 5 x 10’ 6 M, IO’⁶ M, 5 x IO’⁷ M, IO’⁷ M, 5 x 10’⁸ M, 10’⁸ M, 5 x 10’⁹ M, IO’⁹ M, 5 x IO’¹⁰ M, IO’¹⁰ M, 5 x IO ¹ M, IO ¹ M, 5 x 10⁴² M, 10⁴² M, 5 x 10⁴³ M, 10⁴³ M, 5 x 10⁴⁴ M, 10⁴⁴ M, 5 x 10⁴⁵ M, or 10⁴⁵ M.

[0090] Antigen-binding fragments of a binding molecule or antibody as provided herein, including single-chain antibodies or other antigen-binding domains that can exist alone or in combination with one or more of the following: hinge region, CHI, CH2, CH3, or CH4 domains, J-chain, or secretory component. Also included are antigen-binding fragments that can include any combination of variable region(s) sufficient to bind antigen with one or more of a hinge region, CHI, CH2, CH3, or CH4 domains, a J-chain, or a secretory component. Binding molecules, e.g, antibodies or antibody-like molecules can be from any animal origin including birds and mammals. The antibodies can be, e.g., human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from sharks). As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and can in some instances express endogenous immunoglobulins and some not, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. According to embodiments of the present disclosure, an IgM or IgM-like antibody or IgM-derived binding molecule as provided herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgM or IgM-like antibody is able to form a multimer, e.g., a hexamer or a pentamer. [0091] As used herein, the term “heavy chain subunit” includes amino acid sequences derived from an immunoglobulin heavy chain. A binding molecule, e.g., an antibody or antibody-like molecule comprising a heavy chain subunit can include a VH domain and one or more of a CHI domain, a hinge (e.g, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, a p tail-piece (ptp), or a variant or fragment thereof. For example, a binding molecule, e.g., an antibody, antibody-like molecule, or fragment, variant, or derivative thereof can include without limitation, in addition to a VH domain, any combination of a CHI domain, a hinge, a CH2 domain; a CH3 domain; a CH4 domain; or a p tailpiece (ptp) of one or more antibody isotypes and/or species. In certain embodiments, a binding molecule, e.g, an antibody, antibody-like molecule, or fragment, variant, or derivative thereof can include, in addition to a VH domain, one or more of a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, a p-tailpiece (ptp) domain and a J-chain (in the case of IgM), or one or more of a CHI domain, a hinge region, a CH2 domain, a CH3 domain, an a-tailpiece (dtp) domain, and a J-chain (in the case of IgA) . Further, a binding molecule, e.g., antibody or antibody-like molecule provided in the disclosure can lack certain constant region portions, e.g, all or part of a CHI domain, a hinge, a CH2 domain, or a CH3 domain. These domains (e.g. , the heavy chain subunit) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule. According to embodiments of the present disclosure, an IgM or IgM-like antibody as provided herein includes sufficient portions of an IgM heavy chain constant region to allow the IgM or IgM-like antibody to form a multimer, e.g, a hexamer or a pentamer, e.g, the IgM heavy chain constant region includes a “multimerizing fragment” of an IgM heavy chain constant region.

[0092] As used herein, the term “light chain subunit” includes amino acid sequences derived from an immunoglobulin light chain. The light chain subunit includes at least a VL, and can further include a CL (e.g, CK or CL) domain.

[0093] Binding molecules, e.g., antibodies, antibody-like molecules, antigen-binding fragments, variants, or derivatives thereof, or multimerizing fragments thereof can be described or specified in terms of the epitope(s) or portion(s) of an antigen that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an “epitope,” or an “antigenic determinant.” A target antigen can comprise a single epitope or two or more epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.

[0094] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain in IgG, IgA, and IgD heavy chains. This hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0095] As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0096] As used herein, the term “chimeric antibody” refers to an antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial, or modified) is obtained from a second species. In some embodiments the target binding region or site will be from a non-human source (e.g, mouse or primate) and the constant region is human.

[0097] The terms “multispecific antibody” or “bispecific antibody” refer to an antibody or antibody-like molecule that has antigen-binding domains for two or more different epitopes within a single antibody molecule. Other binding molecules in addition to the canonical antibody structure can be constructed with two binding specificities.

[0098] As used herein, the term “engineered antibody” refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more amino acids in either the CDR or framework regions. In certain embodiments, entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody. Although alternate CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, CDRs can also be derived from an antibody of different class, e.g, from an antibody from a different species. An engineered antibody in which one or more “donor” CDRs from a non-human antibody of known specificity are grafted into a human heavy or light chain framework region is referred to herein as a “humanized antibody.” In certain embodiments, not all the CDRs are replaced with the complete CDRs from the donor variable region and yet the antigen-binding capacity of the donor can still be transferred to the recipient variable domains. Exemplary methods of humanization are described in U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370. [0099] As used herein the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g, by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).

[0100] As used herein, the terms “linked,” “fused” or “fusion” or other grammatical equivalents can be used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the “fused” CDRs are co-translated as part of a continuous polypeptide. The term “associated” and grammatical equivalents refers to the interaction of two or more elements function together and that can be linked or fused, but can also be in proximity, e.g., interacting in trans without being connected in any particular way.

[0101] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is “amino-terminal” or “N-terminal” to another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain. Similarly, a portion of a polypeptide that is “carboxy -terminal” or “C- terminal” to another portion of a polypeptide is that portion that comes later in the sequential polypeptide chain. For example, in a typical antibody, the variable domain is “N-terminal” to the constant region, and the constant region is “C-terminal” to the variable domain. [0102] The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g, a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g, polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0103] As used herein the terms “serum half-life” or “plasma half-life” refer to the time it takes (e.g., in minutes, hours, or days) following administration for the serum or plasma concentration of a protein or a drug, e.g, a binding molecule such as an antibody or antibody-like molecule as described herein, to be reduced by 50%. Two half-lives can be described: the alpha half-life, a half-life, or ti/2O, which is the rate of decline in plasma concentrations due to the process of drug redistribution from the central compartment, e.g. , the blood in the case of intravenous delivery, to a peripheral compartment (e.g., a tissue or organ), and the beta half-life, half-life, or ti/2[3 which is the rate of decline due to the processes of excretion or metabolism.

[0104] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.

[0105] As used herein, “capillary action” refers to the flow of a liquid through a material by intermolecular forces between the liquid and the surrounding material and without the assistance of external forces like gravity.

Lateral Flow Devices

[0106] Provided herein are lateral flow devices for detecting one or more analytes. [0107] The term “lateral flow” refers to liquid flow along the plane of a substrate or carrier of a lateral flow device. A lateral flow device is also referred to as a “lateral flow test,” “lateral flow assay” or “lateral flow immunoassay.”

[0108] An exemplary lateral flow device is shown in FIG. 1. The lateral flow device 100 can include a sample loading section 102, a conjugate section 104, and an analysis section 106. The analysis section can include a test section 108 and/or a control section 110. The lateral flow device can further comprise an absorbent section 112. As used herein, a “lateral flow strip” contains a sample loading section 102, a conjugate section 104, an analysis section 106, and optionally, an absorbent section 112, in series. In addition, a lateral flow device can further comprise a non-porous backing layer 122 beneath the lateral flow strip.

[0109] A lateral flow device provided herein can be used to detect the presence or absence of an analyte in a sample. FIG. 1 shows an exemplary embodiment utilizing a liquid sample 114 that contains an analyte 124. After application of the sample to the sample loading section 102, the sample flows by capillary action from the sample loading section 102 to the conjugate section 104. This flow is illustrated in the middle panel of FIG. 1. The conjugate section 104 contains diffusively-bound labeled multimeric binding molecules 116 that specifically bind to the analyte 124 and that comprise a label 126. The diffusively- bound labeled multimeric binding molecules 116 are released from the conjugate section 104 and specifically bind to the analyte 124. The labeled multimeric binding molecules bound to analyte then flow to the analysis section 106, as depicted in the middle panel of FIG. 1.

[0110] FIG. 1 depicts an exemplary embodiment where the analysis section 106 contains a test section 108 and control section 110. The test section 108 contains immobilized test binding molecules 118 that comprise an antigen-binding domain that specifically binds to the analyte 124. When the labeled multimeric binding molecules bound to analyte flow through the test section, they specifically bind to the immobilized test binding molecules, as depicted in the lower panel of FIG. 1. The control section 110 contains immobilized control binding molecules that specifically bind to the labeled multimeric binding molecule 120. When the labeled multimeric binding molecules bound to analyte flow through the control section, they specifically bind to the immobilized control binding molecules, as depicted in the lower panel of FIG. 1. [oni] From there, the presence or amount of analyte 124 can be determined by measuring the presence or activity of the label 126 on the multimeric binding molecules in the test and/or control sections.

Analytes and Samples

[0112] As used herein, the term “analyte” refers to any substance that can be identified or measured, directly or indirectly, using a multimeric binding molecule described herein. Examples of an analyte include, but are not limited to, a protein, nucleic acid, polysaccharide, lipid, antigen, marker, hormone, growth factor, therapeutic drug, hapten, pesticide and the like, a portion thereof, or a combination thereof.

[0113] In some embodiments, the antigen is a tumor antigen. Examples of tumor antigens include, but are not limited to, p53, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and prostate specific antigen (PSA). Additional examples of tumor antigens are described, for example, in Urban et al., Annu. Rev. Immunol. 1992. 10:617-644; Valilou et al., Vaccines Cancer Immunother. Chapter 4 - Tumor Antigens. 2019. 61-74; and Haen et al., Nat. Rev. Clin. Oncol. 2020. 17:595-610.

[0114] In some embodiments, the antigen is an infectious pathogen antigen or variant thereof. Examples of infectious pathogen antigens include, but are not limited to, a virus, bacterium, fungi, and parasite. In some embodiments, the virus is a coronavirus, an influenza virus, Dengue virus, Ebola virus, hepatitis virus, respiratory syncytial virus (RSV), human metapneumovirus, human immunodeficiency virus (HIV), herpes virus, paramyxovirus, or enterovirus. Additional examples of viruses are described, for example, in Woolhouse et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2012. 367(1604):2864- 2871; and Strauss et al., “Viruses and Human Disease,” Chapter 1, pp. 1-33 (2012).

[0115] In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), or Middle East respiratory syndrome-related coronavirus (MERS-CoV). See also, e.g., Cui et al., Nat. Rev. Microbiol. 17:181-192 (2019); and House et al., Microbiol. Insights. 14:1-8 (2021).

[0116] In some embodiments, the influenza virus is influenza A virus (I AV), influenza B virus (IBV), or influenza C virus (ICV). See also, e.g., Javanian et al., J. Med. Virol. 93(8):4638-4646 (2021); and Moghadami, Iran J. Med. Sci. 42(1):2-13 (2017). [0117] In some embodiments, the hepatitis virus is hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E. See also, e.g., Levinson et al., “Review of Medical Microbiology and Immunology,” Chapter 41 - Hepatitis Viruses (2020).

[0118] In some embodiments, the HIV is HIV type 1 (HIV-1) or HIV type 2 (HIV -2). See also, e.g., Seitz et al., Transfus. Med. Hemother. 43(3):203-222 (2016); and Deeks et al., Nat. Rev. Dis. Primers. 1:15035 (2015).

[0119] In some embodiments, the herpes virus is herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein- Barr virus (EBV), roseolovirus, or Kaposi’s sarcoma-associated herpesvirus (KSHV). See also, e.g., Whitley et al., Lancet. 357(9267):1513-1518 (2001); and Sehrawat et al., Frontiers Cell. Infect. Microbiol. 8:177 (2018).

[0120] In some embodiments, the paramyxovirus is a parainfluenza virus (HPIV). In some embodiments, the HPIV is HPIV type 1 (HPIV-1), HPIV type 2 (HPIV-2), HPIV type 3 (HPIV-3), or HPIV type 4 (HPIV-4). See also, e.g., Branche et al., Semin. Respir. Crit. Care Med. 37(4):538-554 (2016); and Henrickson, Clin. Microbiol. Rev. 16(2):242-264 (2020).

[0121] In some embodiments, the enterovirus is a poliovirus, coxsackie virus A virus, coxsackie virus B, echovirus, or EV71. See also, e.g., Lugo et al., Curr. Opin. Pediatri. 28(l):107-l 13 (2016); and Noor et al., Pediatr. Rev. 37(12):505-515 (2016).

[0122] In some embodiments, the bacterium is Helicobacter pylori, Streptococcus pyogenes, Mycobacterium tuberculosis, Bacillus anthracis, Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Salmonella species, Listeria species, or Pseudomonas aeruginosa. See also, e.g., Schulz et al., Ann. Rev. Microbiol. 55:105-137 (2001); and Venkova et al., Front. Microbiol. 9:1702 (2018).

[0123] In some embodiments, the analyte is a variant infectious pathogen antigen, such as a variant coronavirus antigen, variant influenza virus antigen, or variant respiratory syncytial virus (RSV) antigen. For example, in some embodiments, the variant SARS- CoV-2 antigen comprises one or more substitutions found in alpha, beta, gamma, delta, eta, iota, kappa, lambda, 1.617.3, mu, theta, or zeta variant.

