Cross-neutralizing rubulavirus binding domains and uses thereof

Abstract

Binding domains that bind and can neutralize Rubulaviruses such as Human Parainfluenza Virus (HPIV) 2 and HPIV4. The binding domains include monoclonal antibodies: 2x4-Ka and 2x4-La. Each of these binding domains can be engineered into numerous formats for research, detection, and or treatment of Rubulaviruses, individually and in combination.

Description

F053-0199PCT / 25-069-WO-PCTCROSS-NEUTRALIZING RUBULAVIRUS BINDING DOMAINS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 745,015 filed January 14, 2025, the entire contents of which are incorporated by reference herein.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 3K05382.XML. The XML file is 139,264 bytes, was created on January 13, 2026, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The current disclosure provides binding domains that bind and can neutralize Rubulaviruses such as Human Parainfluenza Virus (HPIV) 2 and HPIV4. The binding domains include monoclonal antibodies: 2x4-Ka and 2x4-La. Each of these binding domains can be engineered into numerous formats for research, detection, and or treatment of Rubulaviruses, individually and in combination.BACKGROUND OF THE DISCLOSURE

[0004] Human parainfluenza virus types 2 (HPIV2) and 4 (HPIV4) are ubiquitous and cause acute respiratory illness. HPIV2, HPIV4, and several related viruses in bats and pigs that can spillover into humans make up the Rubulavirinae subfamily. Human disease ranges from mild upper respiratory tract symptoms to life-threatening lower respiratory tract disease. Over 90% of adults have antibodies to the parainfluenza viruses, but these antibodies are not fully protective, and reinfections frequently occur. Severe disease is more common in the elderly, immunocompromised individuals, and patients with cardiac and pulmonary comorbidities. Among immunocompromised patients, the highest morbidity and mortality has been described in hematopoietic cell transplant (HOT) recipients, patients with leukemia, and lung transplant recipients. Over a third of the infections in HCT recipients progress to the lower respiratory tract, and mortality after the virus reaches the lungs ranges from 13-63%.SUMMARY OF THE DISCLOSURE

[0005] The current disclosure provides binding domains that bind and can neutralize Rubulaviruses such as Human Parainfluenza Virus (HPIV) 2 and HPIV4. The binding domains include monoclonal antibodies: 2x4-Ka and 2x4-La. Each of these binding domains can be engineered into numerous formats for research, detection, and or treatment of Rubulaviruses, individually and in combination.

[0006] In particular embodiments, a binding domain from 2x4-Ka includes a variable heavy chain including a complementarity determining region (CDR) heavy (H)1 including the sequence as set forth in SEQ ID NO: 4, a CDRH2F053-0199PCT / 25-069-WO-PCTincluding the sequence as set forth in SEQ ID NO: 5, and a CDRH3 including the sequence as set forth in SEQ ID NO: 6; and a variable light chain including a CDR light (L)1 including the sequence as set forth in SEQ ID NO: 7, a CDRL2 including the sequence as set forth in SEQ ID NO: 8, and a CDRL3 including the sequence as set forth in SEQ ID NO: 9, according to IMGT.

[0007] In particular embodiments, a binding domain from 2x4-Ka includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 48 and a variable light chain including the sequence as set forth in SEQ ID NO: 49.

[0008] In particular embodiments, a binding domain from 2x4-La includes a variable heavy chain including a CDRH1 including the sequence as set forth in SEQ ID NO: 26, a CDRH2 including the sequence as set forth in SEQ ID NO: 27, and a CDRH3 including the sequence as set forth in SEQ ID NO: 28; and a variable light chain including a CDRL1 including the sequence as set forth in SEQ ID NO: 29, a CDRL2 including the sequence as set forth in SEQ ID NO: 30, and a CDRL3 including the sequence as set forth in SEQ ID NO: 31, according to IMGT.

[0009] In particular embodiments, a binding domain from 2x4-La includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 52 and a variable light chain including the sequence as set forth in SEQ ID NO: 53.

[0010] In particular embodiments, the binding domains described herein can be used to neutralize Rubulavirus in a subject in need thereof. In particular embodiments, the binding domains described herein can be used to neutralize HPIV2 in a subject in need thereof. In particular embodiments, the binding domains described herein can be used to neutralize HPIV4 in a subject in need thereof.BRIEF DESCRIPTION OF THE FIGURES

[0011] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0012] FIGs. 1A, 1B Binding kinetics of monoclonal antibodies (mAbs) targeting the Human Parainfluenza Virus 4(HPI V4) fusion (F) protein. Binding was measured using biolayer interferometry and anti-His capture probes to load HPIV4 F protein that was stabilized in the pre-fusion (preF) conformation. Binding between HPIV4 preF and the 2x4-KaF (1A) and 2x4-LaF (1B) mAbs.

[0013] FIG. 2. In vitro neutralization of HPIV2 and HPIV4 by the 2x4 mAbs. Plaques of HPIV2 (solid lines) and HPIV4 (dotted lines) were counted on day 3 or day 4 post-infection of Vero cells and expressed as a percentage of plaques in control wells that did not contain any mAb.

[0014] FIGs. 3A, 3B. Neutralization assay with (3A) 2x4-KaF and (3B) 2x4-LaF of HPIV2, Mumps, and PIV5. 2x4 mAbs were serially diluted with virus, inoculated onto Vero cells and immunostained. 2x4 mAbs bind, but do not neutralize Mumps or PIV5.F053-0199PCT / 25-069-WO-PCT

[0015] FIG. 4. Biolayer interferometry of binding between 2x4-KaF mAb and the Mumps F protein. Binding to the Mumps F protein stabilized in the prefusion conformation.

[0016] FIG. 5. In vitro neutralization potencies of 2x4-KaF and 2x4-LaF against HPIV2 and HPIV4. The following provides mAb / virus / IC50 (ng / mL): 2x4-KaF I HPIV21233; 2x4-LaF I HPIV2 / 1731; 2x4-KaF / HPIV4 / 50; 2x4-LaF I HPIV4 / 48.

[0017] FIG. 6. In vivo efficacy of 2x4 mAb as prophylaxis for HIPV2 in hamsters. Hamsters were injected with 2x4-KaF mAb intramuscularly and inoculated two days later intranasally with 104pfu of HPIV2.

[0018] FIG. 7. Structural analysis by nsEM depicting a three-dimensional (3D) map of HPIV4 preF and 2x4-LaF.

[0019] FIG. 8. The 2x4 mAbs do not neutralize mumps or PIV5 at 200 pig / mL, even with the addition of complement.

[0020] FIG. 9. Binding by ELISA of 2x4 antibodies to viral fusion proteins stabilized in the prefusion conformation.2X4 kappa OD50 for mumps: 404 pig / mL , PIV5400 pig / mL, HPIV4406 pig / mL; 2X4 lambda OD50 for mumps: 407 pig / mL , PIV5403 g / mL, HPIV4404 g / mL.

[0021] FIG. 10. Sequences supporting the disclosure.DETAILED DESCRIPTION

[0022] Human parainfluenza virus types 2 (HPIV2) and 4 (HPIV4) are ubiquitous and cause acute respiratory illness. HPIV2, HPIV4, and several related viruses in bats and pigs that can spillover into humans make up the Rubulavirinae subfamily. Human disease ranges from mild upper respiratory tract symptoms to life-threatening lower respiratory tract disease. Over 90% of adults have antibodies to the parainfluenza viruses, but these antibodies are not fully protective, and reinfections frequently occur. Severe disease is more common in the elderly, immunocompromised individuals, and patients with cardiac and pulmonary comorbidities. Among immunocompromised patients, the highest morbidity and mortality has been described in hematopoietic cell transplant (HCT) recipients, patients with leukemia, and lung transplant recipients. Over a third of the infections in HCT recipients progress to the lower respiratory tract, and mortality after the virus reaches the lungs ranges from 13-63%.

[0023] The current disclosure provides binding domains that bind and can neutralize Rubulaviruses such as Human Parainfluenza Virus (HPIV) 2 and HPIV4. The binding domains include monoclonal antibodies: 2x4-Ka and 2x4-La. Each of these binding domains can be engineered into numerous formats for research, detection, and or treatment of Rubulaviruses, individually and in combination.

[0024] In particular embodiments, a binding domain from 2x4-Ka includes a variable heavy chain including a complementarity determining region (CDR) heavy (H)1 including the sequence as set forth in SEQ ID NO: 4, a CDRH2 including the sequence as set forth in SEQ ID NO: 5, and a CDRH3 including the sequence as set forth in SEQ ID NO: 6; and a variable light chain including a CDR light (L)1 including the sequence as set forth in SEQ ID NO: 7, a CDRL2 including the sequence as set forth in SEQ ID NO: 8, and a CDRL3 including the sequence as set forth in SEQ ID NO: 9, according to IMGT. CDRs according to other models are provided elsewhere herein.F053-0199PCT / 25-069-WO-PCT

[0025] In particular embodiments, a binding domain from 2x4-Ka includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 48 and a variable light chain including the sequence as set forth in SEQ ID NO: 49.

[0026] In particular embodiments, a binding domain from 2x4-La includes a variable heavy chain including a CDRH1 including the sequence as set forth in SEQ ID NO: 26, a CDRH2 including the sequence as set forth in SEQ ID NO: 27, and a CDRH3 including the sequence as set forth in SEQ ID NO: 28; and a variable light chain including a CDRL1 including the sequence as set forth in SEQ ID NO: 29, a CDRL2 including the sequence as set forth in SEQ ID NO: 30, and a CDRL3 including the sequence as set forth in SEQ ID NO: 31, according to IMGT. CDRs according to other models are provided elsewhere herein.

[0027] In particular embodiments, a binding domain from 2x4-La includes a variable heavy chain including the sequence as set forth in SEQ ID NO: 52 and a variable light chain including the sequence as set forth in SEQ ID NO: 53.

[0028] In particular embodiments, the binding domains described herein can be used to neutralize Rubulavirus in a subject in need thereof. In particular embodiments, the binding domains described herein can be used to neutralize HPIV2 in a subject in need thereof. In particular embodiments, the binding domains described herein can be used to neutralize HPIV4 in a subject in need thereof.

[0029] The binding domains disclosed herein can inhibit or neutralize Rubulavirus infection and can be engineered into numerous formats for research, detection, and / or treatment purposes.

[0030] Aspects of the current disclosure are now described in more supporting detail as follows: (I) Antibodies; (II) Antibody Variants; (III) Multi-Domain Binding Molecules; (IV) Recombinant Production; (V) Antibody Conjugates; (VI) Recombinant Receptors; (VII) Compositions or Formulations (VIII) Kits; (IX) Methods of Use; (X) Exemplary Embodiments; (XI) Examples; and (XII) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0031] (I) Antibodies. The present disclosure provides binding domains which can be in the form of antibodies that bind Rubulaviruses. In particular embodiments, the Rubulaviruses include HPIV2 and HPIV4. In particular embodiments, the binding domains disclosed herein bind the fusion protein of HPIV2 and the fusion protein of HPIV4. In particular embodiments, HPIV4 includes HPIV4a and / or HPI V4b.

[0032] In particular embodiments, the fusion protein of HPIV2 [Human orthorubulavirus 2] (GenBank: WMD01087.1) includes the sequence:MYHLHPMIVCIFVMYTGIVGSDAIAGDQLLNVGVIQSKIRSLMYYTDGSASFIWKLLPNLPPSNGTCNITSLDAYNVTLF KLLTPLIENLSKISAVTDTKPRRERFAGWIGLAALGVATAAQITAAVAIVKANANAAAINNLASSIQSTNKAVSDVITASR TIATAVQAIQDHINGAIVNGITSASCRAHDALIGSILNLYLTELTTIFHNQITNPALTPLSIQALRILLGSTLPIVIESKLNTKL NTAELLSSGLLTGQIISISPMYMQMLIQINVPTFIMQPGAKVIDLIAISANHKLQEVWQVPNRILEYANELQNYPANDCV VTPNSVFCRYNEGSPIPESQYQCLRGNLNSCTFTPIIGNFLKRFAFANGVLYANCKSLLCKCADPPHWSQDDTQGISIF053-0199PCT / 25-069-WO-PCTIDIKRCSEMMLDTFSFRITSTFNATYVTDFSMINANIVHLSPLDLSNQINSINKSLKSAEDWIADSNFLANQARTAKTLYS LSAIALILSVITLVWGLLIAYIVKLVSQIHQFRALAATTMFHRENPAVFSKNNHGNIYGIS (SEQ ID NO: 1).

[0033] In particular embodiments, the fusion protein of HPIV4a (Genbank: AGU90035.1) includes the sequence: MGVKGSSLIMIGLLISPITNLDITHLMNLGTVPTAIRSLVYYTYTKPSYLTVDLIPNLKNLDQNCNYSSLNYYNKTALSLIQ PIADNINRLTKPITSSEVQSRFFGAVIGTIALGVATAAQVTAAIGLAKAQENAKLILTLKKAATETNEAVRDLANSNKIWK MISAIQNQINTIIQPAIDQINCQIKDLQVANILNLYLTEITTVFHNQLTNPALESISIQALKSLLGSTLPEVLSKLDLNNISAAS VMASGLIKGQIIAVDIPTMTLVLMVQIPSISPLRQAKIIDLTSITIHTNSQEVQAVVPARVLEIGSEILGFDGSVCQITKDTV FCPYNDAYVLPIQQKRCLQGQTRDCVFTPVAGTFPRRFLTTYGTIVANCRDLVCSCLRPPQIIYQPDENPVTIIDKDLC TTLTLDSITIEIQKSINSTFRREWLESTQVRSLTPLDLSTDLNQYNQLLKSAEDHIQRSTDYLNSINPSIVNNNAIIILIILCI LLILTVTICIIWLKYLTNEVKNVARNQRLNRDADLFHRIPSQIPVPRQ (SEQ ID NO: 2).