[0124] In some embodiments, the marker is a cardiac marker. Examples of cardiac markers include, but are not limited to, troponin C, creatine kinase, and myoglobin. [0125] In some embodiments, the hormone is human chorionic gonadotropin (hCG), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), or follicle-stimulating hormone (FSH). See also, e.g., Hiller-Sturmhofel, Alcohol Health Res. World. 22(3): 153- 164 (1998); and Hull et al., Int. J. Endocrinol. Article ID 234014, 24 pages (2014).

[0126] A multimeric binding molecule can bind to a multivalent antigen through more than one epitope. The spatial distance between multiple epitopes can influence the binding properties of the multimeric binding molecule to the antigen. Accordingly, in some embodiments, an analyte comprises two, three, or more epitopes that have the same or similar spacing between the epitopes on the analyte, and a multimeric binding molecule disclosed herein specifically binds to two, three or more of the epitopes. Examples of such an analyte include, but are not limited to, a polysaccharide, a viral particle, or a trimeric viral antigen.

[0127] In some embodiments, the analyte is present in a sample. In some embodiments, the sample is a biological sample, including, but not limited to, tissue, saliva, tears, urine, blood, sputum, sweat, vaginal sample, nasal sample, skin sample, semen, feces, mucous, breast milk, ascites, lymph, pleural effusion, synovial fluid, bone marrow, spinal fluid, or washings from bodily cavities (e.g., lung washings).

[0128] In some embodiments, the tissue is homogenized tissue or a biopsy sample.

[0129] In some embodiments, the blood is whole blood, serum, plasma or dried blood.

[0130] In some embodiments, the vaginal sample is vaginal fluid or a vaginal swab.

[0131] In some embodiments, the nasal sample is nasal fluid or a nasal swab.

[0132] In some embodiments, the skin sample is a skin scraping or skin swab.

[0133] In some embodiments, the sample is a liquid sample. A liquid sample includes a sample naturally in liquid form (e.g., urine), or a sample made into liquid form by dissolving, dispersing, or suspending a solid or semi-solid sample in a solvent. A liquid sample also includes a sample naturally in liquid form that has been diluted in a solvent or a sample naturally in liquid form that has been concentrated but remains in a liquid form or was dried and resuspended. Examples of solvents include, but are not limited to, water, ethanol, methanol, acetone or a combination thereof.

[0134] In some embodiments, the sample contains a preservative. Examples of preservatives include, but are not limited to, benzoic acid, calcium sorbate, erythorbic acid, potassium nitrate, sodium benzoate, citric acid, or a combination thereof. [0135] Methods for collecting, preparing and storing samples for lateral flow assay are described, for example, in Vaught et al., IARC Sci. Publ. 2011;163:23-42; Lygirou et al., Methods Mol. Biol. 2015; 1243:3-27; and Holland et al., Mutat. Res. 2003;543(3):217- 234. Such methods include, but are not limited to, protein precipitation (optionally followed by centrifugation or another precipitation separation step), liquid-liquid extraction, solid-phase extraction, microextraction, and addition of sorbents (e.g., MIP stable synthetic polymers, immunoaffinity sorbents, aptamers). Accordingly, in other embodiments, a sample (e.g., liquid sample) can be pretreated to remove an existing component and/or add an additional component.

Sample Loading Section

[0136] Lateral flow devices provided herein contain, in general, a sample loading section, a conjugate section, and an analysis section.

[0137] As used herein, the term “sample loading section” refers to the region of a lateral flow device where a sample is introduced to the device. A sample loading section can have any shape or size, and a lateral flow device disclosed herein can have one or more sample loading sections.

[0138] In some embodiments, the sample loading section contains a sample pad that is composed of cellulose (e.g., a cellulose fiber filter), rayon, woven materials (e.g., glass fiber, polyester), or a combination thereof.

[0139] In some embodiments, the sample pad is from about 0.45 mm to about 2.5 mm thick, or any values or range of values thereof, e.g., from about 0.5 mm to about 2.5 mm, from about 1 mm to about 2.5 mm, from about 1.5 mm to about 2.5 mm, from about 2 mm to about 2.5 mm, from about 0.45 mm to about 2 mm, from about 0.5 mm to about 2 mm, from about 1 mm to about 2 mm, from about 1.5 mm to about 2 mm, from about 0.45 mm to about 1.5 mm, from about 0.5 mm to about 1.5 mm, from about 1 mm to about 1.5 mm, from about 0.45 mm to about 1 mm, from about 0.5 mm to about 1 mm, or from about 0.45 mm to about 0.5 mm. In some embodiments, the sample pad is about 0.45 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, or about 2.5 mm thick.

[0140] In some embodiments, the sample loading section comprises a buffer, a non-specific protein, a surfactant, or a combination thereof. [0141] In some embodiments, the buffer is phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane (“Tris,” for example, Tris-acetate, Tris-HCl), borate buffer, or a combination thereof.

[0142] In some embodiments, the non-specific protein is albumin, casein, bovine serum albumin (BSA), milk (e.g., skim milk), or a combination thereof.

[0143] In some embodiments, the surfactant comprises sodium dodecyl sulfate (SDS), polysorbate, Tween (ethoxylated or polyoxyethylene derivatives of sorbitan ester), Triton X-100, or a combination thereof.

[0144] Additional exemplary sample loading mixtures for lateral flow are described, for example, in Vaught et al., I ARC Sci. Publ. 2011;163:23-42; Lygirou etal., Methods Mol. Biol. 2015; 1243:3-27; and Holland et al., Mutat. Res. 2003;543(3):217-234.

Multimeric Binding Molecules and Labels

[0145] In some embodiments, a lateral flow device provided herein comprises a multimeric binding molecule. A multimeric binding molecule comprises two, four, five or six bivalent binding units or variants or fragments thereof.

[0146] In some embodiments, each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or multimerizing variants thereof.

[0147] An IgM heavy chain constant region can include one or more of a Cpl domain or fragment or variant thereof, a Cp2 domain or fragment or variant thereof, a Cp3 domain or fragment or variant thereof, a Cp4 domain or fragment or variant thereof, and/or a C- terminal p-tail piece (ptp) domain or fragment or variant thereof, provided that the constant region can serve a desired function, e.g, associate with a second IgM constant region to form a binding unit with one, two, or more antigen-binding domain(s), and/or associate with other binding units (and in the case of a pentamer, a J-chain) to form a hexamer or a pentamer. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a Cp4 domain or fragment or variant thereof, a ptp domain or fragment or variant thereof, or a combination of a Cp4 domain and a ptp domain or fragment or variant thereof. In certain embodiments, the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each further comprise a Cp3 domain or fragment or variant thereof, a Cp2 domain or fragment or variant thereof, a Cpl domain or fragment or variant thereof, or any combination thereof.

[0148] In some embodiments, the multimeric binding molecule is a hexamer and comprises six bivalent binding units.

[0149] In some embodiments, the multimeric binding molecule is a pentamer and comprises five bivalent binding units. In some embodiments, the multimeric binding molecule is a pentamer and comprises five bivalent binding units and a J-chain or a functional fragment thereof or a functional variant thereof. In some embodiments, the J-chain or functional fragment thereof, or functional variant thereof comprises the amino acid sequence of SEQ ID NOs: 3-47. or a functional fragment thereof, or a functional variant thereof.

[0150] In some embodiments, each heavy chain constant region or fragment or variant thereof is associated with a copy of a heavy chain variable region (VH). In some embodiments, the VH specifically binds to an analyte.

[0151] In some embodiments, each binding unit further comprises two light chains each comprising a light chain constant region or fragment or variant thereof. In some embodiments, the light chain constant regions or fragments or variants thereof are associated with a copy of a light chain variable region (VL). In some embodiments, the VL specifically binds to an analyte.

[0152] In some embodiments, the IgM heavy chain constant regions or fragments or variants thereof each further comprise a Cyl domain, a Cy2 domain, a Cy3 domain, or any combination thereof.

[0153] In some embodiments, the IgM heavy chain constant regions or multimerizing variant or fragment thereof each further comprise a Cpl domain, a Cp2 domain, a Cp3 domain, or any combination thereof.

[0154] In some embodiments, each IgM heavy chain constant region or multimerizing variant or fragment thereof is a human IgM constant region or multimerizing variant or fragment thereof, comprising the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or a multimerizing variant or fragment thereof.

[0155] In some embodiments, each IgM heavy chain constant region or multimerizing variant or fragment thereof is a variant IgM constant region, wherein the multimeric binding molecule has reduced CDC activity relative to a multimeric binding molecule comprising IgM heavy chain constant regions comprising the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.

[0156] In some embodiments, each variant human IgM constant region comprises an amino acid substitution corresponding to position P311 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to positions K315 of SEQ ID NO: 1 or SEQ ID NO: 2, or combinations thereof.

[0157] In other embodiments, the multimeric binding molecules are labeled. Examples of labels that can be used with a lateral flow device have been described, as well as methods for incorporating such labels into a multimeric binding molecule. See Baptista et al., Anal. Bioanal. Chem. 391:943-950 (2008); Baptista etal., Prog. Mol. Biol. Transl. Sci. 104:427- 488 (2011); Wu et al., J. Inorgan. Biochem. 210:111163 (2020); and Kocula eta/., Essays Biochem. 60(1): 111-120 (2016). For example, in some embodiments, the label comprises a gold particle, silver particle, carbon particle, or selenium particle. In other embodiments, the label comprises a colloidal gold nanoparticle, a colloidal silver nanoparticle, a carbon nanoparticle, or a selenium nanoparticle. In some embodiments, the label is a quantum dot, a magnetic particle, an up converting phosphor, a color dye, a fluorescent dye, or an oligonucleotide. In some embodiments, the oligonucleotide is an aptamer. In other embodiments, the label is attached to a latex bead.

[0158] In some embodiments, the J-chain or functional fragment thereof, or functional variant thereof is a modified J-chain further comprises a heterologous moiety. In some embodiments, the heterologous moiety is fused or conjugated to the J-chain or functional fragment or functional variant thereof. In other embodiments, the heterologous moiety comprises a label or a histidine (His) tag. In some embodiments, the His tag comprises from about 6 to about 9 histidine residues.

[0159] In some embodiments, the multimeric binding molecule is an antibody or antigenbinding fragment thereof. In some embodiments, the multimeric binding molecule is an IgM antibody, an IgM-like antibody, an IgM-derived binding molecule, an IgA antibody, an IgA-like antibody, or IgA-derived binding molecule. IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules

[0160] IgM is the first immunoglobulin produced by B cells in response to stimulation by antigen and is naturally present at around 1.5 mg/ml in serum with a half-life of about 5 days. IgM is a pentameric or hexameric molecule and thus includes five or six binding units. An IgM binding unit typically includes two light and two heavy chains. While an IgG heavy chain constant region contains three heavy chain constant domains (CHI, CH2 and CH3), the heavy (p) constant region of IgM additionally contains a fourth constant domain (CH4) and includes a C-terminal p “tailpiece” (ptp). While several human alleles exist, the human IgM constant region typically comprises the amino acid sequence SEQ ID NO: 1 (IMGT allele IGHM*03, identical to, e.g., GenBank AccessionNo. pir| | S37768) or SEQ ID NO: 2 (IMGT allele IGHM*04, identical to, e.g., GenBank Accession No. sp|P01871.4). The human Cpl region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 1 or SEQ ID NO: 2; the human Cp2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 1 or SEQ ID NO: 2, the human Cp3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID NO: 1 or SEQ ID NO: 2, the Cp 4 region ranges from about amino acid 329 to about amino acid

430 of SEQ ID NO: 1 or SEQ ID NO: 2, and the tailpiece ranges from about amino acid

431 to about amino acid 453 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0161] Other forms of the human IgM constant region with minor sequence variations exist, including, without limitation, GenBank Accession Nos. CAB37838.1 and pir||MHHU. The amino acid substitutions, insertions, and/or deletions at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 described and claimed elsewhere in this disclosure can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species.

[0162] Each IgM heavy chain constant region is associated with an antigen-binding domain, e.g., a scFv, or a subunit of an antigen-binding domain, e.g, a VH region.

[0163] Five IgM binding units can form a complex with an additional small polypeptide chain (the J-chain), or a functional fragment, variant, or derivative thereof, to form a pentameric IgM antibody or IgM-like antibody. The precursor form of the human J-chain is presented as SEQ ID NO: 48. The signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 48, and the mature human J-chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO: 48. The mature human J-chain has the amino acid sequence SEQ ID NO: 3.