[0034] In particular embodiments, the fusion protein of HPI V4b (Genbank: WMD01093.1) includes the sequence: MGVKSLSLVMIGLLISPITNLDITHLMNLGTVPTAIRSLVYYTYSKPSYLTVDLIPNLKNLDPNCNYSSLNYYNKTVLSLIQ PIADNINHLTKPITSSEIQSRFFGAVIGTVALGVATAAQVTAAIGLAKAQENARLILKLKKAAEETNDAVRDLIESNKIVAR MISAIQNQINTVIQPAINRIDCQIKDLQAANILNLYLTEITTVFHNQLTNPALESISIQALKSLLGSTLPEVLSKLDLNNISAA SVMASGLIKGQIIAVDIPTMTLVLMVQIPSISPLRQAKIMDLTSITIHTNNQEVQAWPDRVLEIGSEILGFDGSVCQITKD TVFCPYNDAYILPIQQKRCLQGQTRDCVFTPVAGTFPRRFLTTYGTIVANCRNLVCSCLRPPQIIYQPDETPVTIIDKDL CTTLTLDSITIEIQKSINSTFRREWLESTQVRSLTSLDLSTDLSQYNQLLKSAEDHIQRSNDYLNSINPSIVNNNAIIILIILC ILLILTVTICIIWLKYLTKEVRNVARNQRLNRDADLIYKIPSQIPLPR (SEQ ID NO: 3).

[0035] Naturally occurring antibody structural units include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FRA

[0036] The assignment of amino acids to each domain can be in accordance with Kabat numbering (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia" numbering scheme)), Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum ef al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745." (Contact numbering scheme)), IMGT (Lefranc M P ef al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme)), AHo (Honegger A and Pluckthun A, “Yet another numberingF053-0199PCT / 25-069-WO-PCTscheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North etal., J Mol Biol. 406(2):228-256 (2011), "A new clustering of antibody CDR loop conformations”), or other numbering schemes. Software programs, such as ABody Builder can also be used.

[0037] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs and bioinformatical tools, such as ABody Builder and Paratome can also be used to determine CDR sequences.

[0038] In particular embodiments, a binding domain that binds a Rubulavirus includes the binding domain of a 2x4-Ka antibody.

[0039] In particular embodiments, a 2x4-Ka binding domain includes a variable heavy chain including a CDRH1, CDRH2, and CDRH3, and a variable light chain including a CDRL1, CDRL2, and CDRL3 defined in Table 1.

[0040] Table 1. CDR sequences as defined by IMGT, Kabat, Chothia, North, and Contact CDR definitions.F053-0199PCT / 25-069-WO-PCT

[0041] In particular embodiments, 2x4-Ka antibody includes a variable heavy chain including the sequence:QVQLQESGPGLVKPSGTLSLTCTVSGGSISSNNWWTWVRQAPGKGLEWIGEIYHDGRTNYNPSLMSRVTLSVDKSK NQFSLRLSSVTAADTAVYYCVRDQNWMGGMDVWGQGFTVTVSS (SEQ ID NO: 48) and a variable light chain including the sequence: DIQMTQSPSSLSASVGDRVTITCRASQSVITFLNWYQQKPGKAPNLLIYGASSLQGGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQTYRTPYTFGQGTKLEIT (SEQ ID NO: 49).

[0042] In particular embodiments, 2x4-Ka antibody includes a variable heavy chain encoded by the sequence: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACGTGCACTGTC TCTGGAGGGTCCATTAGCAGTAATAATTGGTGGACCTGGGTCCGCCAGGCCCCAGGGAAGGGGCTGGAGTGG ATTGGGGAAATCTATCATGATGGGAGAACCAACTACAACCCGTCCCTCATGAGTCGAGTCACCCTTTCAGTAGA CAAGTCCAAGAATCAGTTCTCCCTGAGGCTGAGTTCTGTGACGGCCGCCGACACGGCCGTGTATTACTGTGTGA GAGATCAGAATTGGATGGGCGGTATGGACGTCTGGGGCCAAGGGTTCACGGTCACCGTCTCCTCAG (SEQ ID NO: 50) and a variable light chain encoded by the sequence:GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGC AAGTCAGAGCGTTATCACCTTTTTAAATTGGTATCAGCAGAAGCCAGGGAAAGCCCCTAACCTCCTGATCTATGG TGCATCCAGTTTGCAAGGTGGGGTCCCATCAAGATTTAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCA TCAGCAGTCTGCAACCTGAAGATTTTGCTACCTACTACTGTCAACAGACTTACAGAACCCCGTACACTTTTGGCC AGGGGACCAAGCTGGAGATCACAC (SEQ ID NO: 51).F053-0199PCT / 25-069-WO-PCT

[0043] In particular embodiments, a binding domain that binds a Rubulavirus includes the binding domain of a 2x4-La antibody.

[0044] In particular embodiments, a 2x4-La binding domain includes a variable heavy chain including a CDRH1, CDRH2, and CDRH3, and a variable light chain including a CDRL1, CDRL2, and CDRL3 defined in Table 2.

[0045] Table 2. CDR sequences as defined by IMGT, Kabat, Chothia, North, and Contact CDR definitions.F053-0199PCT / 25-069-WO-PCT

[0046] In particular embodiments, 2x4-La antibody includes a variable heavy chain including the sequence:QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYSIDWVRQAPGQGLEWVGGIIPLFGATNYAQKFQGRVTITADKST GTAYMTLSSLRSEDTAVYFCARGADSSGPQKNFFDYWGRGTLVTVSS (SEQ ID NO: 52) and a variable light chain including the sequence:SYVLTQPPSVSVAPGQTARITCGGNNIGTKRVHWFQQKPSQAPILWFDDSDRPSGIPERFSGSNSANMATLTISRVE VGDEADYHCQVWDNSDHWVFGGGTKLTVL (SEQ ID NO: 53).

[0047] In particular embodiments, 2x4-La antibody includes a variable heavy chain encoded by the sequence: CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCT TCTGGAGGCACCTTCAGCAGCTATTCTATCGACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGGTGG GAGGGATCATCCCTTTGTTTGGTGCGACAAACTACGCACAGAAATTCCAGGGCAGAGTCACCATTACCGCGGAC AAATCCACGGGCACAGCCTACATGACTCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTTTTGTGCGAG AGGGGCCGATAGTAGTGGCCCCCAAAAGAATTTCTTTGACTACTGGGGCCGGGGAACCTTGGTCACCGTCTCC TCAG (SEQ ID NO: 54) and a variable light chain encoded by the sequence:TCCTATGTGCTGACACAGCCACCCTCGGTGTCCGTGGCCCCAGGACAGACGGCCAGGATTACCTGTGGGGGAA ATAACATTGGAACTAAACGTGTGCACTGGTTCCAGCAGAAGCCAAGTCAGGCCCCTATTCTGGTCGTCTTTGAT GATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGCGAACATGGCCACCCTGACCA TCAGCAGGGTCGAAGTCGGGGATGAGGCCGACTATCACTGTCAGGTGTGGGATAATAGTGATCATTGGGTGTTT GGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO: 55).

[0048] The carboxy-terminal portion of each chain of a naturally occurring antibody defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples of effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.

[0049] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains. In particular embodiments, a human IgK Fc region includes the sequence: TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 68). In particular embodiments, a human lambda light chain (IgA) constant region includes the sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 69).

[0050] Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, lgG1, lgG2, lgG3, and lgG4. IgM hasF053-0199PCT / 25-069-WO-PCTsubclasses including IgM 1 and lgM2. IgA is similarly subdivided into subclasses including lgA1 and lgA2. IgG causes opsonization and cellular cytotoxicity and crosses the placenta, IgA functions on the mucosal surface, IgM is most effective in complement fixation, and IgE mediates degranulation of mast cells and basophils. The function of IgD is still not well understood. Resting B cells, which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies these B cells can express any of the five isotypes. The heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 5, and E chains.

[0051] The constant region of the antibody with multiple binding domains may be of any suitable immunoglobulin subtype. In particular embodiments the subtype of the antibody may be of the class IgG, IgD, IgE, IgA, or IgM. Such an antibody may further belong to any subclass, e.g., lgG1, lgG2a, lgG2b, lgG3 and lgG4. In particular embodiments, a constant region includes a light chain constant region and a heavy chain constant region. A “functional constant heavy chain” or “functional CH” activates an aspect of the immune response.

[0052] In particular embodiments, a human lgG1 Fc region includes the sequence: THTCPPCPAPEFFGGPSVFFFPPKPKDTFMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVETVFHQDWENGKEYKCKVSNKAFPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70).

[0053] In particular embodiments, a human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLT WHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71)

[0054] In particular embodiments, a human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQFNSTFRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESS GQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 72).

[0055] In particular embodiments, a human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 73).

[0056] The human IgD constant region typically includes the amino acid sequence: APTKAPDVFPIISGCRHPKDNSPWLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQW RQGEYKCWQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEE RETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQS QHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMF053-0199PCT / 25-069-WO-PCTWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVSYVTDHGP MK (SEQ ID NO: 74).

[0057] The human IgE constant region typically includes the amino acid sequence: ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSGHYATISLLTVSGAW AKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQV MDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTYTCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIR KSPTITCLWDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRA LMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFF VFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK (SEQ ID NO: 75).

[0058] The human lgA1 constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNWIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQC LAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDA SGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVH LLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKG DTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSWMAEVDGTCY (SEQ ID NO: 76).

[0059] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNVWACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQ CPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSS GKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALN ELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEA LPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 77).

[0060] The human IgM constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 78; identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1) Referring to this SEQ ID NO: 78, the human Cpi1 region ranges from amino acid 5 to amino acid 102; the human Cpi2 region ranges from amino acid 114 to amino acid 205, the human Cpi3 region ranges from amino acid 224 to amino acid 319, the C 4 region ranges from amino acid 329 to amino acid 430, and the tailpiece ranges from amino acid 431 to amino acid 453.

[0061] In particular embodiments, an IgM heavy chain constant region includes the sequence:F053-0199PCT / 25-069-WO-PCTGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 79; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 78 by one amino acid at position 191.

[0062] 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, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I, 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.

[0063] In particular embodiments, human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif” includes the amino acid sequence N-X1-S / T, wherein N is asparagine, X1 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: 78 or SEQ ID NO: 113 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. Each of these sites in the human IgM heavy chain constant region, except for N4, can be mutated to prevent glycosylation at that site, while still allowing IgM expression and assembly into a hexamer or pentamer.

[0064] In certain aspects, a variant human IgM constant region includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311, P313, R344, E345, S401, E402, and / or E403 the corresponding IgM sequence in SEQ ID NO: 78. These positions correspond to the Kabat numbering system as follows: S401 of the corresponding IgM sequence above corresponds to S524 of Kabat; E402 of the corresponding IgM sequence above corresponds to E525 of Kabat; E403 of the corresponding IgM sequence above corresponds to E526 of Kabat; R344 of the corresponding IgM sequence above corresponds to R467 of Kabat; and E345 of the corresponding IgM sequence above corresponds to E468 of Kabat.F053-0199PCT / 25-069-WO-PCT

[0065] Within full-length light and heavy chains, the variable and constant regions are joined by a "J” region of amino acids, with the heavy chain also including a “D” region of amino acids. See, e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

[0066] Antibodies bind epitopes on antigens. The term antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody when in the non-blocked presence of the antibody. An epitope is a region of an antigen that is bound by the variable region of an antibody. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. When the antigen is a protein or peptide, the epitope includes specific amino acids within that protein or peptide that contact the variable region of an antibody.

[0067] In particular embodiments, an epitope denotes the binding site on a viral peptide, bacterial peptide, cancer protein, or other antigen bound by a corresponding variable region of an antibody. The variable region either binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the variable region binds to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target. Three-dimensional epitopes recognized by a variable region, e.g., by the epitope recognition site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely (in)to the corresponding binding site of the variable region and thereby binding between the variable region and its target protein (more generally, antigen) is facilitated. In particular embodiments, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody variable region would cast on the antigen to which it binds; and (ii) the individual participating side chains and backbone residues that facilitate binding. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0068] Epitopes of the currently disclosed antibodies (that is, epitopes to which the antibodies bind) can be found on a virus (e.g., HPIV2 or HPIV4).

[0069] In particular embodiments, “bind” means that the variable regions that form a binding domain associate with their target epitope with a dissociation constant (Kd or KD) of 10'8M or less, in particular embodiments of from 10'6M to 10’13M, in particular embodiments of from 10'5M to 10-10M, in particular embodiments of from 10'5M to 107M, in particular embodiments of from 10-® M to 10-13M, or in particular embodiments of from 10-9M to 10-13M. The term can be further used to indicate that the variable regions do not bind to other biomolecules present (e.g., it binds to other biomolecules with a dissociation constant (Kd) of 1CH M or more, in particular embodiments of from 10~4M to 1 M). A “functional antigen binding domain” is a binding domain that binds its intended antigen.