[0164] Exemplary variant and modified J-chains are provided elsewhere herein. Without the J-chain, an IgM antibody or IgM-like antibody typically assembles into a hexamer, comprising six binding units and up to twelve binding unit-associated antigen-binding domains. With a J-chain, an IgM antibody or IgM-like antibody typically assembles into a pentamer, comprising five binding units and up to ten binding unit-associated antigenbinding domains. The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody or IgM-like antibody is thought to involve interactions between the Cp4 and p tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755- 42762 (2002). Accordingly, the constant regions of a pentameric or hexameric IgM antibody or antibody-like molecule provided in this disclosure typically includes at least the Cp4 and/or p tailpiece domains. A “multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cp4 domain and a ptp domain. An IgM heavy chain constant region can additionally include a Cp3 domain or a fragment thereof, a Cp2 domain or a fragment thereof, and/or a Cpl domain or a fragment thereof. In certain embodiments, a binding molecule, e.g. , an IgM antibody or IgM-like antibody as provided herein can include a complete IgM heavy (p) chain constant domain, e.g, SEQ ID NO: 1 or SEQ ID NO: 2, or a multimerizing variant, derivative, or analog thereof, e.g, as provided herein.

[0165] In certain embodiments, the disclosure provides a pentameric IgM or IgM-like antibody comprising five bivalent binding units, where each binding unit includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an antigen-binding domain or a subunit of an antigen-binding domain. In certain embodiments, the two IgM heavy chain constant regions are human heavy chain constant regions. In certain embodiments, the antigen-binding domain can comprise the extracellular domain of a cell surface molecule (e.g., angiotensin converting enzyme type 2).

[0166] Where the IgM or IgM-like antibody provided herein is pentameric, the IgM or IgM- like antibody typically further includes a J-chain, or functional fragment or variant thereof. In some embodiments, the J-chain is a modified J-chain comprising a heterologous moiety, e.g., a label or tag as described herein. In certain embodiments the J-chain can be mutated to affect glycosylation, as discussed elsewhere in this disclosure.

[0167] In some embodiments, the IgM or IgM-like antibody provided herein is hexameric and comprises six bivalent binding units. In some embodiments, each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof.

[0168] An IgM heavy chain constant region can include one or more of a Cpl domain or fragment or variant thereof, a Cp2 domain or fragment or variant thereof, a Cp3 domain or fragment or variant thereof, a Cp4 domain or fragment or variant thereof, and/or a p tail piece (ptp) or fragment or variant thereof, provided that the constant region can serve a desired function in the IgM or IgM-like antibody, e.g. , associate with second IgM constant region to form a binding unit with one, two, or more antigen-binding domain(s), and/or associate with other binding units (and in the case of a pentamer, a J-chain) to form a hexamer or a pentamer. In certain embodiments the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a Cp4 domain or fragment or variant thereof, a p tailpiece (ptp) or fragment or variant thereof, or a combination of a Cp4 domain and a ptp or fragment or variant thereof. In certain embodiments the two IgM heavy chain constant regions or fragments or variants thereof within an individual binding unit each further comprise a Cp3 domain or fragment or variant thereof, a Cp2 domain or fragment or variant thereof, a Cpl domain or fragment or variant thereof, or any combination thereof.

[0169] In some embodiments, the binding units of the IgM or IgM-like antibody comprise two light chains. In some embodiments, the binding units of the IgM or IgM-like antibody comprise two fragments of light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments, the light chains are hybrid kappa and lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region. IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules with reduced CDC

[0170] Certain IgM-derived multimeric binding molecules, e.g. , antibodies or antibody-like molecules as provided herein can be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity to cells in the presence of complement, relative to a reference IgM antibody or IgM-like antibody with a corresponding reference human IgM constant region identical, except for the mutations conferring reduced CDC activity. These CDC mutations can be combined with any of the mutations to block N-linked glycosylation and/or to confer increased serum half-life as provided herein. By “corresponding reference human IgM constant region” is meant a human IgM constant region or portion thereof, e.g., a Cp3 domain, that is identical to the variant IgM constant region except for the modification or modifications in the constant region affecting CDC activity. In certain embodiments, the variant human IgM constant region includes one or more amino acid substitutions, e.g, in the Cp3 domain, relative to a wild-type human IgM constant region as described, e.g, in PCT Publication No. WO/2018/187702, which is incorporated herein by reference in its entirety. Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g, in US Patent Application Publication No. 2021-0147567, which is incorporated by reference herein in its entirety.

[0171] In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310, P311, P313, and/or K315 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments the variant IgM constant region as provided herein contains an amino acid substitution corresponding to the wild-type human IgM constant region at position P313 of SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments the variant IgM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human IgM constant region at positions P311 of SEQ ID NO: 1 or SEQ ID NO: 2 and/or P313 of SEQ ID NO: 1 or SEQ ID NO: 2. These proline residues can be independently substituted with any amino acid, e.g., with alanine, serine, or glycine. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2. The lysine residue can be independently substituted with any amino acid, e.g, with alanine, serine, glycine, or aspartic acid. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position K315 of SEQ ID NO: 1 or SEQ ID NO: 2 with aspartic acid. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310 of SEQ ID NO: 1 or SEQ ID NO: 2. The lysine residue can be independently substituted with any amino acid, e.g., with alanine, serine, glycine, or aspartic acid. In certain embodiments, a variant human IgM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human IgM constant region at position L310 of SEQ ID NO: 1 or SEQ ID NO: 2 with aspartic acid.

Glyco-modified IgM antibodies, IgM-like antibodies, and IgM-derived binding molecules

[0172] Human and certain non-human primate IgM constant regions typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif’ comprises or consists of the amino acid sequence N- Xi-S/T, where N is asparagine, Xi is any amino acid except proline (P), and S/T is serine (S) or threonine (T). The glycan is attached to the nitrogen atom of the asparagine residue. See, e.g. , Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ ID NO: 1 or SEQ ID NO: 2 starting at positions 46 (“Nl”), 209 (“N2”), 272 (“N3”), 279 (“N4”), and 440 (“N5”). These five motifs are conserved in non- human primate IgM heavy chain constant regions, and four of the five are conserved in the mouse IgM heavy chain constant region. Accordingly, in some embodiments, IgM heavy chain constant regions of a multimeric binding molecule as provided herein comprise 5 N- linked glycosylation motifs: Nl, N2, N3, N4, and N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., Nl, N2, and N3) on each IgM heavy chain constant region are occupied by a complex glycan.

[0173] In certain embodiments, at least one, at least two, at least three, or at least four of the N- Xi-S/T motifs can include an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif. In certain embodiments, the IgM-derived multimeric binding molecule can include an amino acid insertion, deletion, or substitution at motif Nl, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs Nl, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif. In some embodiment, the IgM constant region comprises one or more substitutions relative to a wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 1 (human IgM constant region allele IGHM*03) or SEQ ID NO: 2 (human IgM constant region allele IGHM*04). See, e.g., PCT Application Publication No. WO 2021/041250, which is incorporated herein by reference in its entirety.

IgA antibodies, IgA-like antibodies, and IgA-derived binding molecules

[0174] IgA plays a critical role in mucosal immunity and comprises about 15% of total immunoglobulin produced. IgA can be monomeric or multimeric, forming primarily dimeric molecules, but can also assemble as trimers, tetramers, and/or pentamers. See, e.g. , de Sousa-Pereira, P., and J.M. Woof, Antibodies 8:51 (2019).

[0175] In some embodiments, the multimeric binding molecules are dimeric and comprise two bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecules are dimeric or tetrameric, comprising two or four bivalent binding units or variants or fragments thereof, respectively, and further comprise a J-chain or functional fragment or variant thereof as described herein. In some embodiments, the multimeric binding molecules are dimeric, comprise two bivalent binding units or variants or fragments thereof, and further comprise a J-chain or functional fragment or variant thereof as described herein, where each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof.

[0176] In some embodiments, the multimeric binding molecules are tetrameric and comprise four bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecules are tetrameric, comprise four bivalent binding units or variants or fragments thereof, and further comprise a J-chain or functional fragment or variant thereof as described herein. In some embodiments, the multimeric binding molecules are tetrameric, comprise four bivalent binding units or variants or fragments thereof, and further comprise a J-chain or functional fragment or variant thereof as described herein, where each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof.

[0177] In certain embodiments, the multimeric binding molecule provided by this disclosure is a dimeric binding molecule that includes four IgA heavy chain constant regions, or multimerizing fragments thereof, each associated with an antigen-binding domain for a total of four antigen-binding domains. As provided herein, a dimeric IgA antibody, IgA-derived binding molecule, or IgA-like antibody includes two binding units and a J-chain. Each binding unit as provided comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof. In certain embodiments, at least three or all four antigen-binding domains of the multimeric binding molecule bind to the same target antigen. In certain embodiments, at least three or all four binding polypeptides of the multimeric binding molecule are identical.

[0178] A bivalent IgA-derived binding unit includes two IgA heavy chain constant regions, and a dimeric IgA-derived binding molecule includes two binding units. IgA contains the following heavy chain constant domains, Cal (or alternatively CAI or CHI), a hinge region, Ca2 (or alternatively CA2 or CH2), and Ca3 (or alternatively CA3 or CH3), and a C-terminal “tailpiece.” Human IgA has two subtypes, IgAl and IgA2. The human IgAl constant region typically includes the amino acid sequence SEQ ID NO: 50. The human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 50; the human IgAl hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO: 50, the human Ca2 domain extends from about amino acid 125 to about amino acid 219 of SEQ ID NO: 50, the human Ca3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO: 50, and the tailpiece extends from about amino acid 331 to about amino acid 352 of SEQ ID NO: 50. The human IgA2 constant region typically includes the amino acid sequence SEQ ID NO: 51. The human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 51; the human IgA2 hinge region extends from about amino acid 102 to about amino acid 111 of SEQ ID NO: 51, the human Ca2 domain extends from about amino acid 113 to about amino acid 206 of SEQ ID NO: 51, the human Ca3 domain extends from about amino acid 215 to about amino acid 317 of SEQ ID NO: 51, and the tailpiece extends from about amino acid 318 to about amino acid 340 of SEQ ID NO: 51.

[0179] Two IgA binding units can form a complex with two additional polypeptide chains, the J-chain (e.g., SEQ ID NO: 3) and the secretory component (precursor, SEQ ID NO: 52, mature, from about amino acid 19 to about amino acid 764 of SEQ ID NO: 52) to form a bivalent secretory IgA (slgA)-derived binding molecule as provided herein. The assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the Ca3 and tailpiece domains. See, e.g., Braathen, R., et al., J. Biol. Chem. 277:42755-42762 (2002). Accordingly, a multimerizing dimeric IgA-derived binding molecule provided in this disclosure typically includes IgA constant regions that include at least the Ca3 and a tailpiece domains. Four IgA binding units can likewise form a tetramer complex with a J-chain. An IgA antibody can also form as a higher order multimer, e.g., a tetramer.

[0180] An IgA heavy chain constant region can additionally include a Ca2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a Cal domain or a fragment thereof, and/or other IgA (or other immunoglobulin, e.g., IgG) heavy chain domains, including, e.g., an IgG hinge region. In certain embodiments, a binding molecule as provided herein can include a complete IgA heavy (a) chain constant domain (e.g., SEQ ID NO: 50 or SEQ ID NO: 51), or a variant, derivative, or analog thereof. In some embodiments, the IgA heavy chain constant regions or multimerizing fragments thereof are human IgA constant regions.

[0181] In certain embodiments each binding unit of a multimeric binding molecule as provided herein includes two IgA heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgA Ca3 domain and an IgA tailpiece domain. In certain embodiments the IgA heavy chain constant regions can each further include an IgA Ca2 domain situated N-terminal to the IgA Ca3 and IgA tailpiece domains. For example, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 50 or amino acids 113 to 340 of SEQ ID NO: 51. In certain embodiments the IgA heavy chain constant regions can each further include an IgA or IgG hinge region situated N-terminal to the IgA Ca2 domains. For example, the IgA heavy chain constant regions can include amino acids 102 to 353 of SEQ ID NO: 50 or amino acids 102 to 340 of SEQ ID NO: 51. In certain embodiments the IgA heavy chain constant regions can each further include an IgA Cal domain situated N-terminal to the IgA hinge region.

[0182] In some embodiments, each binding unit of an IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprises two light chains. In some embodiments, each binding unit of an IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprises two fragments light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments the light chains are chimeric kappa-lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region.

Modified and/or Variant J-chains

[0183] In certain embodiments, the multimeric binding molecule, e.g., antibody or antibody-like molecule provided herein comprises a J-chain or functional fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein is a pentameric IgM antibody or IgM antibody-like molecule and comprises a J-chain or functional fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein is a dimeric IgA, IgA antibody or IgA antibody-like molecule and comprises a J-chain or functional fragment or variant thereof. In some embodiments, the multimeric binding molecule can comprise a naturally occurring J-chain, such as a mature human J-chain (e.g., SEQ ID NO: 3). In some embodiments, the multimeric binding molecule can comprise a functional fragment or functional variant of a naturally occurring J-chain.

[0184] In certain embodiments, the J-chain of a pentameric an IgM or IgM-like antibody or a dimeric IgA or IgA-like antibody as provided herein can be modified, e.g., by introduction of a heterologous moiety, or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of the IgM or IgM-like antibody or IgA or IgA-like antibody to assemble and bind to its binding target(s). See U.S. Patent Nos. 9,951,134, 10,975,147, 10,400,038, and 10,618,978, and U.S. Patent Application Publication No. US-2019-0185570, each of which is incorporated herein by reference in its entirety. Accordingly, IgM or IgM-like antibodies or IgA or IgA-like antibodies as provided herein, including bispecific or multispecific IgM or IgM-like antibodies or IgA or IgA-like antibodies as described elsewhere herein, can include a modified J-chain or functional fragment or variant thereof that further includes a heterologous moiety, e.g., a label or tag as described herein.