[0070] In particular embodiments, Kd can be characterized using BIAcore. For example, in particular embodiments, Kd can be measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25°C with immobilized antigen CM5 chips at 10 response units (RU).F053-0199PCT / 25-069-WO-PCT

[0071] Unless otherwise indicated, the term "antibody” includes (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof, examples of which are described below. Furthermore, unless explicitly excluded, antibodies can include monoclonal antibodies (mAbs), human or humanized antibodies, multi-specific antibodies, bi-specific antibodies, tri-specific antibodies, tetraspecific antibodies, penta-specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, single chain variable fragments (scFvs), polyclonal antibodies, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of the antibodies.

[0072] A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies including the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0073] A "human antibody” is one which includes an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences.

[0074] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or V framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. The subgroup of sequences can be a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In particular embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat etal. (supra). In particular embodiments, for the V , the subgroup is subgroup III as in Kabat etal. (supra).

[0075] A neutralizing antibody is an antibody that is responsible for blocking the entry of a pathogen into a cell so that it is firstly unable to infect healthy cells, and secondly, it is unable to replicate and cause severe infection.

[0076] In particular embodiments, the binding domains disclosed herein are part of a full antibody. In particular embodiments, the antibody is an IgG antibody, an IgA antibody, an IgM antibody, an IgE antibody, or an IgD antibody. In particular embodiments, the IgG antibody is an lgG1 antibody, an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody. In particular embodiments, the IgG antibody is an lgG1 antibody. In particular embodiments, the IgA antibodyF053-0199PCT / 25-069-WO-PCTis an lgA1 antibody or an I gA2 antibody. In particular embodiments, the IgA antibody is an lgA1 antibody. In particular embodiments, the IgA antibody is monomeric, dimeric, or polymeric.

[0077] (II) Antibody Variants. Binding domains disclosed herein can be utilized to prepare various forms of relevant binding domain molecules. For example, particular embodiments can include binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to an epitope described herein.

[0078] In particular embodiments, an antibody fragment is used. An “antibody fragment” denotes a portion of a full-length antibody that retains the ability to bind to an epitope. Antibody fragments can be made by various techniques, including proteolytic digestion of an intact antibody as well as production by recombinant host-cells (e.g., mammalian suspension cell lines, E. coli or phage), as described herein. Antibody fragments can be screened for their binding properties in the same manner as intact antibodies. Examples of antibody fragments include Fv, scFv, Fab, Fab', Fab'-SH, F(ab’)2; diabodies; and linear antibodies.

[0079] A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VL and VH domains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, VLand VH, are coded by separate genes, they can be joined, using, for example, recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (single chain Fv (scFv)). For additional information regarding Fv and scFv, see e.g., Bird, et al., Science 242:423-426, 1988; Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO 1993 / 16185; U S Pat. No.5,571,894; and U.S. Pat. No. 5,587,458.

[0080] Linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. Linker sequences of scFv are commonly Gly-Ser linkers, described in more detail elsewhere herein.

[0081] Additional examples of antibody-based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jesperset al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0082] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH1 domains. A F(ab')2 fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab')2 fragments having increased in vivo half-life, see U.S. Patent 5,869,046. Diabodies include two epitopebinding sites that may be bivalent. See, for example, EP 0404097; WO1993 / 01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993. Dual affinity retargeting antibodies (DART™; based on the diabody format butF053-0199PCT / 25-069-WO-PCTfeaturing a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117:4542-51, 2011)) can also be used. Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9:129-134, 2003.

[0083] In particular embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human lgG1, lgG2, lgG3 or lgG4 Fc region) including an amino acid modification (e.g., a substitution) at one or more amino acid positions. Numerous Fc modifications are known in the art, and a representative sampling of such possible modifications are described herein.

[0084] In particular embodiments, variants (including Fc variants) have been modified from a reference sequence to produce an administration benefit. Exemplary administration benefits can include (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for forming protein complexes, (4) altered binding affinities, (5) reduced immunogenicity; and / or (6) extended half-life. While the disclosure below describes these modifications in terms of their application to antibodies, when applicable to another particular anti-Rubulavirus binding domain format (e.g., bispecific antibodies), the modifications can also be applied to these other formats.

[0085] In particular embodiments, the Fc moiety of an antibody includes a substitution at positions CH24, CH25, or both. In general, the amino acid at positions 4 and 5 of CH2 of the wild-type lgG1 and lgG3 is a leucine ("L"). In particular embodiments, the antibody includes an amino acid at position CH2 4, CH2 5, or both, that is not an L. In particular embodiments, an antibody includes an alanine ("A") at position CH2 4, or CH2 5, or both. In particular embodiments, the antibody includes both, a CH2 L4A and a CH2 L5A substitution. Such antibodies are referred to herein as a "LALA" variant. Interestingly, a "LALA" mutation in the Fc moiety does not only result in a lack of contribution of the respective antibody in antibody-dependent enhancement (ADE), but also blocks ADE.

[0086] In particular embodiments, an lgG4 Fc region is mutated to form the lgG4_S228P Fc region. lgG4 antibodies can undergo a process called Fab arm exchange which results in functionally monovalent, bispecific antibodies with unknown specificity and thus potentially reduced therapeutic efficacy. Mutating the wildtype lgG4 serine at position 228 within the core-hinge region to a proline creates the I gG4_S228P mutant. In particular embodiments, the I gG4_S228P mutant prevents Fab arm exchange.

[0087] In particular embodiments the antibodies can be mutated to increase their affinity for Fc receptors. Exemplary mutations that increase the affinity for Fc receptors include: G236A / S239D / A330L / I332E (GASDALIE). Smith et al., Proceedings of the National Academy of Sciences of the United States of America, 109(16), 6181-6186, 2012. In particular embodiments, an antibody variant includes an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). In particular embodiments, alterations are made in the Fc region that result in altered C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184, 2000.F053-0199PCT / 25-069-WO-PCT

[0088] In particular embodiments, it may be desirable to create cysteine engineered antibodies, e.g, “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further below. In particular embodiments, residue 5400 (EU numbering) of the heavy chain Fc region is selected. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.

[0089] Antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e, between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., W02000 / 61739; WO 2001 / 29246; W02002 / 031140; US2002 / 0164328; W02003 / 085119; W02003 / 084570; US2003 / 0115614; US2003 / 0157108; US2004 / 0093621; US2004 / 0110704; US2004 / 0132140; US2004 / 0110282; US2004 / 0109865; W02005 / 035586; W02005 / 035778; W02005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led 3 OHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986, and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614, 2004; Kanda et al, Biotechnol. Bioeng, 94(4):680-688, 2006; and W02003 / 085107).

[0090] In particular embodiments, modified antibodies include those wherein one or more amino acids have been replaced with a non-amino acid component, or where the amino acid has been conjugated to a functional group or a functional group has been otherwise associated with an amino acid. The modified amino acid may be, e.g, a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent. Amino acid(s) can be modified, for example, co-translationally or post-translationally during recombinant production (e.g, N-linked glycosylation at N-X-S / T motifs during expression in mammalian cells) or modified by synthetic means. The modified amino acid can be within the sequence or at the terminal end of a sequence. Modifications also include nitrited constructs.

[0091] In particular embodiments, variants include glycosylation variants wherein the number and / or type of glycosylation site has been altered compared to the amino acid sequences of a reference sequence. In particularF053-0199PCT / 25-069-WO-PCTembodiments, glycosylation variants include a greater or a lesser number of N-linked glycosylation sites than the reference sequence. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (e.g., those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the reference sequence. These cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. These cysteine variants generally have fewer cysteine residues than the reference sequence, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0092] PEGylation particularly is a process by which polyethylene glycol (PEG) polymer chains are covalently conjugated to other molecules such as proteins. Several methods of PEGylating proteins have been reported in the literature. For example, N-hydroxy succinimide (NHS)-PEG was used to PEGylate the free amine groups of lysine residues and N-terminus of proteins; PEGs bearing aldehyde groups have been used to PEGylate the amino-termini of proteins in the presence of a reducing reagent; PEGs with maleimide functional groups have been used for selectively PEGylating the free thiol groups of cysteine residues in proteins; and site-specific PEGylation of acetylphenylalanine residues can be performed.

[0093] Covalent attachment of proteins to PEG has proven to be a useful method to increase the half-lives of proteins in the body (Abuchowski, A. et al., Cancer Biochem. Biophys., 1984, 7:175-186; Hershfield, M. S. et al., N. Engl. J. Medicine, 1987, 316:589-596; and Meyers, F. J. et al., Clin. Pharmacol. Then, 49:307-313, 1991). The attachment of PEG to proteins not only protects the molecules against enzymatic degradation, but also reduces their clearance rate from the body. The size of PEG attached to a protein has significant impact on the half-life of the protein. The ability of PEGylation to decrease clearance is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Usually the larger the PEG is, the longer the in vivo half-life of the attached protein. In addition, PEGylation can also decrease protein aggregation (Suzuki et al., Biochem. Bioph. Acta 788:248, 1984), alter protein immunogenicity (Abuchowski et al., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92 / 16221).

[0094] Several sizes of PEGs are commercially available (Nektar Advanced PEGylation Catalog 2005-2006; and NOF DDS Catalogue Ver 7.1), which are suitable for producing proteins with targeted circulating half-lives. A variety of active PEGs have been used including mPEG succinimidyl succinate, mPEG succinimidyl carbonate, and PEG aldehydes, such as mPEG-propionaldehyde.F053-0199PCT / 25-069-WO-PCT

[0095] In particular embodiments, the antibody can be fused or coupled to an Fc polypeptide that includes amino acid alterations that extend the in vivo half-life of an antibody that contains the altered Fc polypeptide as compared to the half-life of a similar antibody containing the same Fc polypeptide without the amino acid alterations. In particular embodiments, Fc polypeptide amino acid alterations can include M252Y, S254T, T256E, M428L, and / or N434S and can be used together, separately or in any combination. For example, M428L / N434S is a pair of mutations that increase the half-life of antibodies in serum, as described in Zalevsky et al., Nature Biotechnology 28, 157-159, 2010. Other alterations that can be helpful are described in US Patent No. 7,083,784, US Patent No. 7,670,600, US Publication No.2010 / 0234575, PCT / US2012 / 070146, and Zwolak, Scientific Reports 7: 15521, 2017. In particular embodiments, any substitution at one of the following amino acid positions in an Fc polypeptide can be considered an Fc alteration that extends half-life: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428, 430, 434, 436. Each of these alterations or combinations of these alterations can be used to extend the half-life of a bispecific antibody as described herein.

[0096] In particular embodiments, Fc modifications include hulgG4 ProAlaAla, hulgG2m4, and / or hulgG2sigma mutations. In particular embodiments, one or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For additional information regarding Fc mutations that create administration benefits, see Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Frontiers in Immunology (2019) Vol. 10, Article 1296.

[0097] (III) Multi-Domain Binding Molecules. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes a binding domain disclosed herein. In particular embodiments, a multidomain binding molecule includes at least one, at least two, at least, three, at least four binding domains that bind an epitope on a Rubulavirus (e.g., HPIV2 and / or HPI V4). In particular embodiments, all of the binding domains of a multidomain binding molecule bind a Rubulavirus.

[0098] Multi-domain binding molecules include bispecific antibodies which bind at least two epitopes wherein at least one of the epitopes is located on a Rubulavirus. Multi-domain binding molecules include trispecific antibodies which binds at least 3 epitopes, wherein at least one of the epitopes is located on a Rubulavirus, and so on.

[0099] Bispecific antibodies can be prepared utilizing antibody fragments (for example, F(ab')2 bispecific antibodies). For example, WO 1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma Rill antibody; US Pat. No. 5,837,234 describes a bispecific anti-ErbB2 / anti-Fc gamma Rl antibody; WO 1998 / 002463 describes a bispecific anti-ErbB2 / Fc alpha antibody; and US 5,821,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody.[OO1OO] Some additional exemplary bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain), and twoF053-0199PCT / 25-069-WO-PCTimmunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including bi-specific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.

[0101] Two antibodies or fragments thereof can be linked through a linker to form a bispecific antibody. In particular embodiments, the two antibodies or fragments thereof can bind the same epitope or different epitopes. Examples of linkerscan be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0102] Commonly used flexible linkers include a linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 80), (Gly3Ser)n(Gly4Ser)n (SEQ ID NO: 81), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 82), and (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 83). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 84), (Gly4Ser)3(SEQ ID NO: 85), (Gly4Ser)2(SEQ ID NO: 86), (Gly4Ser)i (SEQ ID NO: 87), (Gly3Ser)2(SEQ ID NO: 88), (Gly3Ser)i (SEQ ID NO: 89), (Gly2Ser)2(SEQ ID NO: 90) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 91), GGSGGGSGSG (SEQ ID NO: 92), or GGSGGGSG (SEQ ID NO: 93).