[0185] In certain embodiments, the J-chain of an IgM antibody, IgM-like antibody, IgA antibody, IgA-like antibody, or IgM- or IgA-derived binding molecule as provided herein is a variant J-chain.

[0186] In some embodiments, the multimeric binding molecule can comprise a variant J- chain sequence, such as a variant sequence described herein with reduced glycosylation. See, e.g., U.S. Patent No. 10,899,835, which is incorporated herein by reference in its entirety. In certain embodiments, the variant J-chain can comprise an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 3). By “an amino acid corresponding to amino acid Y102 of the mature wild-type human J-chain” is meant the amino acid in the sequence of the J-chain of any species which is homologous to Y102 in the human J-chain. See U.S. Patent No. 10,899,835. The position corresponding to Y 102 in SEQ ID NO: 3 is conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S. Patent No. 9,951,134, which is incorporated by reference herein. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 3 can be substituted with any amino acid. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 3 can be substituted with alanine (A), serine (S) or arginine (R). In a particular embodiment, the amino acid corresponding to Y102 of SEQ ID NO: 3 can be substituted with alanine. In a particular embodiment the J-chain or functional fragment or variant thereof is a variant human J-chain and comprises the amino acid sequence SEQ ID NO: 49, a J-chain referred to herein as “J*”.

[0187] Wild-type J-chains typically include one N-linked glycosylation site. In certain embodiments, a variant J-chain or functional fragment thereof of a multimeric binding molecule as provided herein includes a mutation within the asparagine(N)-linked glycosylation motif N-Xi-S/T, e.g., starting at the amino acid position corresponding to amino acid 49 (motif N6) of the mature human J-chain (SEQ ID NO: 3) or J* (SEQ ID NO: 49), where N is asparagine, Xi is any amino acid except proline, and S/T is serine or threonine, and where the mutation prevents glycosylation at that motif as descrbied in U.S. Patent No. 10,899,835.

[0188] For example, in certain embodiments the variant J-chain or functional fragment thereof of a binding molecule comprising a J-chain as provided herein can include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 3 or SEQ ID NO: 49, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or where the variant J-chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 3 or SEQ ID NO: 49. In certain embodiments, the position corresponding to N49 of SEQ ID NO: 3 or SEQ ID NO: 49 is substituted with any amino acid, e.g., alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular embodiment, the position corresponding to N49 of SEQ ID NO: 3 or SEQ ID NO: 49 can be substituted with alanine (A). In another embodiment, the position corresponding to N49 of SEQ ID NO: 3 or SEQ ID NO: 49 can be substituted with aspartic acid (D). In some embodiments, the position corresponding to S51 of SEQ ID NO: 3 or SEQ ID NO: 49 is substituted with alanine (A) or glycine (G). In some embodiments, the position corresponding to S51 of SEQ ID NO: 3 or SEQ ID NO: 49 is substituted with alanine (A).

[0189] Multimeric binding molecules have several advantages as analyte detection molecules in a lateral flow device, including stronger binding to the analyte and greater sensitivity for detecting the analyte. As described herein, a multimeric binding molecule having multiple valencies has high avidity for an analyte. Accordingly, in some embodiments, the multimeric binding molecules used in the lateral flow devices described herein have greater avidity for an analyte than an IgG molecule that specifically binds to the same analyte. In some embodiments, the IgG molecule comprises the same VH and VL sequences as the multimeric binding molecule.

[0190] Binding strength of a multimeric binding molecule to an analyte can also be evaluated by the rate that the molecule binds to the analyte (the association rate constant, or “Kon”) and the rate that the molecule dissociates from the analyte (the dissociation rate constant, or “Koff’). In some embodiments, multimeric binding molecules used in the lateral flow devices described herein have a lower Koff rate for binding of the multimeric binding molecule to the analyte, than the Koff rate for binding of an IgG molecule to the same analyte. FIG. 2 illustrates this concept.

[0191] In addition, the multimeric binding molecules disclosed herein can specifically bind a variant infectious pathogen antigen, such as a variant coronavirus antigen, variant influenza virus antigen, or variant respiratory syncytial virus (RSV) antigen. For example, in some embodiments, the variant SARS-CoV-2 antigen comprises one or more substitutions found in alpha, beta, gamma, delta, eta, iota, kappa, lambda, 1.617.3, mu, theta, or zeta variant.

Conjugate Section

[0192] As used herein, the term “conjugate section” refers to the region of a lateral flow device containing multimeric binding molecules that specifically bind to the analyte. The conjugate section delivers such binding molecules, directly or indirectly, to the analysis section (e.g., the test section and/or control section).

[0193] Multimeric binding molecules can be introduced to the conjugate section by introducing the binding molecules directly onto the conjugate pad or by introducing the binding molecules to the sample loading section and moving the binding molecules to the conjugate section with capillary action.

[0194] A conjugate section can have any shape or size, and a lateral flow device disclosed herein can have one or more conjugate sections.

[0195] In other embodiments, the conjugate section comprises a carbohydrate. In other embodiments, the carbohydrate is sucrose.

[0196] In some embodiments, the conjugate section comprises a conjugate pad. In some embodiments, the conjugate pad is composed of cellulose (e.g., a cellulose fiber filter), rayon, woven materials (e.g, glass fiber, polyester), or a combination thereof. In some embodiments, the conjugate pad is from about 0.45 mm to about 2.5 mm thick, or any values or range of values thereof, e.g., from about 0.5 mm to about 2.5 mm, from about 1 mm to about 2.5 mm, from about 1.5 mm to about 2.5 mm, from about 2 mm to about 2.5 mm, from about 0.5 mm to about 2 mm, from about 1 mm to about 2 mm, from about 1.5 mm to about 2 mm, from about 0.5 mm to about 1.5 mm, from about 1 mm to about 1.5 mm, or from about 0.5 mm to about 1 mm. In other embodiments, the conjugate pad is about 0.45 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, or about 2.5 mm thick.

[0197] In some embodiments, the conjugate pad is pretreated with a carbohydrate, a nonspecific protein, a surfactant, or a combination thereof. In some embodiments, the carbohydrate is sucrose. In some embodiments, the non-specific protein is albumin, casein, bovine serum albumin (BSA), milk (e.g., skim milk), or a combination thereof. In some embodiments, the surfactant comprises sodium dodecyl sulfate (SDS), polysorbate, Tween (ethoxylated or polyoxyethylene derivatives of sorbitan ester), Triton X-100, or a combination thereof.

[0198] In some embodiments, the multimeric binding molecules are diffusively-bound to the conjugate section (e.g., conjugate pad). As used herein, “diffusively-bound” multimeric binding molecules are reversibly joined to the conjugate section (e.g., conjugate pad) and can be released without substantial denaturation or aggregation. In some embodiments, a diffusively-bound multimeric binding molecule can be joined to the conjugate section by contacting a solution containing the multimeric binding molecule with the conjugate pad, thereby soaking up the solution and joining the multimeric binding molecule with the conjugate section. In other embodiments, a diffusively-bound multimeric binding molecule can be joined to the conjugate section by contacting a solution containing the multimeric binding molecule with the conjugate pad, thereby soaking up the solution, and then drying the solution-containing conjugate pad. In other embodiments, the diffusively-bound multimeric binding molecule can be released by contacting the conjugate section with the same or a different solution. In some embodiments, the diffusively-bound multimeric binding molecule can be released by contacting the conjugate pad with the same or a different solution, thereby solubilizing the multimeric binding molecule, and then wicking the solution from the conjugate pad, or from one region of the conjugate pad to another.

Analysis Section

[0199] As used herein, the “analysis section” refers to the region of a lateral flow device that contains a test binding molecule comprising an antigen-binding domain that specifically binds to an analyte, and optionally, a control binding molecule that specifically binds to a multimeric binding molecule. The analysis section can have any shape or size, and a lateral flow device disclosed herein can have one or more analysis sections.

[0200] In some embodiments, the analysis section (e.g., the test section and/or control section) comprises a porous membrane. In some embodiments, the porous membrane contains nitrocellulose, nylon, polyvinylidene fluoride, cellulose acetate, or a combination thereof. In some embodiments, the porous membrane is from about 100 mm to 150 mm thick, or any values or range of values therein, e.g., from about 110 mm to about 150 mm, from about 120 mm to about 150, from about 130 mm to about 150 mm, from about 140 mm to about 150 mm, from about 110 mm to about 140 mm, from about 120 mm to about 140 mm, from about 130 mm to about 140 mm, from about 110 mm to about 130 mm, from about 120 mm to about 130 mm, or from about 110 to about 120 mm. In some embodiments, the porous membrane is about 100 mm, about 110 mm, about 120 mm, about 130 mm, about 140 mm, or about 150 mm thick.

[0201] In other embodiments, the porous membrane has a pore size of from about 0.1 pm to about 12 pm, or any values or ranges of values therein, e.g., from about 0.5 pm to about 12 pm, from about 1 pm to about 12 pm, from about 2 pm to about 12 pm, from about 5 pm to about 12 pm, from about 10 pm to about 12 pm, from about 0.1 pm to about 10 pm, from about 0.5 pm to about 10 pm, from about 1 pm to about 10 pm, from about 2 pm to about 10 pm, from about 5 pm to about 10 pm, from about 0. 1 pm to about 5 pm, from about 0.5 pm to about 5 pm, from about 1 pm to about 5 pm, from about 2 pm to about 5 pm, from about 0.1 pm to about 2 pm, from about 0.5 pm to about 2 pm, from about 1 pm to about 2 pm, from about 0. 1 pm to about 1 pm, from about 0.5 pm to about 1 pm, or from about 0. 1 pm to about 0.5 pm. In some embodiments, the porous membrane has a pore size of about 0. 1 pm, about 0.5 pm, about 1 pm, about 2 pm, about 5 pm, about 10 pm, or about 12 pm.

[0202] In other embodiments, the porous membrane has a capillary flow time of from about

60 seconds to about 240 seconds to traverse about 4 cm, or any values of range or values therein, e.g., from about 100 seconds to about 240 seconds, from about 120 seconds to about 240 seconds, from about 180 seconds to about 240 seconds, from about 60 seconds to about 180 seconds, from about 100 seconds to about 180 seconds, from about 120 seconds to about 180 seconds, from about 60 seconds to about 120 seconds, from about 100 seconds to about 120 seconds, or from about 60 seconds to about 100 seconds to traverse about 4 cm. In some embodiments, the porous membrane has a capillary flow time of about 60 seconds, about 120 seconds, about 180 seconds, or about 240 seconds to traverse about 4 cm.

[0203] In some embodiments, the analysis section comprises one or more test sections. In some embodiments, the analysis section comprises one or more control sections. In some embodiments, the analysis section comprises one or more test section and one or more control sections.

[0204] As used herein, a “test section” refers to the region of a lateral flow device that contains a test binding molecule comprising an antigen-binding domain that specifically binds to an analyte. The test section can have any shape or size, and a lateral flow device disclosed herein can have one or more test sections.

[0205] In some embodiments, the test binding molecules are immobilized to the analysis section and/or test section. In some embodiments, the test binding molecules are immobilized to the surface of the porous membrane in the analysis section and/or test section. As used herein, “immobilized” test binding molecules are irreversibly joined to the analysis section and/or test section and are not removed or substantially removed from their location under standard lateral flow conditions.

[0206] In some embodiments, the test binding molecule is a monomeric antibody. In some embodiments, the test binding molecule is an IgG antibody. In some embodiments, the test binding molecule is a multimeric antibody.

[0207] Methods for immobilizing a binding molecule include, but are not limited to, binding the molecules to the porous membrane of the analysis section and/or test section by standard blotting techniques (e.g., electroblotting to nitrocellulose, poly vinylidene fluoride or nylon).

[0208] As used herein, a “control section” refers to the region of a lateral flow device that contains a control binding molecule that specifically binds to a multimeric binding molecule. The control section can have any shape or size, and a lateral flow device disclosed herein can have one or more control sections.

[0209] In some embodiments, the control binding molecules are immobilized to the analysis section and/or control section. In some embodiments, the control binding molecules are immobilized to the surface of the porous membrane in the analysis section and/or test section. As used herein, “immobilized” control binding molecules are irreversibly j oined to the analysis section and/or control section and are not removed or substantially removed from their location under standard lateral flow conditions. Methods for immobilizing a binding molecule include, but are not limited to, binding the molecules to the porous membrane of the analysis section and/or control section by standard blotting techniques (e.g., electroblotting to nitrocellulose, polyvinylidene fluoride or nylon).

[0210] In some embodiments, the control binding molecule is a monomeric antibody. In some embodiments, the control binding molecule is an IgG antibody. In some embodiments, the control binding molecule is a multimeric antibody.