[0103] Linkers that include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0104] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of binding domains needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a prolinerich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0105] In particular embodiments, binding domains disclosed herein can be used to create bi-, tri, (or more) specific immune cell engaging molecules. Immune cell engaging molecules have at least one binding domain that binds a receptor on an immune cell and alters the activation state of the immune cell.

[0106] Bispecific binding molecules with extended half-lives are described in, for example, US Patent No. 8,921 ,528 and US Patent Publication No. 2014 / 0308285.F053-0199PCT / 25-069-WO-PCT

[0107] In particular embodiments, multi-domain binding molecules are multimers of an antibody disclosed herein. Multimerization strategies include formation of a fusion protein using protein linkers or use of IgA or IgM constant regions as a multimerization scaffold. In certain aspects, multimerization is achieved by linking antibodies or binding domains of antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0108] Certain examples include fusion protein with two or three copies of an antibody or binding domain disclosed herein, each linked with the Gly-Ser linker (Gly4Ser)4 (SEQ ID NO: 84).

[0109] A “multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains are highly conserved protein sequences that can include different types of sequence motifs such as leucine zipper, helix loop-helix, ankyrin and PAS (Feuerstein et al, Proc. Natl. Acad. Sci. USA, 91:10655-10659, 1994). Multimerization domains present in proteins can bind to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer, and / or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type (US Patent No. 10030065).

[0110] Dimerization domains can include protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an "interface" with an engineered "knob" and / or "protruberance" (US 5821333), leucine zippers (US 5932448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336- 44, 2004), PTB (Zhou et al., Nature, 378:584-592, 1995), WW (Sudol Prog Biochys MoL Bio, 65: 113-132, 1996), PDZ (Kim et al. , Nature, 378: 85-88, 1995; Komau et al„ Science, 269:1737-1740, 1995) and WD40 (Hu et al., J Biol Chem., 273:33489- 33494, 1998). Additional examples of molecules that contain dimerization domains / motifs are receptor dimer pairs such as the interleukin-8 receptor (IL-8R), integrin heterodimers such as LFA-l and GPIIIb / llla, dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993) and variants of some of these domains with modified affinities (PCT Publication No. WO 2012 / 001647).

[0111] In particular embodiments, the sequence corresponding to a dimerization motif / domain includes the leucine zipper domain of Jun (US5932448;RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN (SEQ ID NO: 94)), the dimerization domain of Fos (US 5932448; LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAA (SEQ ID NO: 95)), a consensus sequence for a WW motif (PCT Publication No. WO 1997 / 037223), the dimerization domain of the SH2B adapter protein from GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621, 2005;WREFCESHARAAALDFARRFRLYLASHPQYAGPGAEAAFSRRFAELFLQHFEAEVARAS (SEQ ID NO: 96)), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006;THRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIE (SEQ ID. NO: 97)), the PTBF053-0199PCT / 25-069-WO-PCTdomain of human DOK-7 from GenBank Accession no. NP_005535.1 (Wagner et al., Cold Spring Harb Perspect Biol.5: a008987, 2013;LGEVHRFHVTVAPGTKLESGPATLHLCNDVLVLARDIPPAVTGQWKLSDLRRYGAVPSGFIFEGGTRCGYWAGVFFL SSAEGEQISFLFDCIVRGISPTKG (SEQ ID NO: 98)), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21: 5591-5604, 2001;DCKEEHAEFVLVRKDMLFNQLIEMALLSLGYSHSSAAQAKGLIQVGKWNPVPLSYVTDAPDATVADMLQDVYHWTL KIQLHSCPKLEDLPPEQWSHTTVRNALKDLLKDMNQSS (SEQ ID NO: 99)), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463;CAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRNASIWLGCGHTDRGQ LSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINTEDGKKRHTLEKMTDSVTCLYCNSFSKQS KQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIGNVSTPLMCLSESTNSTERNVMWGGCGSQLFSYAAFSDSNIIT VWDTALYIAKQNSPVVEVWDKKTEKLCGLIDCVHFLREVMVKETKIFSFSNDFTIQKLIETRTNKESKHKMSYSGRVK TLCLQKNTALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQKEIQSCLT VWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE (SEQ ID NO: 100)), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079-4088, 1998;DQELKHLILEAADGFLFIVSCETGRWYVSDSVTPVLNQQQSEWFGSTLYDQVHPDDVDKLREQLSTSENALTGR(SEQ ID NO: 101)) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014;LSAKETKMLMAAGDKDGDGKIGVDEFSTLVAES (SEQ ID NO: 102)).

[0112] In particular embodiments, the dimerization domain can be a dimerization and docking domain (DDD) on one antibody and an anchoring domain (AD) on another antibody to facilitate a stably tethered structure. In particular embodiments, the DDD (DDD1 and DDD2) are derived from the regulatory subunits of a cAMP-dependent protein kinase (PKA), and the AD (AD1 and AD2) are derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. In particular embodiments, DDD1 includes the amino acid sequence: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 103). In particular embodiments, DDD2 includes the amino acid sequence: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 104). In particular embodiments, AD1 includes the amino acid sequence: QIEYLAKQIVDNAIQQA (SEQ ID NO: 105). In particular embodiments, AD2 includes the amino acid sequence: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 106). However, one skilled in the art will realize that other DDDs and ADs are known and can be used such as: the 4-helix bundle type DDD domains may be obtained from p53, DCoH (pterin 4 alpha carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1)) and HNF-1 (hepatocyte nuclear factor 1). Other AD sequences of potential use may be found in Patent Publication No. US2003 / 0232420A1.

[0113] The X-type four-helix bundle dimerization motif that is a structural characteristic of the DDD (Newlon, et al.F053-0199PCT / 25-069-WO-PCTEMBO J. 2001; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) is found in other classes of proteins, such as the S100 proteins (for example, SWOB and calcyclin), and the hepatocyte nuclear factor (HNF) family of transcriptional factors (for example, HNF-1a and HNF-1|3). Over 300 proteins that are involved in either signal transduction or transcriptional activation also contain a module of 65-70 amino acids termed the sterile a motif (SAM) domain, which has a variation of the X-type four-helix bundle present on its dimerization interface. For SWOB, this X-type four-helix bundle enables the binding of each dimer to two p53 peptides derived from the c-terminal regulatory domain (residues 367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998; 37: 1951-1960). Similarly, the N-terminal dimerization domain of HNF-1a (HNF-p1) was shown to associate with a dimer of DCoH (dimerization cofactor for HNF-1) via a dimer of HNF-p1 (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). In alternative embodiments, these naturally occurring systems can also be used to provide stable multimeric structures with multiple functions or binding specificities. Other binding events such as those between an enzyme and its substrate / inhibitor, for example, cutinase and phosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052), may also be utilized to generate the two associating components (the ''docking” step), which are subsequently stabilized covalently (the "lock” step).

[0114] In particular embodiments, dimerization of antibodies can be induced by a chemical inducer. This method of dimerization requires one antibody to contain a chemical inducer of dimerization binding domain 1 (CBD1) and the second antibody to contain the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). If the CID is rapamycin, CBD1 and CBD2 can be the rapamycin binding domain of FK-binding protein 12 (FKBP12) and the FKBP12-Rapamycin Binding (FRB) domain of mTOR. In particular embodiments, FKBP12 includes the sequence:MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 107).

[0115] In particular embodiments, FRB includes the sequence:MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK SGNVKDLTQAWDLYYHVFRRISKLES (SEQ ID NO: 108). If the CID is FK506 / cyclosporin fusion protein or a derivative thereof, CBD1 and CBD2 can be the FK506 (Tacrolimus) binding domain of FK-binding protein 12 (FKBP12) and the cyclosporin binding domain of cylcophilin A. If the CID is estrone / biotin fusion protein or a derivative thereof, CBD1 and CBD2 can be an oestrogen-binding domain (EBD) and a streptavidin binding domain. If the CID is dexamethasone / methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain. If the CID is O6-benzylguanine derivative / methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and a dihydrofolate reductase (DHFR) binding domain. If the CID is RSL1 or a derivative thereof, CBD1 and CBD2 can be a retinoic acid receptor domain and an ecodysone receptor domain. If the CID is AP1903 or a derivative thereof, CBD1 and CBD2 can be the FK506 binding protein (FKBP12) binding domainsF053-0199PCT / 25-069-WO-PCTincluding a F36V mutation. Use of the CID binding domains can also be used to alter the affinity to the CID. For instance, altering amino acids at positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin: KTW has high, KHF intermediate and PLW is low (Bayle et al, Chemistry & Biology 13, 99-107, January 2006).

[0116] In particular embodiments, antibodies can multimerize using a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, an antibody can include a part of a FCERI alpha chain and another antibody can include a partofan FCERI beta chain or variant thereof such that said FCERI chains spontaneously dimerize together to form a dimeric antibody. In particular embodiments, antibodies can include a part of a FCERI alpha chain and a part of a FCERI gamma chain or variant thereof such that said FCERI chains spontaneously trimerize together to form a trimeric antibody, and in another embodiment the multi-chain antibody can include a part of FCERI alpha chain, a part of FCERI beta chain and a part of FCERI gamma chain or variants thereof such that said FCERI chains spontaneously tetramerize together to form a tetrameric antibody.

[0117] In particular embodiments, additional methods of causing dimerization can be utilized. Additional modifications to generate a dimerization domain in antibody could include: replacing the C-terminus domain with murine counterparts; generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both antibodies; swapping interacting residues in each of the antibodies in the C-terminus domains ("knob-in-hole”); and fusing the variable domains of the antibodies directly to CD3 (CD3 fusion) (Schmitt et al., Hum. Gene Then 2009.20:1240-1248).

[0118] Particular embodiments can utilize multimerization domains, such as C4b multimerization domains or ferritin multimerization domains. Full-length native C4b includes seven o-chains linked together by a multimerization (i.e., heptamerization) domain at the C-terminus of the a-chains. Blom et al., (2004) Mol Immunol 40: 1333-1346. Ferritin is an iron storage protein found in almost all living organisms, and has been extensively studied and engineered for a number of biochemical / biomedical purposes (US 20090233377; Meldrum, et al. Science 257, 522-523 (1992); U.S.20110038025; Yamashita, Biochim Biophys Acta 1800, 846-857 (2010), including as a multimerizing vaccine platform for displaying peptide epitopes (US 20060251679 (2006); Li, et al. Industrial Biotechnol 2, 143-147 (2006)).

[0119] Mutlimerization with encapsulin and lumazine synthase can also be performed. Both can be linked to antibodies to create self-assembling 60mer particles (Jardine et al., 2013, Science 340, 711-716 and Kanekiyo et al., 2015, Cell 162, 1090-1100).

[0120] Multimerized antibodies and antibody-like molecules such as IgA and IgM antibodies have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos. 9,951,134, 10,400,038, and 9,938,347, U.S. Patent Application Publication Nos. US20190100597A1 , US20180118814A1, US20180118816A1, US20190185570A1, and US20180265596A1, and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, and WO 2019 / 165340.

[0121] Particular embodiments include using IgA and IgM constant region domains to allow the binding portion ofF053-0199PCT / 25-069-WO-PCTmolecules provided herein to readily multimerize into dimers, pentamers or hexamers. Basic immunoglobulin structures in vertebrate systems are described above and are well understood. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0122] Immunoglobulin A (IgA), as the major class of antibody present in the mucosal secretions of most mammals, represents a key first line of defense against invasion by inhaled and ingested pathogens. IgA is also found at significant concentrations in the serum of many species, where it functions as a second line of defense mediating elimination of pathogens that have breached the mucosal surface. Receptors specific for the Fc region of IgA, FcaR, are key mediators of IgA effector function. Native IgA is a tetrameric protein including two identical light chains (K or A) and two identical heavy chains. IgA, similarly to IgG, contains three constant domains (CA1-CA3), with a hinge region between the CA1 and CA2 domains. The main difference between lgA1 and lgA2 resides in the hinge region that lies between the two Fab arms and the Fc region. lgA1 has an extended hinge region due to the insertion of a duplicated stretch of amino acids, which is absent in lgA2. Both forms of IgA have the capacity to form dimers, in which two monomer units, are arranged in an end-to-end configuration stabilized by disulfide bridges and incorporation of a J-chain. J-chains are also part of IgM pentamers and are discussed in more detail below In particular embodiments, binding domains disclosed herein can be expressed as an IgA antibody. In particular embodiments, binding domains disclosed herein can be expressed as an IgM antibody. In particular embodiments, binding domains disclosed herein can be expressed as an IgG antibody.

[0123] Both IgA and IgM (discussed further below in relation to pentamers and hexamers) possess an 18-amino acid extension in the C terminus called the "tail-piece" (tp). The IgA and IgM tp is highly conserved among various animal species. The conserved penultimate cysteine residue in the IgA and IgM tp has been demonstrated to be involved in multimerization by forming a disulfide bond between heavy chains to permit formation of a multimer. Both tp contain an N-linked carbohydrate addition site, the presence of which is required for dimer formation in IgA and J-chain incorporation and pentamer formation in IgM. However, the structure and composition of the N-linked carbohydrates in the tp differ, suggesting differences in the accessibility of the glycans to processing by glycosyltransferases. Particularly, the IgA (atp) and IgM (ptp) tp differ at seven amino acid positions.

[0124] The human lgA1 constant region typically includes the amino acid sequence as set forth in SEQ ID NO: 76. Referring to this SEQ ID NO: 76, the human CA1 domain extends from amino acid 6 to amino acid 98; the human lgA1 hinge region extends from amino acid 102 to amino acid 124, the human CA2 domain extends from amino acid 125 to amino acid 219, the human CA3 domain extends from amino acid 228 to amino acid 330, and the tp extends from amino acid 331 to amino acid 352.

[0125] The human lgA2 constant region typically includes the amino acid sequence as set forth in SEQ ID NO: 77. Referring to this SEQ ID NO: 77, the human CA1 domain extends from amino acid 6 to amino acid 98, the human I g A2 hinge region extends from amino acid 102 to amino acid 111, the human CA2 domain extends from amino acid 113 to amino acid 206, the human CA3 domain extends from amino acid 215 to amino acid 317, and the tp extends fromF053-0199PCT / 25-069-WO-PCTamino acid 318 to amino acid 340.

[0126] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 121, the mature human J chain) and the secretory component to form a bivalent secretory IgA (slgA)-derived binding molecule. An exemplary precursor secretory component includes the sequence MLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYA GRANLTNFPENGTFWNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTE NAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADL QVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVWNTLGKRAPAFEGRILLNPQDKDGSFSWIT GLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTWKGVAGGSVAVLCPYNRKESKSIKYWCLWEG AQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVP GNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWY WCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQA DGSRASVDSGSSEEQGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGA NDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEAQDGPQEA (SEQ ID NO: 109). An exemplary mature secretory component includes KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITUSSEGYVSSKYAGRANLTNFPENGTFW NIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYP VLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSWINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRG SVTFHCALGPEVANVAKFLCRQSSGENCDVWNTLGKRAPAFEGRILLNPQDKDGSFSWITGLRKEDAGRYLCGAH SDGQLQEGSPIQAWQLFVNEESTIPRSPTWKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGW VKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCH FPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAA VYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPR (SEQ ID NO: 110). While not wishing to be bound by theory, and as indicated above, the assembly of two IgA binding units into a dimeric IgA-derived binding molecule is thought to involve the CA3 and tp domains. See, e.g., Braathen, R., el 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 tp domains.

[0127] An engineered IgA heavy chain constant region can additionally include a CA2 domain or a fragment thereof, an IgA hinge region or fragment thereof, a CA1 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 chain constant region (e.g., SEQ ID NO: 76 or SEQ ID NO: 77), or a variant, derivative, or analog thereof.

[0128] In particular embodiments, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 76 or amino acids 113 to 340 of SEQ ID NO: 77. In particular embodiments, the IgA heavy chain constantF053-0199PCT / 25-069-WO-PCTregions 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: 76 or amino acids 102 to 340 of SEQ ID NO: 77. In particular embodiments, the IgA heavy chain constant regions can each further include an IgA CA1 domain situated N-terminal to the IgA hinge region.

[0129] Each of the strategies discussed above can be used to create IgA antibody-based dimers.

[0130] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0131] Particular embodiments include IgM constant regions (or variants thereof). These embodiments have the ability to form hexamers, or in association with a J-chain, form pentamers. Embodiments with an IgM constant region typically include at least the Cpi4-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. In particular embodiments, one or more constant region domains can be deleted so long as the IgM antibody is capable of forming hexamers and / or pentamers. Thus, an IgM antibody can be, e.g., a hybrid IgM / IgG antibody or can be a “multimerizing fragment" of an IgM-derived binding molecule.

[0132] The assembly of five or six IgM binding units into a pentameric or hexameric IgM antibody is thought to involve the Cpi4 and tp domains. See, e.g., Braathen, R., et al., J Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody described in this disclosure typically includes at least the C|d4 and / or tp domains (also referred to herein collectively as Cpi4-tp). A "multimerizing fragment” of an IgM heavy chain constant region thus includes at least the Cpi4-tp domains. An IgM heavy chain constant region can additionally include a Cpi3 domain or a fragment thereof, a Cpi2 domain or a fragment thereof, a Cpi 1 domain or a fragment thereof, and / or other IgM heavy chain domains.

[0133] Five IgM monomers form a complex with a J-chain to form a native IgM molecule. The J-chain is considered to facilitate polymerization of chains before IgM is secreted from antibody-producing cells. Sequences for the human IGJ gene are known in the art, for example, (IGMT Accession: J00256, X86355, M25625, AJ879487). The J chain establishes the disulfide bridges between IgM antibodies to form multimeric structures such as pentamers. See, for example, Sorensen et al. International Immunology, (2000), pages 19-27. While crystallization of IgM has proved to be notoriously challenging, Czajkowsky and Shao (PNAS 106(35): 14960-14965, 2009) published a homology-based structural model of IgM, based on the structure of the IgE Fc domain and the known disulfide pairings. The authors report that the human IgM pentamer is a mushroom-shaped molecule with a flexural bias. The IgM heavy (p) chain contains five N-linked glycosylation sites: Asn-171, Asn-332, Asn-395, Asn-402 and Asn-563. In an IgM antibody where each binding unit is bivalent, the binding molecule itself can have 10 or 12 valencies.

[0134] 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, b-2 Microglobulins, Major Histocompatibility Antigens, Thy-I,F053-0199PCT / 25-069-WO-PCTComplement, 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.

[0135] 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 orC I), an antibody heavy chain constant domain 2 (CM2 or C 2), an antibody heavy chain constant domain 3 (CM3 or C 3), and an antibody heavy chain constant domain 4 (CM4 or C 4) that can include a tp, as indicated above.

[0136] In particular embodiments, each binding unit of a multimeric binding molecule as provided herein includes two IgM heavy chain constant regions or multimerizing fragments or variants thereof, each including at least an IgM C 4 domain and an IgM tp domain. In certain embodiments the IgM heavy chain constant regions can each further include an IgM C i3 domain situated N-terminal to the IgM C 4 and IgM tp domains.

[0137] In particular embodiments, the IgM heavy chain constant regions can each further include an IgM C 2 domain situated N-terminal to the IgM C 3 domain. Exemplary multimeric binding molecules provided herein include human IgM constant regions that include the wild-type human C|J2, C 3, and C 4-tp domains as follows:VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTST LTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWT RQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAR EQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCW AHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 111).

[0138] In certain IgM-derived multimeric binding molecules as provided herein each IgM constant region can include, instead of, or in addition to an IgM CJJ2 domain, an IgG hinge region or functional variant thereof situated N-terminal to the IgM C 3 domain. An exemplary variant human lgG1 hinge region amino acid sequence in which the cysteine at position 6 is substituted with serine is VEPKSSDKTHTCPPCPAP (SEQ ID NO: 140). An exemplary IgM constant region of this type includes the variant human IgG 1 hinge region fused to a multimerizing fragment of the human IgM constant region including the C 3, C 4, and tp domains, and includes the amino acid sequence:VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHP NATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFS PADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKST GKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 112).

[0139] Human IgM constant regions, and also certain non-human primate IgM constant regions, as provided herein typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites. As used herein “an N-linked glycosylation motif” includes the amino acid sequence N-X1-S / T, wherein N is asparagine, X1 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 asparagineF053-0199PCT / 25-069-WO-PCTresidue. 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: 78 or SEQ ID NO: 113 starting at positions 46 (“N1”), 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. Each of these sites in the human IgM heavy chain constant region, except for N4, can be mutated to prevent glycosylation at that site, while still allowing IgM expression and assembly into a hexamer or pentamer.

[0140] The human IgM heavy chain constant region typically includes the amino acid sequence as set forth in SEQ ID NO: 78; identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1, and CAA47714.1). Referring to this SEQ ID NO: 78, the human Cpi1 region ranges from amino acid 5 to amino acid 102; the human Cpi2 region ranges from amino acid 114 to amino acid 205, the human Cpi3 region ranges from amino acid 224 to amino acid 319, the Cpi4 region ranges from amino acid 329 to amino acid 430, and the tp ranges from amino acid 431 to amino acid 453.

[0141] In particular embodiments, an IgM heavy chain constant region includes the sequence:GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 113; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 78 by one amino acid at position 191.

[0142] Other forms of the human IgM constant region with minor sequence variations exist, including GenBank Accession Nos. P01871.4, CAB37838.1, and pir||MHHU. The amino acid substitutions, insertions, and / or deletions at positions corresponding to SEQ ID NO: 78 described herein can likewise be incorporated into alternate human IgM sequences, as well as into IgM constant region amino acid sequences of other species, e.g., those shown in FIG. 1 of PCT / US2019 / 020374.

[0143] In certain aspects, a variant human IgM constant region includes an amino acid substitution corresponding to the wild-type human IgM constant region at position P311, P313, R344, E345, S401, E402, and / or E403 of SEQ ID NO: 78. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 78 corresponds to S524 of Kabat; E402 of SEQ ID NO: 78 corresponds to E525 of Kabat; E403 of SEQ ID NO: 78 corresponds to E526 of Kabat; R344 of SEQ ID NO: 78 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 78 corresponds to E468 of Kabat.

[0144] In particular embodiments, "corresponds to” means the designated position of SEQ ID NO: 78 and the amino acid in the sequence of the IgM constant region of any species which is homologous to the specified position. See FIG.F053-0199PCT / 25-069-WO-PCT1 of PCT / US2019 / 020374.

[0145] In particular embodiments, P311 of SEQ ID NO: 78 can be substituted, e.g., with alanine (P311A), serine (P311S), or glycine (P311G) and / or P313 of SEQ ID NO: 78 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 78 can be substituted with alanine (P311A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL ASSLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 114).

[0146] In certain aspects, S401 of SEQ ID NO: 78 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 78 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVAEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 115).

[0147] In certain aspects, E402 of SEQ ID NO: 78 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 78 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSAEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 116).

[0148] In certain aspects, E403 of SEQ ID NO: 78 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 78 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQF053-0199PCT / 25-069-WO-PCTVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEAEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 117).

[0149] In certain aspects, R344 of SEQ ID NO: 78 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 78 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 118).

[0150] In certain aspects, E345 of SEQ ID NO: 78 can be substituted with any amino acid. In certain aspects, E345 of SEQ ID NO: 78 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline): GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDV MQGTDEHWCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDL PSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRASATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQ APGRYFAHSILTVSEEEWNTGETYTCWAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 119).

[0151] As indicated, five IgM binding units can form a complex with a J-chain to form a pentameric IgM antibody. The precursor form of the human J-chain includes:MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRT RFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD(SEQ ID NO: 120). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 120and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 120.

[0152] The mature human J-chain includes the amino acid sequence QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 121).

[0153] The term “J-chain” as used herein refers to the J-chain of native sequence IgM or IgA antibodies of any animal species. When specified, it can also refer to any functional fragment thereof, derivative thereof, and / or variant thereof,F053-0199PCT / 25-069-WO-PCTincluding a mature human J-chain amino acid sequence provided herein as SEQ ID NO: 121. A functional fragment, derivative, and / or variant of a J-chain has at least 90% sequence identity to the reference J-chain and retains the multimerizing function of the reference J-chain.

[0154] In certain aspects, the J-chain of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid Y102, T103, N49 or S51 of SEQ ID NO: 121.

[0155] By "an amino acid corresponding to” a position of SEQ ID NO: 121 is meant the amino acid in the sequence of the J-chain of any species which is homologous to the referenced residue in the human J-chain. For example, the position corresponding to Y102 in SEQ ID NO: 121 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 121 is conserved in the J-chain amino acid sequences of at least 37 other species. The positions corresponding to N49 and S51 in SEQ ID NO: 121 are 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 and FIG. 2 of PCT / US2019 / 020374.

[0156] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 121 can be substituted with any amino acid In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 121 can be substituted with alanine (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCATYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 122),With serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCSTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 123),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCRTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 124).

[0157] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 121 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 121 can be substituted with alanine as follows (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCYAYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 125).

[0158] In certain aspects, the variant J-chain or functional fragment thereof of the IgM antibody as provided herein includes an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 121, provided that S51 is not substituted with threonine (T), or wherein the J-chain includes amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and S51 of SEQ ID NO: 121.

[0159] The amino acids corresponding to N49 and S51 of SEQ ID NO: 121 along with the amino acid corresponding to 150 of SEQ ID NO: 121 include an N-linked glycosylation motif in the J-chain. Accordingly, mutations at N49 and / orF053-0199PCT / 25-069-WO-PCTS51 (with the exception of a single threonine substitution at S51) can prevent glycosylation at this motif. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 121 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 121 can be substituted with alanine (A), glycine (G), threonine (T), serine (S) or aspartic acid (D). In a particular aspect the position corresponding to N49 of SEQ ID NO: 121 can be substituted with alanine (A). In a particular aspect the J-chain is a variant human J-chain and includes the amino acid sequence:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNREAISDPTSPLRTRFVYHLSDLCKKCDPTEVELDN QIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 126).

[0160] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 121 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 121 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 121 can be substituted with alanine (A). In a particular aspect the variant J-chain or functional fragment thereof is a variant human J-chain and includes the amino acid sequence:EDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENIADPTSPLRTRFVYHLSDLCKKCDPTEVELDNQI VTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 127).