[0211] In other embodiments, the control binding molecule specifically binds to the label, the light chain, the J-chain, and/or the heavy chain of the labeled multimeric binding molecule. In other embodiments, the labeled multimeric binding molecule comprises a tag and the control binding molecule specifically binds to the tag. In other embodiments, the tag is a histidine (His) tag attached to the J-chain. In some embodiments, the His tag comprises from about 6 to about 9 histidine residues.

[0212] In some embodiments, the control binding molecule and/or the test binding molecule is a monomeric antibody, an IgG antibody or a multimeric antibody.

[0213] In some embodiments, the control binding molecule and/or test binding molecule is a multimeric binding molecule. In some embodiments, the control binding molecule and/or test binding molecule is a multimeric binding molecule comprising a J-chain.

Other General Embodiments

[0214] In some embodiments, a lateral flow device provided herein further comprises an absorbent section. As used herein, “absorbent section” refers to the region of a lateral flow device that helps maintain the flow rate of liquid through the device and stops back flow of a sample. An absorbent section can have any shape or size, and a lateral flow device disclosed herein can have one or more absorbent sections.

[0215] In some embodiments, the absorbent section is located in series after the analysis section.

[0216] In other embodiments, the absorbent section comprises an absorbent pad. In some embodiments, the absorbent pad comprises cellulose (e.g., a cellulose fiber filter), rayon, woven materials (e.g., glass fiber, polyester), or a combination thereof. [0217] In some embodiments, the absorbent pad is from about 0.45 mm to about 2.5 mm thick, or any value or range of values therein, e.g., from about 0.45 mm to about 2 mm, from about 0.45 mm to about 1 mm, from about 0.45 mm to about 0.5 mm, from about 0.5 mm to about 2.5 mm, from about 1 mm to about 2.5 mm, from about 1.5 mm to about 2.5 mm, from about 2 mm to about 2.5 mm, from about 1 mm to about 2.5 mm, from about 1 mm to about 2 mm, from about 1 mm to about 1.5 mm, from about 1.5 mm to about 2.5 mm, from about 1.5 mm to about 2 mm, or from about 2 mm to about 2.5 mm thick. In some embodiments, the absorbent pad is about 0.45 mm, about 0.5 mm, about 1 mm, about 1.5 mm, about 2 mm, or about 2.5 mm thick.

[0218] In some embodiments, a lateral flow device provided herein comprises a sample loading section, a conjugate section, and an analysis section in series. In other embodiments, a lateral flow device provided herein comprises a sample loading section, a conjugate section, an analysis section, and an absorbent section in series. In some embodiments, the analysis section comprises a test section and a control section in series.

[0219] In some embodiments, the sample loading section, the conjugate section, and the test section comprise a single monolithic hydrophilic matrix. In some embodiments, the single monolithic hydrophilic matrix comprises a network of fibers comprising a mixture of polymer and glass fiber or glass microfiber. In some embodiments, the control binding molecules and/or test binding molecules are immobilized on the surface of the monolithic hydrophilic matrix. In some embodiments, the control binding molecules and/or test binding molecules are indirectly immobilized on the surface. In some embodiments, the control binding molecule and/or test binding molecule is indirectly attached to the surface through an antibody that binds the J-chain or a moiety attached to the J-chain.

[0220] In some embodiments, one or more sections of a lateral flow device provided herein overlap or partially overlap with one or more other sections. In some embodiments, the sample loading section and the conjugate section overlap or partially overlap. In some embodiments, the conjugate section and the analysis section (including the test section and/or the control section) overlap or partially overlap. In some embodiments, the sample loading section and the conjugate section overlap or partially overlap, and the conjugate section and the analysis section (including the test section and/or the control section) overlap or partially overlap. In other embodiments, the test section and the control section overlap or partially overlap. In other embodiments, the analysis section and the absorbent section overlap or partially overlap. In some embodiments, the sample loading section and the conjugate section overlap or partially overlap, the conjugate section and the analysis section (including the test section and/or the control section) overlap or partially overlap, and the analysis section (including the test section and/or the control section) and the absorbent section overlap or partially overlap.

[0221] In some embodiments, the lateral flow strip further comprises a non-porous backing layer beneath the sample loading section, the conjugate section, and the analysis section. In some embodiments, the non-porous backing layer comprises polystyrene, polyvinyl chloride, polyester, or a combination thereof.

[0222] In some embodiments, a lateral flow device provided herein further comprises an external casing. In some embodiments, the external casing is composed of a plastic or plastic-like material. In some embodiments, the external casing does not fully enclose the sample loading section. In some embodiments, the external casing is transparent above the test and/or control sections. In other embodiments, the external case does not full enclose the sample loading section and is transparent above the test and/or control sections.

[0223] In some embodiments, a kit for detecting an analyte in a liquid sample is provided herein comprising a lateral flow device provided herein, and instructions for use. In some embodiments, the kit further comprises a sample buffer. In some embodiments, the sample buffer is water, ethanol, methanol, acetone or a combination thereof.

Methods of Use and Manufacturing

[0224] Methods of using a lateral flow device or kit described herein are also provided. Such methods comprise, in general, applying a sample to the sample loading section of the device and detecting the presence or absence of a label in the test section of the device.

[0225] In some embodiments, a lateral flow device provided herein is used to detect an analyte in a sample (e.g., liquid sample). In such embodiments, a sample (e.g., liquid sample) is applied to the sample loading section (e.g., sample pad). In some embodiments, the sample is allowed to stay in contact with the sample loading section (e.g., sample pad) for a brief period of time, such as from about 1 minute to about 10 minutes, or for an amount of time necessary for the sample to be distributed into the sample loading section (e.g., sample pad). [0226] After application of the sample to the sample loading section, the sample moves by capillary action from the sample loading section to the conjugate section. Capillary action can result from having a liquid sample and/or adding a running buffer to the lateral flow device. In some embodiments, the running buffer is phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane (“Tris”, for example, Tris-acetate, Tris-HCl), borate buffer, or a combination thereof. In other embodiments, the running buffer is introduced sequentially or simultaneously with the sample. In other embodiments, the running buffer is introduced to the lateral flow device and then dried before introduction of the sample.

[0227] In some embodiments of the methods provided herein, the conjugate section contains diffusively-bound labeled multimeric binding molecules that specifically bind to the analyte. The diffusively-bound labeled multimeric binding molecules are released from the conjugate section as described herein and specifically bind to the analyte, if present in the sample. The labeled multimeric binding molecules bound to analyte then flow to the analysis section (e.g., test section and/or control section).

[0228] In some embodiments of the methods provided herein, the test section contains immobilized test binding molecules that comprise an antigen-binding domain that specifically binds to the analyte. As such, when the labeled multimeric binding molecules bound to analyte flow through the test section, they specifically bind to the immobilized test binding molecules.

[0229] In some embodiments of the methods provided herein, the control section contains immobilized control binding molecules that specifically bind to the labeled multimeric binding molecule. As such, when the labeled multimeric binding molecules bound to analyte flow through the test section, they specifically bind to the immobilized control binding molecules.

[0230] In some embodiments, the presence or amount of analyte is then determined by measuring the presence or activity of the label on the multimeric binding molecules in the test and/or control sections. Methods for measuring such labels are described, for example, in Baptista et al., Anal. Bioanal. Chem. 391:943-950 (2008); Baptista et al., Prog. Mol. Biol. Transl. Sci. 104:427-488 (2011); Wuetal., J. Inorgan. Biochem. 210: 111163 (2020); and Kocula et al., Essays Biochem. 60(1): 111-120 (2016). For example, a label that is a color dye or fluorescent dye can be detected by visual inspection, scanner, light microscopy, or electron microscopy. Likewise metal particle labels (e.g., gold particle or colloidal gold) are also colored and no development process is needed for visualization. Such labels can be measured by visual inspection or a scanner, or by measuring the thermal contrast under laser light irradiation. In addition, latex (e.g., latex bead) can be used and tagged with a variety of detector reagents such as a color dye or fluorescent dye. Carbon and fluorescent labels, or enzymatic modification of a label can also improve the sensitivity of the method.

[0231] In some embodiments, detection of the label in the test section indicates the analyte is present in the sample. In some embodiments, detection of the label in the test section and the control section indicates the analyte is present in the sample.

[0232] In some embodiments, no detection or no significant detection of the label in the test section indicates no analyte or significant amount of analyte is present in the sample. In some embodiments, no detection or significant detection of the label in the test section and detection of the label in the control section indicates no analyte or significant amount of analyte is present in the sample.

[0233] Accordingly, in certain embodiments, provided herein is a method of detecting an analyte comprising introducing a sample (e.g., liquid sample) to a sample loading section of a lateral flow device described herein, and detecting the presence or absence of a label in the test and/or control sections of the device.

[0234] In certain embodiments, provided herein is a method of detecting an analyte comprising (1) applying a sample to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test section of the device.

[0235] In certain embodiments, provided herein is a method of detecting an analyte comprising (1) applying a sample to the sample loading section of lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device.

[0236] In some embodiments, prior to step (1), the method comprises removing a component from the sample, adjusting the pH of the sample, and/or diluting the sample, thereby generating the liquid sample.

[0237] In other embodiments, provided herein is a method of detecting an analyte in a sample, comprising detecting the presence or absence of a label in the test and/or control sections of a lateral flow device described herein, wherein the sample was applied to the sample loading section and allowed to flow through the lateral flow strip of the device.

[0238] In other embodiments, provided herein is a method of diagnosing a disease or disorder or determining an increased risk of developing a disease or disorder in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is a marker for the disease or disorder. In some embodiments, presence of the label in the test and/or control sections indicates the subject has the disease or disorder or an increased risk of developing the disease or disorder. In some embodiments, absence of the label in the test and/or control sections indicates the subject does not have the disease or disorder or an increased risk of developing the disease or disorder.

[0239] In other embodiments, provided herein is a method for diagnosing cancer or cancer metastasis or determining an increased risk of developing cancer or cancer metastasis in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is a tumor antigen. In some embodiments, presence of the label in the test and/or control sections indicates the subject has cancer or cancer metastasis or an increased risk of developing cancer or cancer metastasis. In some embodiments, absence of the label in the test and/or control sections indicates the subject does not have cancer or cancer metastasis or an increased risk of developing cancer or cancer metastasis. In some embodiments, the tumor antigen is p53, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), or prostate specific antigen (PSA).

[0240] In other embodiments, provided herein is a method for diagnosing infection or determining an increased risk of infection in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is an infectious pathogen antigen. In some embodiments, presence of the label in the test and/or control sections indicates the subject has an infection or an increased risk of infection. In some embodiments, absence of the label in the test and/or control sections indicates that subject does not have an infection or an increased risk of infection. In some embodiments, the infectious pathogen antigen is a virus, bacterium, fungi, or parasite.

[0241] In other embodiments, provided herein is a method for diagnosing viral infection or an increased risk of viral infection in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is a viral antigen. In some embodiments, presence of the label in the test and/or control sections indicates the subject has a viral infection or an increased risk of viral infection. In some embodiments, absence of the label in the test and/or control sections indicates the subject does not have a viral infection or an increased risk of viral infection.

[0242] In some embodiments, the virus is coronavirus, an influenza virus, Dengue virus, Ebola virus, hepatitis virus, respiratory syncytial virus (RSV), human metapneumo virus, human immunodeficiency virus (HIV), herpes virus, paramyxovirus, or enterovirus.

[0243] In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), or Middle East respiratory syndrome-related coronavirus (MERS-CoV).

[0244] In some embodiments, the influenza virus is influenza A virus (IAV), influenza B virus (IBV), or influenza C virus (ICV).

[0245] In some embodiments, the hepatitis virus is hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E.

[0246] In some embodiments, the HIV is HIV type 1 (HIV-1) or HIV type 2 (HIV -2).

[0247] In some embodiments, the herpes virus is herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein- Barr virus (EBV), roseolovirus, or Kaposi’s sarcoma-associated herpesvirus (KSHV).

[0248] In some embodiments, the paramyxovirus is a parainfluenza virus (HPIV). In some embodiments, the HPIV is HPIV type 1 (HPIV-1), HPIV type 2 (HPIV-2), HPIV type 3 (HPIV-3), or HPIV type 4 (HPIV-4). [0249] In some embodiments, the enterovirus is a poliovirus, coxsackie virus A virus, coxsackie virus B, echovirus, or EV71.

[0250] In other embodiments, provided herein is a method for diagnosing bacterial infection or an increased risk of bacterial infection in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is a bacterial antigen. In some embodiments, presence of the label in the test and/or control sections indicates the subj ect has a bacterial infection or an increased risk of bacterial infection. In some embodiments, absence of the label in the test and/or control sections indicates the subject does not have a bacterial infection or an increased risk of bacterial infection. In some embodiments, the bacteria is Helicobacter pylori, Streptococcus pyogenes, Mycobacterium tuberculosis, Bacillus anthracis, Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Salmonella, Listeria, or Pseudomonas aeruginosa.