[0161] Particular embodiments include a heterologous polypeptide (e.g., a single-domain antibody binding domain) fused to the J-chain or functional fragment thereof via a peptide linker, e.g., a peptide linker including at least 5 amino acids, but no more than 25 amino acids. In certain aspects, the peptide linker includes (GGGGS)n (SEQ ID NO: 80) wherein n is 1-5.

[0162] A single-domain antibody binding domain can be introduced into the J-chain at any location that allows the binding of the binding domain to its binding target without interfering with J-chain function or the function of an associated IgA, IgM, or hybrid IgG antibody. Insertion locations include at or near the C- terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible. In certain aspects, the antigen-binding domain can be introduced into the mature human J-chain of SEQ ID NO: 121 between cysteine residues 92 and 101 of SEQ ID NO: 121. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 121 at or near a glycosylation site. In a further aspect, the antigen-binding domain can be introduced into the human J-chain of SEQ ID NO: 121 within 10 amino acid residues from the C-terminus, or within 10 amino acids from the N-terminus.

[0163] In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 121 by chemical or chemo-enzymatic derivatization. In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 121 by a chemical linker. In some embodiments, the chemical linker is a cleavable or non-cleavable linker. In particular embodiments, the cleavable linker is a chemically labile linker or an enzyme-labile linker. In some embodiments, the linker is selected from the group including N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-F053-0199PCT / 25-069-WO-PCTcarboxylate (SMCC), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), iminothiolane (IT), afunctional derivatives of imidoesters, active esters, aldehydes, bis-azido compounds, bis-diazonium derivatives, diisocyanates, and bis-active fluorine compounds. In particular embodiments, the modified J-chain is modified by insertion of an enzyme recognition site, and by post-translationally attaching a binding moiety at the enzyme recognition site through a peptide or nonpeptide linker.

[0164] In certain aspects the modified J-chain can include the formula X[Ln]J or J[Ln]X, where J includes a mature native J-chain or functional fragment thereof, X includes a heterologous binding domain, and [Ln] is a linker sequence including n amino acids, where n is a positive integer from 1 to 100, 1 to 50, or 1 to 25. In certain aspects N is 5, 10, 15, or 20.

[0165] J-chains from the following species can also be used in certain embodiments: Pan troglodytes, Pongo abelii, Callithrix jacchus, Macaca mulatta, Papio Anubis, Saimiri boliviensis, Tupaia chinensis, Tursiops truncatus, Orcinus orca, Loxodonta Africana, Leptonychotes weddellii, Ceratotherium simum, Felis catus, Canis familiaris, Ailuropoda melanoleuca, Mustela furo, Equus caballus, Cavia porcellus, Camelus ferus, Capra hircus, Chinchilla lanigera, Mesocricetus auratus, Ovis aries, Myotis lucifugus, Pantholops hodgsonii, Bos taurus, Mus musculus, Rattus norvegicus, Echinops telfairi, Oryctolagus cuniculus, Monodelphis domestica, Alligator mississippiensis, Chrysemys picta, Sarcophilus harrisii, Ornithorhynchus anatinus, Melopsittacus undulatus, Anas platyrhynchos, Gallus gallus, Meleagris gallopavo, Falco peregrinus, Zonotrichia albicollis, and Pteropus alecto.

[0166] (IV) Recombinant Production. In particular embodiments, the binding domains disclosed herein are produced from a gene using a protein expression system. Protein expression systems can utilize DNA constructs (e.g. , chimeric genes, expression cassettes, expression vectors, recombination vectors) including a nucleic acid sequence encoding the protein or proteins of interest operatively linked to appropriate regulatory sequences. In particular embodiments, such DNA constructs are not naturally-occurring DNA molecules and are useful for introducing DNA into host-cells to express selected proteins of interest. In particular embodiments, a DNA construct that encodes a vaccine protein can be inserted into cells (e.g., bacterial, mammalian, insect, etc.), which can produce the vaccine protein encoded by the DNA construct.

[0167] Operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded protein is expressed. Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e.g., Maniatisef al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989.

[0168] Expression control sequences are DNA sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art. Expression control sequences generally include a promoter. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totallyF053-0199PCT / 25-069-WO-PCTsynthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res , 15, 2343-2361, 1987. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al., Proc. Natl. Acad. Sci. USA, 76:760-764, 1979.

[0169] The promoter may include, or be modified to include, one or more enhancer elements. In particular embodiments, the promoter will include a plurality of enhancer elements. Promoters including enhancer elements can provide for higher levels of transcription as compared to promoters that do not include them.

[0170] For efficient expression, the coding sequences can be operatively linked to a 3' untranslated sequence. In particular embodiments, the 3' untranslated sequence can include a transcription termination sequence and a polyadenylation sequence. The 3' untranslated region can be obtained, for example, from the flanking regions of genes.

[0171] In particular embodiments, a 5' untranslated leader sequence can also be employed. The 5' untranslated leader sequence is the portion of an mRNA that extends from the 5' CAP site to the translation initiation codon.

[0172] In particular embodiments, a “hisavi” tag can be added to the N-terminus or C-terminus of a gene by the addition of nucleotides coding for the Avitag amino acid sequence, ‘‘GLNDIFEAQKIEWHE” (SEQ ID NO: 128), as well as the 6xhistidine tag “HHHHHH" (SEQ ID NO: 129). The Avitag avidity tag can be biotinylated by a biotin ligase to allow for biotin-avidin or biotin-streptavidin based interactions for protein purification, as well as for immunobiology (such as immunoblotting or immunofluorescence) using anti-biotin antibodies. The 6xhistidine tag allows for protein purification using Ni-2+affinity chromatography. Other tags include: Flag tag (DYKDDDDK; SEQ ID NO: 130), Xpress tag (DLYDDDDK; SEQ ID NO: 131), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 132), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 133), Myc tag (EQKLISEEDL; SEQ ID NO: 134), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 135), STREP® tag II (WSHPQFEK SEQ ID NO: 136 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 137), Softag 3 (TQDPSRVG; SEQ ID NO: 138), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 139).

[0173] The binding domains disclosed herein can be produced using, for example, human suspension cells and / or the Daedalus expression system as described in Pechman etal., Am J Physiol 294: R1234-R1239, 2008. The Daedalus system utilizes inclusion of minimized ubiquitous chromatin opening elements in transduction vectors to reduce or prevent genomic silencing and to help maintain the stability of decigram levels of expression. This system can bypass tedious and time-consuming steps of other protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg / l of correctly folded and post-translationally modified, endotoxin-free protein of up to 70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications. Bandaranayake etal., Nucleic Acids Res., 2011 (Nov); 39(21). In some instances, purification by chromatography may not be needed due to the purity of manufacture according the methods described herein.

[0174] In particular embodiments, the DNA constructs can be introduced into a cell by transfection, a technique thatF053-0199PCT / 25-069-WO-PCTinvolves introduction of foreign DNA into the nucleus of eukaryotic cells. In particular embodiments, the proteins can be synthesized by transient transfection (DNA does not integrate with the genome of the eukaryotic cells, but the genes are expressed for 24-96 hours). Various methods can be used to introduce the foreign DNA into the host-cells, and transfection can be achieved by chemical-based means including by the calcium phosphate, by dendrimers, by liposomes, and by the use of cationic polymers. Non-chemical methods of transfection include electroporation, sono-poration, optical transfection, protoplast fusion, and hydrodynamic delivery. In particular embodiments, transfection can be achieved by particle-based methods including gene gun where the DNA construct is coupled to a nanoparticle of an inert solid which is then "shot" directly into the target-cell's nucleus Other particle-based transfection methods include magnet assisted transfection and impalefection.

[0175] Nucleic acid sequences encoding proteins disclosed herein can be derived by those of ordinary skill in the art. Nucleic acid sequences can also include one or more of various sequence polymorphisms, mutations, and / or sequence variants (e.g., splice variants or codon optimized variants). In particular embodiments, the sequence polymorphisms, mutations, and / or sequence variants do not affect the function of the encoded protein.

[0176] Sequence information provided by public databases can be used to identify additional gene and protein sequences that can be used with the systems and methods disclosed.

[0177] (V) Antibody Conjugates. Antibody conjugates include binding domains disclosed herein linked to another molecule, other than an additional binding domain. Examples of antibody conjugates include antibody-drug conjugates (ADCs), antibody-detectable label conjugates, and antibody-particle conjugates.

[0178] Antibody-drug conjugates allow for the targeted delivery of a drug moiety to an infected cell or viral particle, in particular embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0179] In particular embodiments, antibody-drug conjugates refer to targeted molecules which combine properties of both antibodies and drugs by targeting potent drugs to sites of infection. The drug moiety (D) of an antibody-drug conjugate may include any compound, moiety or group that has a toxic effect. Exemplary drugs include antivirals or anti-infection agents. The drug may be obtained from essentially any source; it may be synthetic or a natural product isolated from a selected source, e.g., a plant, bacterial, insect, mammalian or fungal source. The drug may also be a synthetically modified natural product or an analogue of a natural product.

[0180] In particular embodiments, the antibody-drug conjugates include an antibody conjugated, i.e., covalently attached, to the drug moiety. In particular embodiments, the antibody is covalently attached to the drug moiety through a linker. A linker can include any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavableF053-0199PCT / 25-069-WO-PCTlinker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker. The antibody-drug conjugate selectively delivers an effective dose of a drug to cells whereby greater selectivity, i.e. , a lower efficacious dose, may be achieved while increasing the therapeutic index ("therapeutic window").

[0181] To prepare antibody-drug conjugates, linker-toxin conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 114-124, 2006). Antibody-drug conjugates with multiple (e.g., four) drugs per antibody can be prepared by partial reduction of the antibody with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA in Dulbecco's phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of the linker-cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting ADC mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cyto toxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 02 m filter, and can be lyophilized if desired for storage.

[0182] Antibody-detectable label conjugates include an antibody linked to a detectable label. Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In particular embodiments, detectable labels can include fluorescent labels, chemiluminescent labels, spectral colorimetric labels, enzymatic labels, and affinity tags.

[0183] Fluorescent labels can be particularly useful in cell staining, identification, imaging, and isolation uses. Exemplary fluorescent labels include blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™(Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g, YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0184] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester

[0185] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.F053-0199PCT / 25-069-WO-PCT

[0186] Enzymatic labels can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes can include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-Vl-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0187] Affinity tags are described elsewhere herein.

[0188] Antibody-particle conjugates include an antibody linked to a particle. In particular embodiments, particles include microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, and / or colored beads, and can be made from organic matter and / or inorganic matter. They can be made of any suitable materials that allow for the conjugation of capture proteins, such as antibodies made from the binding domains disclosed herein, to their surface. Examples of suitable materials include: ceramics, glass, polymers, and magnetic materials. Suitable polymers include polystyrene, poly-(methyl methacrylate), poly-(lactic acid), (poly-(lactic-co -glycolic acid)), polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon cross- linked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Additional examples include carbon graphited, titanium dioxide, and paramagnetic materials. See, e.g., ''Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. In particular embodiments, microparticles can be made of one or more materials. In particular embodiments, microparticles are paramagnetic microparticles. Particular embodiments utilize carboxy-modified polystyrene latex (CML) flow cytometry beads and / or magnetic MagPlex® (Luminex, Austin, TX) flow cytometry beads. In particular embodiments, particles can carry a payload.

[0189] In particular embodiments, an antibody as disclosed herein can be linked to a conjugate by any method known in the art. In particular embodiments, the constant region can be modified to allow for site specific conjugation. Such techniques include the use of naturally occurring or engineered cysteine residues, disulfide bridges, poly-histidine sequences, glycoengineering tags, and transglutaminase recognition sequences. Antibody fragments can also be modified for site-specific conjugation, see for example, Kim et al., Mol Cancer Ther 2008;7(8).

[0190] (VI) Recombinant Receptors. Rubulavirus binding domains disclosed herein can be utilized within recombinant receptors such as chimeric antigen receptors (CAR) and / or engineered T cell receptors (eTCR).

[0191] CAR include several distinct subcomponents that allow genetically modified cells (e.g., regulatory T cells) to recognize and kill Rubulaviruses. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a Rubulavirus epitope that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such epitopes, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one orF053-0199PCT / 25-069-WO-PCTmore linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain's ability to bind the targeted epitope.

[0192] eTCR disclosed herein include a Rubulavirus binding domain disclosed herein linked to the Caand / or Cp chains of a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and p chain. Each chain includes a variable region (Vaand Vp) and a constant region (Caand Cp). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes an binding domain that binds Rubulaviruses as the variable region of the a and p chain. In particular embodiments, eTCR include a Caand / or Cp chain sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Caor Cp.

[0193] In particular embodiments, the extracellular component of a recombinant receptor includes a binding domain that binds Rubulaviruses. Particular embodiments of binding domains include a 2x4-Ka antibody, a 2x4-La antibody, and / or the CDRs thereof as disclosed herein.

[0194] Recombinant receptors can additionally include spacer regions, transmembrane domains, intracellular effector domains, transduction markers, and tags.

[0195] Spacer regions are used to create appropriate distances and / or flexibility between sub-components of a protein. Spacer regions typically include 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. Exemplary spacer regions include all or a portion of an immunoglobulin hinge region.

[0196] Transmembrane domains typically have a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD45, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0197] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the expressed protein (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the expressed protein (e.g., up to 15 amino acids of the intracellular components).

[0198] Intracellular effector domains activate the expressing cell when the binding domain binds the antigen. The term "effector domain" is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0199] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CD3y, CD35, CD3e, CD3<(,F053-0199PCT / 25-069-WO-PCTCD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRo, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD19, CD4, CD8a, CD8|3, IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11 b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0200] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3s, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1G), FcyRlla, FcR|3 (Fes Rib), DAP10, and DAP12. In particular embodiments, variants of CD3( retain at least one, two, three, or all ITAM regions.

[0201] A co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD137), 0X40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 ), NKG2C, and a ligand that specifically binds with CD83.

[0202] Transduction markers may be selected from, for example, at least one of a truncated CD 19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR; see Wang et al., Blood 118: 1255, 2011); an extracellular domain of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1(5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278). Methods to genetically modify cells to express CAR are well-known in the art.

[0203] Recombinant receptors can additionally include tags, such as the affinity tags elsewhere herein.

[0204] (VII) Compositions or Formulations for Administration. Any of the binding domains described herein (e.g., antibodies, multi-domain binding molecules, antibody conjugates, therapeutics) in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the antibodies can also be formulated into compositions for administration (e.g., nucleic acids encapsulated within nanoparticles (e.g., liposomes or polymer-based nanoparticles) and / or as part of a vector delivery system (e.g., a viral vector or plasmid). Binding domains (e.g., antibodies, multi-domain binding molecules, antibody conjugates) and / or nucleic acids encoding antibodies are collectively referred to herein as "active ingredients”. Certain examples may include formulations. Formulations include cells genetically modified to express an antibody disclosed herein within a pharmaceutically acceptable carrier.

[0205] Salts and / or pro-drugs of active ingredients can also be used.

[0206] A pharmaceutically acceptable salt includes any salt that retains the activity of the active ingredient and isF053-0199PCT / 25-069-WO-PCTacceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0207] Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0208] Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

[0209] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of an active ingredient or by hydrolysis of a biologically labile group.

[0210] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0211] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0212] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0213] An exemplary chelating agent is EDTA.

[0214] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0215] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldi methyl benzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0216] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose,F053-0199PCT / 25-069-WO-PCTfructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0217] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous injection.

[0218] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the composition can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0219] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid compositions such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0220] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredient and a suitable powder base such as lactose or starch.

[0221] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.F053-0199PCT / 25-069-WO-PCT

[0222] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more active ingredients following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0223] Depot compositions can include a variety of bioerodible polymers including poly (lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0224] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0225] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0226] Excipients that partition into the external phase boundary of nanoparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0227] Additional processing of the disclosed sustained release depot compositions can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0228] In particular embodiments, compositions include active ingredients of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0229] In certain examples, cells are genetically modified to express a protein including a disclosed binding domain. In particular embodiments, cells genetically modified to express the binding domains described herein includeF053-0199PCT / 25-069-WO-PCTgenetically modified B cells. In particular embodiments, the modified B cells are modified according to the teachings of International Publication No. WO2019079772. In these embodiments, genetically modified cells can be prepared as formulations for delivery in buffers such as Hanks' solution, Ringer's solution, or physiological saline. Cells can be genetically modified using methods known in the art. Exemplary targeted genetic engineering approaches include the use of CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), and / or transcription activator like effector nucleases (TALENs). In particular embodiments, the cells are B cells genetically modified to express an antibody. Methods to genetically modify a B cell to express an antibody are described in PCT / US2018 / 056789.

[0230] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0231] In particular embodiments, cells are in a formulation volume of a liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Hence, the density of administered cells is typically greater than 104cells / ml, 105cells / ml, 106cells / ml, 107cells / ml, or 108cells / ml.

[0232] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions and formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0233] (VIII) Kits. Also provided herein are kits including at least one antibody or sequences encoding at least one binding domain disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. In particular embodiments, the kit includes a Rubulavirus binding domain, an anti-Rubulavirus antibody, a multidomain binding molecule, or an antibody conjugate, or sequences encoding an antibody, a multi-domain binding molecule, an or an antibody conjugate as described herein. In particular embodiments, the kit includes cells expressing a recombinant receptor or formulation to modify cells to express a recombinant receptor. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or other material useful in sample processing, washing, or conducting any other step of the method described herein.

[0234] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.F053-0199PCT / 25-069-WO-PCT

[0235] (IX) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions and / or formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0236] An “effective amount” is the amount of a composition or formulation necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic effect. Effective amounts are often administered for research purposes Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model or clinical study relevant to the assessment of an infection's development, progression, and / or resolution, as well as the effects of the infection. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0237] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of an infection or displays only early signs or symptoms of an infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the infection. Thus, a prophylactic treatment functions as a preventative treatment against an infection. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of an infection.

[0238] Therefore, a "therapeutic" can refer to a treatment that reduces the severity of infection and / or induces an immune response in a subject to Rubulaviruses. In particular embodiments, a therapeutic may be administered to a subject who has been exposed to a Rubulavirus. Thus, a therapeutic can be used to ameliorate a symptom and / or complication associated with Rubulaviruses.

[0239] In particular embodiments, a therapeutic is a therapeutically effective composition including binding domains disclosed herein that bind the fusion protein of a Rubulavirus (e.g., HPIV2 and / or HPIV4) that neutralizes Rubulaviruses and / or induces an immune response in a subject against Rubulaviruses. The skilled artisan will appreciate that the immune system generally is capable of producing an innate immune response and an adaptive immune response. An innate immune response generally can be characterized as not being substantially antigen specific and / or not generating immune memory. An adaptive immune response can be characterized as being substantially antigen specific, maturing over time (e.g., increasing affinity and / or avidity for antigen), and in general can produce immunologic memory. Even though these and other functional distinctions between innate and adaptive immunity can be discerned, the skilled artisan will appreciate that the innate and adaptive immune systems can be integrated and therefore can act in concert.

[0240] In particular embodiments, administration of a therapeutic can further include administration of one or more adjuvants. The term "adjuvant” refers to material that enhances an immune response and is used herein in the customary use of the term. The precise mode of action is not understood for all adjuvants, but such lack of understanding does not prevent their clinical use for a wide variety of therapeutics.F053-0199PCT / 25-069-WO-PCT

[0241] Exemplary adjuvants, include any kind of Toll-like receptor ligand or combinations thereof (e.g. CpG, Cpg-28 (a TLR9 agonist), polyriboinosinic polyribocytidylic acid (Poly(l:C)), a-galactoceramide, MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081 ), TMX-101 (imiquimod), MGN1703 (a TLR9 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), Entolimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and Imiquimod), and / or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0242] In particular embodiments a squalene-based adjuvant can be used. Squalene is part of the group of molecules known as triterpenes, which are all hydrocarbons with 30 carbon molecules. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seeds, and olives, as well as from animal sources, such as shark liver oil. In particular embodiments, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant that is similar to MF59® but is designed for preclinical research use is Addavax™ (InvivoGen, San Diego, CA). MF59 has been FDA approved for use in an influenza vaccine, and studies indicate that it is safe for use during pregnancy (Tsai et al. Vaccine. 2010. 17:28(7): 1877-80; Heikkinen et al. Am J Obstet Gynecol. 2012. 207(3): 177). In particular embodiments, squalene based adjuvants can include 0.1 %-20% (v / v) squalene oil. In particular embodiments, squalene based adjuvants can include 5%(v / v) squalene oil.

[0243] In particular embodiments the adjuvant alum can be used. Alum refers to a family of salts that contain two sulfate groups, a monovalent cation, and a trivalent metal, such as aluminum or chromium. Alum is an FDA approved adjuvant. In particular embodiments, therapeutics can include alum in the amounts of 1-1000|jg / dose or 0.1mg-Wmg / dose.

[0244] In particular embodiments, one or more STING agonists are used as an adjuvant. "STING" is an abbreviation of "stimulator of interferon genes", which is also known as "endoplasmic reticulum interferon stimulator (ERIS)", "mediator of IRF3 activation (MITA)", "MPYS" or "transmembrane protein 173 (TM173)".

[0245] In particular embodiments, STING agonists include cyclic molecules with one or two phosphodiester linkages, and / or one or two phosphorothioate diester linkages, between two nucleotides. This includes (3',5')-(3',5') nucleotide linkages (abbreviated as (3', 3')); (3',5')-(2',5') nucleotide linkages (abbreviated as (3',2')); (2',5')-(3',5') nucleotide linkages (abbreviated as (2', 3')); and (2',5')-(2',5') nucleotide linkages (abbreviated as (2', 2')). "Nucleotide" refers to any nucleoside linked to a phosphate group at the 5', 3' or 2' position of the sugar moiety.

[0246] In particular embodiments, STING agonists include c-AIMP; (3',2')c-AIMP; (2’,2’)c-AIMP; (2’,3’)c-AIMP; c-AIMP(S); c-(dAMP-dlMP); c-(dAMP-2’FdlMP); c-(2’FdAMP-2’FdlMP); (2’,3’)c-(AMP-2’FdlMP); c-[2’FdAMP(S)-2’FdlMP(S)j; c-[2’ FdAMP(S)-2’Fdl MP(S)](POM)2; and DMXAA. Additional examples of STING agonists are described in W02016 / 145102.

[0247] Other immune stimulants can also be used as adjuvants. Additional exemplary small molecule immune stimulants include TGF-p inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNA capable of down-regulating immune-suppressive signals or oncogenic pathways (such as kras) can be used whereasF053-0199PCT / 25-069-WO-PCTany plasmid DNA (such as minicircle DNA) encoding immune-stimulatory proteins can also be used.

[0248] In particular embodiments, the immune stimulant may be a cytokine and or a combination of cytokines, such as IL-2, IL-12 or IL-15 in combination with IFN-a, IFN-|3 or IFN-y, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The above-identified cytokines stimulate TH1 responses, but cytokines that stimulate TH2 responses may also be used, such as IL-4, IL-10, IL-11, or any effective combination thereof. Also, combinations of cytokines that stimulate TH1 responses along with cytokines that stimulate TH2 responses may be used.

[0249] 11Immune response" refers to a response of the immune system to produce antibodies to neutralize and / or destroy a Rubulavirus. In particular embodiments, an immune response can be an innate and / or adaptive response. In particular embodiments, the therapeutics described herein are responsible for blocking the entry of a pathogen into a cell so that it is firstly unable to infect healthy cells, and secondly, it is unable to replicate and cause severe infection. Furthermore, in particular embodiments, therapeutics described herein mark Rubulaviruses for destruction by immune cells such as macrophages and neutrophils through opsonization.

[0250] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of an infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the infection or effects of the infection. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the infection and / or reduce, control or eliminate side effects of the infection.

[0251] In particular embodiments a therapeutic treatment can reduce, control, or eliminate a primary infection with a Rubulavirus. In particular embodiments a therapeutic treatment can reduce or eliminate the symptoms of a Rubulavirus. In particular embodiments, a therapeutically effective amount reduces or prevents transmission of a Rubulavirus.

[0252] In particular embodiments, a therapeutically effective amount alleviates or reduces the severity or occurrence of symptoms and / or complications associated with Rubulavirus infection. In particular embodiments, a Rubulavirus infection includes an infection with HPIV2, HPIV4, mumps virus, simian parainfluenza virus 5(SV5), SV41, porcine rubulavirus, and the avian paramyxoviruses 2 to 9. In particular embodiments, a Rubulavirus infection includes an HPIV2 infection or an HPIV4 infection. In particular embodiments, HPIV4 infection includes HPIV4a infection and H PI V4b infection. Exemplary symptoms of infection with HPIV2 include fever, runny nose, sore throat, sneezing, cough, and / or wheezing. Exemplary symptoms of infection with HPIV4 include runny nose, fever, cough, sore throat, sneezing, wheezing, croup, bronchitis, bronchiolitis, pneumonia, ear pain, irritability, decreased appetite, redness or swelling of the eyes, noisy breathing, hoarse voice, and / or rattling in the chest or back when breathing.

[0253] In particular embodiments, a therapeutically effective amount reduces the duration of hospitalization for a subject infected with the Rubulavirus as compared to a subject that has not received a therapeutic disclosed herein.

[0254] In particular embodiments, a therapeutically effective amount reduces the time to sustained non-detectable Rubulavirus in the blood or urine in a patient infected with the virus as compared to a subject that has not received a therapeutic disclosed herein.F053-0199PCT / 25-069-WO-PCT

[0255] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0256] In particular embodiments, therapeutically effective amounts provide anti-infection effects. Anti-infection effects include a reducing or preventing a virus from infecting a cell, decreasing the number of infected cells, decreasing the volume of infected tissue, increasing lifespan, increasing life expectancy, reducing or eliminating infection-associated symptoms. In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against Rubulavirus infection.

[0257] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of infection, stage of infection, effects of infection, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0258] Useful doses can range from 0 1 to 5 pig / kg or from 0.5 to 1 pig / kg. In other examples, a dose can include 1 pig / kg, 15 pig / kg, 30 pig / kg, 50 pig / kg, 55 pig / kg, 70 pig / kg, 90 pig / kg, 150 pig / kg, 350 pig / kg, 500 pig / kg, 750 pig / kg, 1000 pig / kg, 0.1 to5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0259] Exemplary doses of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0260] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0261] The pharmaceutical compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual administration and more particularly by intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual injection.