[0251] In other embodiments, provided herein is a method for diagnosing heart injury or determining an increased risk of heart injury in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is a cardiac marker. In some embodiments, presence of the label in the test and/or control sections indicates the subject has heart injury or an increased risk of heart injury. In some embodiments, absence of the label in the test and/or control sections indicates the subject does not have heart injury or an increased risk of heart injury. In some embodiments, the heart injury indicates the subject had a heart attack or is at increased risk for a heart attack. In some embodiments, the cardiac marker is troponin C, creatine kinase, or myoglobin.

[0252] In other embodiments, provided herein is a method for determining pregnancy in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is human chorionic gonadotropin (hCG). In some embodiments, presence of the label in the test and/or control sections indicates the subject is pregnant. In some embodiments, absence of the label in the test and/or control sections indicates the subject is not pregnant. In some embodiments, the sample is urine.

[0253] In other embodiments, provided herein is a method for determining ovulation in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is luteinizing hormone (LH). In some embodiments, presence of the label in the test and/or control sections indicates the subject is ovulating. In some embodiments, absence of the label in the test and/or control sections indicates the subject is not ovulating. In some embodiments, the sample is urine.

[0254] In other embodiments, provided herein is a method for determining hypothyroidism or an increased risk of hypothyroidism in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is a thyroid-stimulating hormone (TSH). In some embodiments, presence of label in the test and/or control sections of the device indicates the subject does not have hypothyroidism or an increased risk of hypothyroidism. In some embodiments, absence of the label in the test and/or control sections indicates the subject has hypothyroidism or an increased risk of hypothyroidism.

[0255] In other embodiments, provided herein is a method for determining menopause or pre-menopause in a subject, comprising (1) applying a sample from the subject to the sample loading section of a lateral flow device described herein, (2) allowing the sample to flow through the lateral flow strip of the device, and (3) detecting the presence or absence of a label in the test and/or control sections of the device; wherein the analyte is follicle-stimulating hormone (FSH). In some embodiments, presence of the label in the test and/or control sections indicates the subject is menopausal or pre-menopausal. In some embodiments, absence of the label in the test and/or control sections indicates the subject is not menopausal or pre-menopausal. In some embodiments, the sample is urine. [0256] In some embodiments of any of the methods described herein, prior to step (1), the method comprises collecting a sample from a subject.

[0257] In some embodiments, the methods provided herein have greater sensitivity for detecting an analyte than a method using an IgG molecule that specifically binds to the same analyte.

[0258] In some embodiments, the methods provided herein have greater avidity for the analyte than a method using an IgG molecule that specifically binds to the same analyte.

[0259] In some embodiments of the methods provided herein, the binding molecules have a lower Koff rate for binding of the multimeric binding molecule to the analyte, than the Koff rate for binding of an IgG molecule to the same analyte.

[0260] In some embodiments of any of the methods disclosed herein, the IgG molecule comprises the same VH and VL sequences as the multimeric binding molecule.

[0261] In some embodiments of the methods provided herein, In some embodiments, the analyte is a variant infectious pathogen antigen, such as a variant coronavirus antigen, variant influenza virus antigen, or variant respiratory syncytial virus (RSV) antigen. For example, in some embodiments, the variant SARS-CoV-2 antigen comprises one or more substitutions found in alpha, beta, gamma, delta, eta, iota, kappa, lambda, 1.617.3, mu, theta, or zeta variant. In some embodiments, the multimeric binding molecules specifically bind to the variant infectious pathogen antigen, while an IgG molecule that comprises the same VH and VL sequences as the multimeric binding molecule does not.

[0262] In other embodiments, provided herein is a method for manufacturing a lateral flow device, comprising (1) assembling a lateral flow strip comprising a sample loading section, a conjugate section, an analysis section, and optionally an absorbent section; (2) optionally disposing a diffusively bound labeled multimeric binding molecule that specifically binds an analyte on the conjugate section; (3) immobilizing a test binding molecule that comprises an antigen binding domain that specifically binds the analyte on the test section of the analysis section; and (4) optionally immobilizing a control binding molecule that specifically binds the labeled multimeric binding molecule to the control section of the analysis section. In some embodiments, a sample pad is added to the sample loading section. In some embodiments, a conjugate pad is added to the conjugate section. In some embodiments, an absorbent pad is added to the absorbent section. In some embodiments, the lateral flow strip is disposed on a non-porous backing layer. In some embodiments, the lateral flow device is placed in an external casing.

[0263] This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL Press); Mullis etal. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1985) Nucleic Acid Hybridization (IRL Press); Hames and Higgins, eds. (1984) Transcription And Translation (IRL Press); Freshney (2016) Culture Of Animal Cells, 7th Edition (Wiley-Blackwell); Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985); Perbal (1988) A Practical Guide To Molecular Cloning; 2d Edition (Wiley-Interscience); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); S.C. Makrides (2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science); Methods in Enzymology, Vols. 151-155 (Academic Press, Inc., N.Y.); Mayer and Walker, eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Weir and Blackwell, eds.; and in Ausubel et al. (1995) Current Protocols in Molecular Biology (John Wiley and Sons).

[0264] General principles of antibody engineering are set forth, e.g., in Strohl, W.R., and L.M. Strohl (2012), Therapeutic Antibody Engineering (Woodhead Publishing). General principles of protein engineering are set forth, e.g., in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). General principles of immunology are set forth, e.g., in: Abbas and Lichtman (2017) Cellular and Molecular Immunology 9th Edition (Elsevier). Additionally, standard methods in immunology known in the art can be followed, e.g., in Current Protocols in Immunology (Wiley Online Library); Wild, D. (2013), The Immunoassay Handbook 4th Edition (Elsevier Science); Greenfield, ed. (2013), Antibodies, a Laboratory Manual, 2d Edition (Cold Spring Harbor Press); and Ossipow and Fischer, eds., (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press). [0265] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

Embodiments

[0266] Embodiment 1. A lateral flow device for detecting an analyte in a sample, comprising a lateral flow strip that comprises in series: a sample loading section, a conjugate section comprising a diffusively-bound labeled multimeric binding molecule that specifically binds to the analyte; an analysis section comprising in series; a test section comprising an immobilized test binding molecule that comprises an antigen-binding domain that specifically binds to the analyte, and a control section comprising an immobilized control binding molecule that specifically binds to the labeled multimeric binding molecule, wherein the labeled multimeric binding molecule comprises a label and five or six bivalent binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or multimerizing variants thereof, each associated with an antigen-binding domain, wherein the IgM heavy chain constant regions each comprise a Cp4 domain, and a p-tail piece (ptp) domain.

[0267] Embodiment 2. The lateral flow device of embodiment 1, wherein the labeled multimeric binding molecule is a hexamer and comprises six bivalent binding units.

[0268] Embodiment 3. The lateral flow device of embodiment 1, wherein the labeled multimeric binding molecule is a pentamer and comprises five bivalent binding units and a J-chain or a functional fragment thereof or a functional variant thereof.

[0269] Embodiment 4. The lateral flow device of embodiment 3, wherein the J-chain or functional fragment thereof, or functional variant thereof comprises the amino acid sequence of SEQ ID NOs: 3-47 or a functional fragment thereof, or a functional variant thereof.

[0270] Embodiment 5. The lateral flow device of embodiment 3 or embodiment 4, wherein the J-chain or functional fragment thereof, or functional variant thereof is a modified J- chain further comprising a heterologous moiety, wherein the heterologous moiety is fused or conjugated to the J-chain or functional fragment or functional variant thereof.

[0271] Embodiment 6. The lateral flow device of embodiment 5, wherein the heterologous moiety comprises the label. [0272] Embodiment 7. The lateral flow device of embodiment 5, wherein the heterologous moiety comprises a histidine (His) tag.

[0273] Embodiment 8. The lateral flow device of any one of embodiments 1 to 7, wherein the label comprises a colloidal gold nanoparticle, a colloidal silver nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a quantum dot, a magnetic particle, an up converting phosphor, a color dye, a fluorescent dye, or an oligonucleotide.

[0274] Embodiment 9. The lateral flow device of embodiment 8, wherein the label is attached to latex bead.

[0275] Embodiment 10. The lateral flow device of any one of embodiments 1 to 9, wherein each heavy chain constant region or fragment or variant thereof is associated with a copy of a heavy chain variable region (VH).

[0276] Embodiment 11. The lateral flow device of any one of embodiments 1 to 10, wherein each binding unit further comprises two light chains each comprising a light chain constant region or fragment or variant thereof, and wherein the light chain constant regions or fragments or variants thereof are associated with a copy of a light chain variable region (VL).

[0277] Embodiment 12. The lateral flow device of any one of embodiments 1 to 11, wherein the IgM heavy chain constant regions or fragments or variants thereof each further comprise a Cyl domain, a Cy2 domain, a Cy3 domain, or any combination thereof.

[0278] Embodiment 13. The lateral flow device of any one of embodiments 1 to 11, wherein the IgM heavy chain constant regions or multimerizing variant or fragment thereof each further comprise a Cpl domain, a Cp2 domain, a Cp3 domain, or any combination thereof.

[0279] Embodiment 14. The lateral flow device of any one of embodiments 1 to 11 or 13, wherein each IgM heavy chain constant region or multimerizing variant or fragment thereof is a human IgM constant region or multimerizing variant or fragment thereof, comprising the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or a multimerizing variant or fragment thereof.

[0280] Embodiment 15. The lateral flow device of any one of embodiments 1 to 11 or 13 to 14, each IgM heavy chain constant region or multimerizing variant or fragment thereof is a variant IgM constant region, wherein the multimeric binding molecule has reduced CDC activity relative to a multimeric binding molecule comprising IgM heavy chain constant regions comprising the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. [0281] Embodiment 16. The lateral flow device of embodiment 15, wherein each variant human IgM constant region comprises an amino acid substitution corresponding to position P311 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 of SEQ ID NO: 1 or SEQ ID NO: 2, or amino acid substitutions corresponding to positions K315 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0282] Embodiment 17. The lateral flow device of any one of embodiments 1 to 16, wherein the control and/or the test binding molecule is a monomeric antibody.

[0283] Embodiment 18. The lateral flow device of any one of embodiments 1 to 17, wherein the control and/or the test binding molecule is an IgG antibody.

[0284] Embodiment 19. The lateral flow device of any one of embodiments 1 to 16, wherein the control and/or the test binding molecule is a multimeric antibody.

[0285] Embodiment 20. The lateral flow device of any one of embodiments 1 to 19, wherein the control binding molecule specifically binds to the label, the light chain, the J-chain, or the heavy chain of the labeled multimeric binding molecule, or wherein the labeled multimeric binding molecule comprises a tag and the control binding molecule specifically binds to the tag.

[0286] Embodiment 21. The lateral flow device of embodiment 20, wherein the tag is a His tag attached to the J-chain.

[0287] Embodiment 22. The lateral flow device of any one of embodiments 1 to 21, wherein the sample is tissue, saliva, tears, urine, blood, sputum, sweat, vaginal sample, nasal sample, skin sample, semen, feces, mucous, or spinal fluid.

[0288] Embodiment 23. The lateral flow device of any one of embodiments 1 to 22, wherein the sample is a liquid sample.

[0289] Embodiment 24. The lateral flow device of any one of embodiments 1 to 23, wherein the analyte is a tumor antigen, a cardiac marker, an infectious pathogen antigen, a hormone, or a therapeutic drug.

[0290] Embodiment 25. The lateral flow device of embodiment 24, wherein the analyte is a tumor antigen, and wherein the tumor antigen is p53, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), or prostate specific antigen (PSA).

[0291] Embodiment 26. The lateral flow device of embodiment 24, wherein the analyte is a cardiac marker, and wherein the cardiac marker is troponin C, creatine kinase, or myoglobin. [0292] Embodiment 27. The lateral flow device of embodiment 24, wherein the analyte is an infectious pathogen antigen, and the infectious pathogen is a virus, bacterium, fungi, or parasite.

[0293] Embodiment 28. The lateral flow device of embodiment 24, wherein the infectious pathogen is a virus, and the virus is a coronavirus, an influenza virus, Dengue virus, Ebola virus, hepatitis virus, respiratory syncytial virus (RSV), human metapneumovirus, human immunodeficiency virus (HIV), herpes virus, paramyxovirus, or enterovirus.

[0294] Embodiment 29. The lateral flow device of embodiment 28, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2), or Middle East respiratory syndrome-related coronavirus (MERS-CoV).

[0295] Embodiment 30. The lateral flow device of embodiment 28, wherein the influenza virus is influenza A virus (IAV), influenza B virus (IBV), or influenza C virus (ICV).

[0296] Embodiment 31. The lateral flow device of embodiment 28, wherein the hepatitis virus is hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E.

[0297] Embodiment 32. The lateral flow device of embodiment 28, wherein the herpes virus is herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), roseolovirus, or Kaposi’s sarcoma-associated herpesvirus (KSHV).

[0298] Embodiment 33. The lateral flow device of embodiment 28, wherein the paramyxovirus is a parainfluenza virus (HPIV).

[0299] Embodiment 34. The lateral flow device of embodiment 28, wherein the enterovirus is a poliovirus, coxsackie virus A virus, coxsackie virus B, echovirus, or EV71.

[0300] Embodiment 35. The lateral flow device of embodiment 24, wherein the infectious pathogen is a bacterium, and the bacterium is Helicobacter pylori, Streptococcus pyogenes. Mycobacterium tuberculosis, Bacillus anthracis, Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Salmonella, Listeria, or Pseudomonas aeruginosa.

[0301] Embodiment 36. The lateral flow device of embodiment 24, wherein the analyte is a hormone, and the hormone is human chorionic gonadotropin (hCG) or luteinizing hormone. [0302] Embodiment 37. The lateral flow device of any one of embodiments 1 to 3636, wherein the liquid sample was pretreated to remove an existing component and/or add an additional component.

[0303] Embodiment 38. The lateral flow device of any one of embodiments 1 to 37, wherein the sample loading section comprises a buffer, non-specific protein, and/or a surfactant.

[0304] Embodiment 39. The lateral flow device of embodiment 38, wherein the buffer in the sample loading section comprises borate buffer.

[0305] Embodiment 40. The lateral flow device of embodiment 38 or embodiment 39, wherein the surfactant in the sample loading section comprises sodium dodecyl sulfate (SDS) and/or polysorbate.

[0306] Embodiment 41. The lateral flow device of any one of embodiments 38 to 40, wherein the non-specific protein in the sample loading section comprises albumin and/or casein.

[0307] Embodiment 42. The lateral flow device of any one of embodiments 1 to 41, wherein the conjugate section comprises a carbohydrate.

[0308] Embodiment 43. The lateral flow device of embodiment 42, wherein the carbohydrate is sucrose.

[0309] Embodiment 44. The lateral flow device of any one of embodiments 1 to 43, wherein the lateral flow strip further comprises an absorbent section after the analysis section.

[0310] Embodiment 45. The lateral flow device of embodiment 44, wherein the absorbent section comprises an absorbent pad comprising cellulose.

[0311] Embodiment 46. The lateral flow device of embodiment 45, wherein the absorbent pad is 0.45 mm to 2.50 mm thick.

[0312] Embodiment 47. The lateral flow device of any one of embodiments 44 to 46, wherein the analysis section and the absorbent section partially overlap.

[0313] Embodiment 48. The lateral flow device of any one of embodiments 1 to 47, wherein the sample loading section and the conjugate section and/or the conjugate section and the test section partially overlap.

[0314] Embodiment 49. The lateral flow device of embodiment 48, wherein the sample loading section comprises a sample pad comprising cellulose, rayon, and/or glass fiber.

[0315] Embodiment 50. The lateral flow device of embodiment 49, wherein the sample pad is 0.45 mm to 2.50 mm thick. [0316] Embodiment 51. The lateral flow device of any one of embodiments 1 to 50, wherein the conjugate section comprises a conjugate pad comprising glass fiber, cellulose, rayon, and/or polyester.

[0317] Embodiment 52. The lateral flow device of embodiment 51, wherein the conjugate pad is 0.45 mm to 2.50 mm thick.

[0318] Embodiment 53. The lateral flow device of any one of embodiments 1 to 52, wherein the analysis section comprises a porous membrane.

[0319] Embodiment 54. The lateral flow device of embodiment 53, wherein the porous membrane comprises nitrocellulose, nylon, polyvinylidene fluoride, or cellulose acetate.

[0320] Embodiment 55. The lateral flow device of embodiment 54, wherein the porous membrane comprises nitrocellulose.

[0321] Embodiment 56. The lateral flow device of any one of embodiments 53 to 55, wherein the porous membrane is 100 mm to 150 mm thick.

[0322] Embodiment 57. The lateral flow device of any one of embodiments 53 to 56, wherein the porous membrane has a pore size of 0.10 pm to 12 pm.

[0323] Embodiment 58. The lateral flow device of any one of embodiments 53 to 57, wherein the porous membrane has a capillary flow time of 60 to 240 seconds to traverse 4 cm.

[0324] Embodiment 59. The lateral flow device of any one of embodiments 53 to 58, wherein the control and test binding molecules are immobilized on the surface of the porous membrane.

[0325] Embodiment 60. The lateral flow device of any one of embodiments 1 to 59, wherein the sample loading section, the conjugate section, and the test section comprise a single monolithic hydrophilic matrix.

[0326] Embodiment 61. The lateral flow device of embodiment 60, wherein the single monolithic hydrophilic matrix comprises a network of fibers comprising a mixture of polymer and glass fiber or glass microfiber.

[0327] Embodiment 62. The lateral flow device of embodiment 60 or embodiment 61, wherein the control and test binding molecules are immobilized on the surface of the monolithic hydrophilic matrix.

[0328] Embodiment 63. The lateral flow device of embodiment 62, wherein the control and/or test binding molecules are indirectly immobilized on the surface. [0329] Embodiment 64. The lateral flow device of embodiment 63, wherein the control and/or test binding molecules are multimeric binding molecules comprising a J-chain, and the control and/or test binding molecules are indirectly attached to the surface through an antibody that bindings the J-chain or a moiety attached to the J-chain.

[0330] Embodiment 65. The lateral flow device of any one of embodiments 1 to 64, wherein the lateral flow strip further comprises a non-porous backing layer beneath the sample loading section, the conjugate section, and the analysis section.

[0331] Embodiment 66. The lateral flow device of embodiment 65, wherein the non-porous backing layer comprises polystyrene, polyvinyl chloride, and/or polyester.

[0332] Embodiment 67. The lateral flow device of any one of embodiments 1 to 66, further comprising an external casing.

[0333] Embodiment 68. The lateral flow device of embodiment 67, wherein the external casing does not fully enclose the sample loading section.

[0334] Embodiment 69. The lateral flow device of embodiment 67 or embodiment 68, wherein the external casing does not fully enclose the analysis section or is transparent above the test and control sections.

[0335] Embodiment 70. A method of detecting an analyte in a liquid sample comprising (1) applying a liquid sample to the sample loading section of the lateral flow device of any one of embodiments 1 to 69, (2) allowing the liquid sample to flow through the lateral flow strip of the lateral flow device, and (3) detecting the presence or absence of the label in the test and control sections of the lateral flow device.

[0336] Embodiment 71. The method of embodiment 70, wherein the liquid sample comprises urine, saliva, sputum, whole blood, plasma, serum, or an extract or a dilution thereof.

[0337] Embodiment 72. The method of embodiment 70 or embodiment 71, wherein prior to step (1), the method comprises removing a component from a sample, adjusting the pH of the sample, and/or diluting the sample, thereby generating the liquid sample.

[0338] Embodiment 73. A kit for detecting an analyte in a liquid sample comprising the lateral flow device of any one of embodiments 1 to 69, and instructions for use.

[0339] Embodiment 74. The kit of embodiment 73, further comprising a sample buffer. Examples

Example 1: Production and Labeling of IgG and IgM Antibodies

[0340] Ab2 (SEQ ID NOs: 53/54, Pinto et al. (2020) “Structural and functional analysis of a potent sarbecovirus neutralizing antibody” bioRxiv doi: 10.1101/2020.04.07.023903), AblO (SEQ ID NOs: 55/56, Liu et al. (2020) Nature 584:450-56), and AB14 (SEQ ID NOs: 57/58, Cao et al. (2022) “Omicron BA.2 specifically evades broad sarbecovirus neutralizing antibodies” bioRxiv doi: 10.1101/2022.02.07.479349) are three antibodies that specifically bind to the RBD domain of SARS-CoV-2 Spike Protein. IgG and IgM versions of Ab2 (FIG. 4B), AblO (FIG. 5 A), and Ab 14 (FIG. 4A) were produced and purified using recombinant protein techniques. See, e.g., PCT Publication No. WO 2022026475, which is incorporated herein by reference in its entirety. Ab2-IgG, Ab2-IgM, AblO-IgG, AblO-IgM, Abl4-IgG, and Abl4-IgM were labeled with horseradish peroxidase (HRP) using Mix-n-Stain HRP Antibody Labeling Kit (Biotium, catalog number 92302) following the manufacturer’s protocol. The efficiency of the HRP-labeling on each antibody was tested in ELISA assay to ensure comparable labeling efficiency.

Example 2: Virion Capture ELISA Assay

[0341] To demonstrate binding of multimeric IgM antibodies specifically to a viral antigen as they would in a lateral flow device, a virus binding ELISA assay (FIG. 6) was developed using HRP-labeled (126) IgM antibodies (116). The ELISA assay was first applied to the detection of SARS-CoV-2 Spike protein where VSVM-S2 (VectorBuilder, catalog number VB010000-9314gcp), a pseudotyped VSV (vesicular stomatitis virus) overexpressing the wild-type SARS-CoV-2 Spike protein, was used as the representation of a viral analyte (124). The ELISA plate was coated (FIG. 6A, 118) with a rabbit polyclonal anti-Spike antibody (Invitrogen, catalog number PA5-120736) overnight. Subsequently 1:2 serial dilutions of approximately 4.60*10⁸ pfu/mL of VSVM-S2 (FIG. 6A, 124) was added to the ELISA plate for capture (FIG. 6A, third analyte from the left) on the polyclonal anti-Spike coating. The ELISA plate was then washed to remove the unbound analytes. HRP-labeled (FIG. 6B, 126) Ab2-IgG, Ab2-IgM, AblO-IgG, AblO-IgM, Abl4- IgG or Abl4-IgM (116) was added to the plate for detection of captured viral particles. Bound HRP (FIG. 6C) was visualized using ADHP substrate, QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit (Thermo Scientific, 15159). For all three antibodies tested, the IgM version of the antibody produced a stronger signal than the corresponding IgG version (FIGs. 3 & 4).

[0342] The virion capture ELISA assay developed in this study was also applied using chimeras of antgiotension-converting enzyme 2 (ACE2) fused with IgG or IgM constant domains, to demonstrate improved avidity binding of IgM based detection agents. The ACE2-h-IgM (SEQ ID NO:65) chimera is described in WO2022026475. The ACE-IgG (SEQ ID NO:66) comprises the extracellular domain of ACE2 fused to the CH2-CH3 domains of a human IgGl , with a mutation in the y hinge domain. The viral antigen applied to ACE2 fusion protein binding ELISA was a pseudotyped VSV expressing SARS-CoV2 Spike protein beta variant, VSVM-S2J3 (Vector Builder, VB010000-9315gcp beta). The ELISA plate was coated with a rabbit polyclonal anti-Spike antibody (Invitrogen, catalog number PA5-120736) overnight. Subsequently 1:2 serial dilutions of approximately 4.70*10⁸ pfu/mL of VSVM-S2J3 as added to the ELISA plate and captured by the coated polyclonal anti-Spike antibody. The ELISA plate was then washed to remove the unbound analytes. HRP-labeled ACE2-IgG or ACE2-h-IgM was then added to the plate for the detection of captured virial particles and visualized as above. As with the antibodies above, the IgM chimera produced a stronger ELISA signal than the corresponding IgG chimera (FIG. 4B).

[0343] The virion capture ELISA assay developed in this study was also applied for detecting other viruses, for example, using RSV (respiratory syncytial virus) and PIV3 (parainfluenza virus type 3) as viral analytes. RSV-RFP1 virus (ViraTree, R131) was used as the viral agent in the RSV capture ELISA assay. The ELISA plate was coated with an RSV glycoprotein G monoclonal antibody 8C5 (Invitrogen MAI-83436) overnight. Subsequently 1:2 serial dilutions of approximately 6.52* 10⁶ pfu/mL of RSV-RFP1 was added to the ELISA plate and captured by the coated monoclonal anti-RSV glycoprotein G antibody. The ELISA plate was then washed to remove the unbound analytes. HRP- labeled anti-RSV antibody, Abl5 (SEQ ID NOs: 59/60, Tang etal. (2019) Nature Comm. 10:4153) in an IgGl or IgM isotype was then added to the plate for the detection of captured virial particles using ADHP substrate, QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit (Thermo Scientific, 15159). The IgM version of Abl5 produced a stronger signal than the corresponding IgG version (FIG. 5A).

[0344] PIV3-GFP virus (ViraTree, P3231) was used as the viral agent in the PIV3 capture ELISA assay. The ELISA plate was coated with an anti-PIV3 monoclonal antibody Abl7 (SEQ ID NOs: 63/64, WO 22/183018) overnight. Subsequently 1:2 serial dilutions of approximately 8.30*10⁸ pfu/mL of PIV3-GFP as added to the ELISA plate and captured by the coated Abl7 monoclonal antibody. The ELISA plate was then washed to remove the unbound analytes. An HRP-labeled version of Abl6 (SEQ ID NOs: 61/62, WO 22/183018) — a second, anti-PIV3 monoclonal antibody, which does not compete with the capture by the Abl7 — was then added to the plate for the detection of captured virial particles using ADHP substrate, QuantaRed™ Enhanced Chemifluorescent HRP Substrate Kit (Thermo Scientific, 15159). The IgM version of Abl6 produced a stronger signal than the corresponding IgG version (FIG. 5B).

Example 3 : Chimera Binding Kinetics

[0345] The improved avidity binding of IgM antibody constructs over corresponding IgG constructs on the recombinant viral protein targets was also demonstrated with Bio-Layer Interferometry (BLI) Octet assay. 500 ng/mL of SARS-CoV2 B.1.1.529 Omicron Spike RBD protein with His tag (Sino Biological, 40592-V08H121) was immobilized onto the surface of HIS IK sensors, and then exposed to 3-fold serial dilutions — starting from 200 pg/mL — of ACE2-h-IgM (FIG. 7A labels the 200 pg/mL sample as “59.3,” the 66.7 pg/mL sample as “19.8,” etc.) and ACE2-IgG (FIG. 7B labels the 200 pg/mL sample as “303,” the 66.7 pg/mL sample as “101,” etc.). Dissociation was performed in lx PBS with 0.1% BSA and 0.02% Tween 20 (10x Kinetic buffer). Sensors were regenerated in 10 mM Glycine pH 3.0. A bivalent model was used for kinetic curve fitting. ACE2-h-IgM has > 5 -fold faster Ka and > 1000-fold slower K is compared to ACE2-IgG. The results are shown in Table 2 below.

Table 3: Sequences in the Disclosure

Claims

WHAT IS CLAIMED IS:

1. A lateral flow device for detecting an analyte in a sample, comprising a lateral flow strip that comprises in series:

(a) a sample loading section,

(b) a conjugate section comprising a diffusively-bound labeled multimeric binding molecule that specifically binds to the analyte;

(c) an analysis section comprising in series;

(i) a test section comprising an immobilized test binding molecule that comprises an antigen-binding domain that specifically binds to the analyte, and

(ii) a control section comprising an immobilized control binding molecule that specifically binds to the labeled multimeric binding molecule, wherein the labeled multimeric binding molecule comprises a label and five or six bivalent binding units, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or multimerizing variants thereof, each associated with an antigen-binding domain, wherein the IgM heavy chain constant regions each comprise a Cp4 domain, and a pi-tail piece (ptp) domain.

2. The lateral flow device of claim 1, wherein the labeled multimeric binding molecule is a hexamer and comprises six bivalent binding units.

3. The lateral flow device of claim 1, wherein the labeled multimeric binding molecule is a pentamer and comprises five bivalent binding units and a J-chain or a functional fragment thereof or a functional variant thereof.

4. The lateral flow device of claim 3, wherein the J-chain or functional fragment thereof, or functional variant thereof comprises the amino acid sequence of SEQ ID NOs: 3-47 or a functional fragment thereof, or a functional variant thereof.

5. The lateral flow device of claim 3 or claim 4, wherein the J-chain or functional fragment thereof, or functional variant thereof is a modified J-chain further comprising a heterologous moiety, wherein the heterologous moiety is fused or conjugated to the J-chain or functional fragment or functional variant thereof.

6. The lateral flow device of claim 5, wherein the heterologous moiety comprises the label.

7. The lateral flow device of claim 5, wherein the heterologous moiety comprises a histidine (His) tag.

8. The lateral flow device of any one of claims 1 to 7, wherein the label comprises a colloidal gold nanoparticle, a colloidal silver nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a quantum dot, a magnetic particle, an up converting phosphor, a color dye, a fluorescent dye, or an oligonucleotide.

9. The lateral flow device of claim 8, wherein the label is attached to latex bead.

10. The lateral flow device of any one of claims 1 to 9, wherein each heavy chain constant region or fragment or variant thereof is associated with a copy of a heavy chain variable region (VH).

11. The lateral flow device of any one of claims 1 to 10, wherein each binding unit further comprises two light chains each comprising a light chain constant region or fragment or variant thereof, and wherein the light chain constant regions or fragments or variants thereof are associated with a copy of a light chain variable region (VL).

12. The lateral flow device of any one of claims 1 to 11, wherein the IgM heavy chain constant regions or fragments or variants thereof each further comprise a Cyl domain, a Cy2 domain, a Cy3 domain, or any combination thereof.

13. The lateral flow device of any one of claims 1 to 11, wherein the IgM heavy chain constant regions or multimerizing variant or fragment thereof each further comprise a Cpl domain, a Cp2 domain, a Cp3 domain, or any combination thereof.

14. The lateral flow device of any one of claims 1 to 11 or 13, wherein each IgM heavy chain constant region or multimerizing variant or fragment thereof is a human IgM constant region or multimerizing variant or fragment thereof, comprising the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or a multimerizing variant or fragment thereof.

15. The lateral flow device of any one of claims 1 to 11 or 13 to 14, each IgM heavy chain constant region or multimerizing variant or fragment thereof is a variant IgM constant region, wherein the multimeric binding molecule has reduced CDC activity relative to a multimeric binding molecule comprising IgM heavy chain constant regions comprising the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.

16. The lateral flow device of claim 15, wherein each variant human IgM constant region comprises an amino acid substitution corresponding to position P311 of SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid substitution corresponding to position P313 of SEQ ID NO: 1 or SEQ ID NO: 2, or amino acid substitutions corresponding to positions K315 of SEQ ID NO: 1 or SEQ ID NO: 2.

17. The lateral flow device of any one of claims 1 to 16, wherein the control and/or the test binding molecule is a monomeric antibody.

18. The lateral flow device of any one of claims 1 to 17, wherein the control and/or the test binding molecule is an IgG antibody.

19. The lateral flow device of any one of claims 1 to 16, wherein the control and/or the test binding molecule is a multimeric antibody.

20. The lateral flow device of any one of claims 1 to 19, wherein the control binding molecule specifically binds to the label, the light chain, the J-chain, or the heavy chain of the labeled multimeric binding molecule, or wherein the labeled multimeric binding molecule comprises a tag and the control binding molecule specifically binds to the tag.

21. The lateral flow device of claim 20, wherein the tag is a His tag attached to the J-chain.

22. The lateral flow device of any one of claims 1 to 21, wherein the sample is tissue, saliva, tears, urine, blood, sputum, sweat, vaginal sample, nasal sample, skin sample, semen, feces, mucous, or spinal fluid.

23. The lateral flow device of any one of claims 1 to 22, wherein the sample is a liquid sample.

24. The lateral flow device of any one of claims 1 to 23, wherein the analyte is a tumor antigen, a cardiac marker, an infectious pathogen antigen, a hormone, or a therapeutic drug.

25. The lateral flow device of claim 24, wherein the analyte is a tumor antigen, and wherein the tumor antigen is p53, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), or prostate specific antigen (PSA).

26. The lateral flow device of claim 24, wherein the analyte is a cardiac marker, and wherein the cardiac marker is troponin C, creatine kinase, or myoglobin.

27. The lateral flow device of claim 24, wherein the analyte is an infectious pathogen antigen, and the infectious pathogen is a virus, bacterium, fungi, or parasite.

28. The lateral flow device of claim 24, wherein the infectious pathogen is a virus, and the virus is a coronavirus, an influenza virus, Dengue virus, Ebola virus, hepatitis virus, respiratory syncytial virus (RSV), human metapneumovirus, human immunodeficiency virus (HIV), herpes virus, paramyxovirus, or enterovirus.

29. The lateral flow device of claim 28, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2), or Middle East respiratory syndrome-related coronavirus (MERS-CoV).

30. The lateral flow device of claim 28, wherein the influenza virus is influenza A virus (IAV), influenza B virus (IBV), or influenza C virus (ICV).

31. The lateral flow device of claim 28, wherein the hepatitis virus is hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E.

32. The lateral flow device of claim 28, wherein the herpes virus is herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), roseolovirus, or Kaposi’s sarcoma- associated herpesvirus (KSHV).

33. The lateral flow device of claim 28, wherein the paramyxovirus is a parainfluenza virus (HPIV).

34. The lateral flow device of claim 28, wherein the enterovirus is a poliovirus, coxsackie virus A virus, coxsackie virus B, echovirus, or EV71.

35. The lateral flow device of claim 24, wherein the infectious pathogen is a bacterium, and the bacterium is Helicobacter pylori, Streptococcus pyogenes. Mycobacterium tuberculosis, Bacillus anthracis, Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Salmonella, Listeria, or Pseudomonas aeruginosa.

36. The lateral flow device of claim 24, wherein the analyte is a hormone, and the hormone is human chorionic gonadotropin (hCG) or luteinizing hormone.

37. The lateral flow device of any one of claims 1 to 3636, wherein the liquid sample was pretreated to remove an existing component and/or add an additional component.

38. The lateral flow device of any one of claims 1 to 37, wherein the sample loading section comprises a buffer, non-specific protein, and/or a surfactant.

39. The lateral flow device of claim 38, wherein the buffer in the sample loading section comprises borate buffer.

40. The lateral flow device of claim 38 or claim 39, wherein the surfactant in the sample loading section comprises sodium dodecyl sulfate (SDS) and/or polysorbate.

41. The lateral flow device of any one of claims 38 to 40, wherein the non-specific protein in the sample loading section comprises albumin and/or casein.

42. The lateral flow device of any one of claims 1 to 41, wherein the conjugate section comprises a carbohydrate.

43. The lateral flow device of claim 42, wherein the carbohydrate is sucrose.

44. The lateral flow device of any one of claims 1 to 43, wherein the lateral flow strip further comprises an absorbent section after the analysis section.

45. The lateral flow device of claim 44, wherein the absorbent section comprises an absorbent pad comprising cellulose.

46. The lateral flow device of claim 45, wherein the absorbent pad is 0.45 mm to 2.50 mm thick.

47. The lateral flow device of any one of claims 44 to 46, wherein the analysis section and the absorbent section partially overlap.

48. The lateral flow device of any one of claims 1 to 47, wherein the sample loading section and the conjugate section and/or the conjugate section and the test section partially overlap.

49. The lateral flow device of claim 48, wherein the sample loading section comprises a sample pad comprising cellulose, rayon, and/or glass fiber.

50. The lateral flow device of claim 49, wherein the sample pad is 0.45 mm to 2.50 mm thick.

51. The lateral flow device of any one of claims 1 to 50, wherein the conjugate section comprises a conjugate pad comprising glass fiber, cellulose, rayon, and/or polyester.

52. The lateral flow device of claim 51, wherein the conjugate pad is 0.45 mm to 2.50 mm thick.

53. The lateral flow device of any one of claims 1 to 52, wherein the analysis section comprises a porous membrane.

54. The lateral flow device of claim 53, wherein the porous membrane comprises nitrocellulose, nylon, polyvinylidene fluoride, or cellulose acetate.

55. The lateral flow device of claim 54, wherein the porous membrane comprises nitrocellulose.

56. The lateral flow device of any one of claims 53 to 55, wherein the porous membrane is 100 mm to 150 mm thick.

57. The lateral flow device of any one of claims 53 to 56, wherein the porous membrane has a pore size of 0.10 pm to 12 pm.

58. The lateral flow device of any one of claims 53 to 57, wherein the porous membrane has a capillary flow time of 60 to 240 seconds to traverse 4 cm.

59. The lateral flow device of any one of claims 53 to 58, wherein the control and test binding molecules are immobilized on the surface of the porous membrane.

60. The lateral flow device of any one of claims 1 to 59, wherein the sample loading section, the conjugate section, and the test section comprise a single monolithic hydrophilic matrix.

61. The lateral flow device of claim 60, wherein the single monolithic hydrophilic matrix comprises a network of fibers comprising a mixture of polymer and glass fiber or glass microfiber.

62. The lateral flow device of claim 60 or claim 61, wherein the control and test binding molecules are immobilized on the surface of the monolithic hydrophilic matrix.

63. The lateral flow device of claim 62, wherein the control and/or test binding molecules are indirectly immobilized on the surface.

64. The lateral flow device of claim 63, wherein the control and/or test binding molecules are multimeric binding molecules comprising a J-chain, and the control and/or test binding molecules are indirectly attached to the surface through an antibody that bindings the J-chain or a moiety attached to the J-chain.

65. The lateral flow device of any one of claims 1 to 64, wherein the lateral flow strip further comprises a non-porous backing layer beneath the sample loading section, the conjugate section, and the analysis section.

66. The lateral flow device of claim 65, wherein the non-porous backing layer comprises polystyrene, polyvinyl chloride, and/or polyester.

67. The lateral flow device of any one of claims 1 to 66, further comprising an external casing.

68. The lateral flow device of claim 67, wherein the external casing does not fully enclose the sample loading section.

69. The lateral flow device of claim 67 or claim 68, wherein the external casing does not fully enclose the analysis section or is transparent above the test and control sections.

70. A method of detecting an analyte in a liquid sample comprising (1) applying a liquid sample to the sample loading section of the lateral flow device of any one of claims 1 to 69, (2) allowing the liquid sample to flow through the lateral flow strip of the lateral flow device, and (3) detecting the presence or absence of the label in the test and control sections of the lateral flow device.

71. The method of claim 70, wherein the liquid sample comprises urine, saliva, sputum, whole blood, plasma, serum, or an extract or a dilution thereof.

72. The method of claim 70 or claim 71, wherein prior to step (1), the method comprises removing a component from a sample, adjusting the pH of the sample, and/or diluting the sample, thereby generating the liquid sample.

73. A kit for detecting an analyte in a liquid sample comprising the lateral flow device of any one of claims 1 to 69, and instructions for use.

74. The kit of claim 73, further comprising a sample buffer.