[0262] The Exemplary Embodiments and Examples below are included to demonstrate particular embodiments ofF053-0199PCT / 25-069-WO-PCTthe disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0263] (X) Exemplary Embodiments.1. A binding domain that binds a Rubulavirus, wherein the binding domain includes a variable heavy chain including a complementarity determining region (CDR) heavy (H)1 , a CDRH2, and a CDRH3 and a variable light chain including a CDR light (L)1 , CDRL2, and CDRL3; wherein:the CDRH1 includes the sequence as set forth in SEQ ID NO: 4, the CDRH2 includes the sequence as set forth in SEQ ID NO: 5, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 6, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 7, the CDRL2 includes the sequence as set forth in SEQ ID NO: 8, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 9;the CDRH1 includes the sequence as set forth in SEQ ID NO: 10, the CDRH2 includes the sequence as set forth in SEQ ID NO: 11, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 12, and the CDRLI includes the sequence as set forth in SEQ ID NO: 13, the CDRL2 includes the sequence as set forth in SEQ ID NO: 14, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 9;the CDRH1 includes the sequence as set forth in SEQ ID NO: 15, the CDRH2 includes the sequence as set forth in SEQ ID NO: 16, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 12, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 13, the CDRL2 includes the sequence as set forth in SEQ ID NO: 14, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 9;the CDRH1 includes the sequence as set forth in SEQ ID NO: 17, the CDRH2 includes the sequence as set forth in SEQ ID NO: 18, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 6, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 13, the CDRL2 includes the sequence as set forth in SEQ ID NO: 19, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 9; orthe CDRH1 includes the sequence as set forth in SEQ ID NO: 20, the CDRH2 includes the sequence as set forth in SEQ ID NO: 21, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 22, and the CDRLI includes the sequence as set forth in SEQ ID NO: 23, the CDRL2 includes the sequence as set forth in SEQ ID NO: 24, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 25.2. The binding domain of embodiment 1, wherein the variable heavy chain includes a sequence as set forth in SEQ ID NO: 48 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 48; and the variable light chain includes a sequence as set forth in SEQ ID NO: 49 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 49.3. The binding domain of embodiments 1 or 2, wherein the variable heavy chain is encoded by a sequence as set forth in SEQ ID NO: 50 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 50; and the variable light chain is encoded by a sequence as set forth in SEQ ID NO: 51 or a sequenceF053-0199PCT / 25-069-WO-PCThaving at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 51.A binding domain that binds a Rubulavirus, wherein the binding domain includes a variable heavy chain including a complementarity determining region (CDR) heavy (H)1 , a CDRH2, and a CDRH3 and a variable light chain including a CDR light (L)1, CDRL2, and CDRL3; wherein:the CDRH1 includes the sequence as set forth in SEQ ID NO: 26, the CDRH2 includes the sequence as set forth in SEQ ID NO: 27, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 28, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 29, the CDRL2 includes the sequence as set forth in SEQ ID NO: 30, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 31 ;the CDRH1 includes the sequence as set forth in SEQ ID NO: 32, the CDRH2 includes the sequence as set forth in SEQ ID NO: 33, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 34, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 35, the CDRL2 includes the sequence as set forth in SEQ ID NO: 36, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 31 ;the CDRH1 includes the sequence as set forth in SEQ ID NO: 37, the CDRH2 includes the sequence as set forth in SEQ ID NO: 38, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 34, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 35, the CDRL2 includes the sequence as set forth in SEQ ID NO: 36, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 31 ;the CDRH1 includes the sequence as set forth in SEQ ID NO: 39, the CDRH2 includes the sequence as set forth in SEQ ID NO: 40, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 28, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 35, the CDRL2 includes the sequence as set forth in SEQ ID NO: 41, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 31 ; orthe CDRH1 includes the sequence as set forth in SEQ ID NO: 42, the CDRH2 includes the sequence as set forth in SEQ ID NO: 43, and the CDRH3 includes the sequence as set forth in SEQ ID NO: 44, and the CDRL1 includes the sequence as set forth in SEQ ID NO: 45, the CDRL2 includes the sequence as set forth in SEQ ID NO: 46, and the CDRL3 includes the sequence as set forth in SEQ ID NO: 47.The binding domain of embodiment 4, wherein the variable heavy chain includes a sequence as set forth in SEQ ID NO: 52 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 52; and the variable light chain includes a sequence as set forth in SEQ ID NO: 53 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 53.The binding domain of embodiments 4 or 5, wherein the variable heavy chain is encoded by a sequence as set forth in SEQ ID NO: 54 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 54; and the variable light chain is encoded by a sequence as set forth in SEQ ID NO: 55 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 55.A binding molecule including the binding domain of any of embodiments 1-3 or any of embodiments 4-6.The binding molecule of embodiment 7, wherein the binding molecule further includes a human constant region.F053-0199PCT / 25-069-WO-PCTThe binding molecule of embodiment 8, wherein the human constant region includes a human light chain constant region and / or a human heavy chain constant region.The binding molecule of embodiment 9, wherein the human light chain constant region includes a human IgK light chain constant region or a human IgA light chain constant region.The binding molecule of embodiment 10, wherein the human IgK light chain constant region includes the sequence as set forth in SEQ ID NO: 68.The binding molecule of embodiment 10, wherein the human IgA light chain constant region includes the sequence as set forth in SEQ ID NO: 69.The binding molecule of any of embodiments 7-12, wherein the binding molecule is an IgG antibody, an IgA antibody, an IgM antibody, an IgD antibody, or an IgE antibody.The binding molecule of embodiment 13, wherein the IgG antibody includes an lgG1 antibody, an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody.The binding molecule of any of embodiments 7-14, wherein the binding molecule is a neutralizing antibody. The binding molecule of any of embodiments 7-15, wherein the binding molecule is an scFV or a Fab.The binding molecule of any of embodiments 7-16, including one or more modified amino acids.The binding molecule of embodiment 17, wherein the one or more modified amino acids include a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent. The binding molecule of any of embodiments 7-18, wherein the binding molecule includes one or more Fc modifications.The binding molecule of any of embodiments 7-19, wherein the binding molecule is a part of a multi-domain binding molecule.A multi-domain binding molecule including at least two binding domains wherein at least one binding domain includes the binding domain of any of embodiments 1-3 or 4-6.The multi-domain binding molecule of embodiment 21, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer.The multi-domain binding molecule of embodiments 21 or 22, including an Fc region.The multi-domain binding molecule of embodiment 23, wherein the Fc region is an IgA Fc region or an IgM Fc region.The multi-domain binding molecule of embodiments 23 or 24, wherein the Fc region is an IgA Fc region having the sequence as set forth in SEQ ID NOs: 76 and 77.The multi-domain binding molecule of embodiments 23 or 24, wherein the Fc region is an IgM Fc region having the sequence as set forth in SEQ ID NOs: 78, 79, or 111-119.The multi-domain binding molecule of any of embodiments 23-26, wherein the Fc region includes a multimerizingF053-0199PCT / 25-069-WO-PCTfragment of the IgA Fc region or a multi merizi ng fragment of the IgM Fc region.The multi-domain binding molecule of embodiment 27, wherein the multimerizing fragment of the IgA Fc region includes the IgA tailpiece.The multi-domain binding molecule of embodiment 28, wherein the IgA tailpiece has the sequence of residues 331-352 as set forth in SEQ ID NO: 76 or the sequence of residues 318-340 as set forth in SEQ ID NO: 77. The multi-domain binding molecule of any of embodiments 27-29, wherein the multimerizing fragment of the IgA Fc region includes the IgA CA3 domain and the IgA tailpiece.The multi-domain binding molecule of any of embodiments 27-30, wherein the multimerizing fragment of the IgA Fc region includes the IgA CA2 domain, the IgA CA3 domain, and the IgA tailpiece.The multi-domain binding molecule of embodiment 27-31, wherein the multimerizing fragment of the IgA Fc region includes the IgA CA1 domain, the IgA CA2 domain, the IgA CA3 domain, and the IgA tailpiece.The multi-domain binding molecule of embodiment 27, wherein the multimerizing fragment of the IgM Fc region includes the IgM tailpiece.The multi-domain binding molecule of any of embodiments 27-33, wherein the multimerizing fragment of the IgM Fc region includes the C i4 domain and the IgM tailpiece.The multi-domain binding molecule of any of embodiments 27-34, wherein the multimerizing fragment of the IgM Fc region includes the C i3 domain, the C i4 domain, and the IgM tailpiece.The multi-domain binding molecule of any of embodiments 27-35, wherein the multimerizing fragment of the IgM Fc region includes the C 2 domain, the C 3 domain, the Cpi4 domain, and the IgM tailpiece.The multi-domain binding molecule of any of embodiments 27-36, wherein the multimerizing fragment of the IgM Fc region includes the C i 1 domain, the C i2 domain, the Cpi3 domain, the C i4 domain, and the IgM tailpiece. The multi-domain binding molecule of any of embodiments 27-37, wherein the multimerizing fragment of the IgM Fc region has the sequence as set forth in any of SEQ ID NOs: 111-119.A single-chain variable fragment (scFv) including the binding domain of any of embodiments 1-6, wherein the binding domain includes a humanized light chain variable region and / or a humanized heavy chain variable region and lacks a constant region.A conjugate including the binding domain of any of embodiments 1-3 or 4-6, linked to a drug, a detectable label, or a particle.A composition including the binding domain of any of embodiments 1-3 or 4-6, the binding molecule of any of embodiments 7-20, the multi-domain binding molecule of any of embodiments 21 -38, the scFv of embodiment 39, or the conjugate of embodiment 40; and a pharmaceutically acceptable carrier.The composition of embodiment 41, further including one or more adjuvants.The composition of embodiments 41 or 42, further including a second type of binding molecule.A nucleic acid sequence encoding the binding domain of any of embodiments 1-3 or 4-6, the binding molecule ofF053-0199PCT / 25-069-WO-PCTany of embodiments 7-20, or the scFv of embodiment 39.45 A vector including the nucleic acid sequence of embodiment 44.46. A cell genetically modified to express the binding molecule of any of embodiments 7-20.47. A method of treating a subject in need thereof including administering to the subject a therapeutically effective amount of a composition of any of embodiments 41-43 thereby treating the subject in need thereof.48. The method of embodiment 47, wherein the subject has been infected with a Rubulavirus.49. The method of embodiment 48, wherein the Rubulavirus includes Human Parainfluenza Virus (HPIV) 2.50 The method of embodiments 48 or 49, wherein the Rubulavirus includes HPIV4.51. The method of embodiment 50, wherein the HPIV4 includes HPIV4a or H P I V4b.52. The method of any of embodiments 47-51, wherein the subject has previously been infected with a Rubulavirus.53. The method of embodiment 47, wherein the administering is prophy lactical ly administering.54. The method of any of embodiments 47-52, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

[0264] (XI) Examples.

[0265] Example 1. Cross-protective antibodies to common, emerging, and re-emerging respiratory viruses related to the parainfluenza viruses.

[0266] Abstract. Human parainfluenza virus types 2 (HPIV2) and 4 (HPIV4) are ubiquitous and cause acute respiratory illness. HPIV2, HPIV4, and several related viruses in bats and pigs that can spillover into humans make up the Rubulavirinae subfamily. Human disease ranges from mild cold symptoms to life-threatening lower respiratory tract disease. Over 90% of adults have antibodies to the parainfluenza viruses, but these antibodies are not fully protective, and reinfections frequently occur. Severe disease is more common in the elderly, immunocompromised individuals, and patients with cardiac and pulmonary comorbidities. Among immunocompromised patients, the highest morbidity and mortality has been described in hematopoietic cell transplant (HCT) recipients, patients with leukemia, and lung transplant recipients Over a third of the infections in HCT recipients progress to the lower respiratory tract, and mortality after the virus reaches the lungs ranges from 13-63%. Parainfluenza viral infections in the post-lung transplant setting are associated with respiratory failure, bronchiolitis obliterans, and chronic allograft dysfunction. Steroid use has also been linked to severe disease and mortality. Since there are no vaccines or antivirals available for the prevention or treatment of any of the parainfluenza viruses, clinicians are limited to providing supportive care. The goal of this project is to provide a safe and effective intervention that can prevent and treat multiple rubulaviruses simultaneously. Novel cross-neutralizing monoclonal antibodies (mAbs) that target a conserved site on a surface protein of both HPIV2 and HPIV4 have been discovered. This was unexpected, given that the amino acid homology between HPIV2 and HPIV4 is 35%. This development of mAbs provides for prophylaxis and / or therapy of respiratory infections caused by HPIV2, HPIV4, and potentially other zoonotic rubulaviruses with spillover potential.F053-0199PCT / 25-069-WO-PCT

[0267] Summary. Significance and impact. A 67-year-old woman with multiple myeloma was evaluated. She had acquired an HPIV4 upper respiratory tract infection from her great grandchildren a few days after receiving an autologous HCT for multiple myeloma. Within a week, the virus gained a foothold in her lungs. She unfortunately developed hypoxic respiratory failure and died two weeks later. HPIV2 and HPIV4 are related viruses that cause cold symptoms in healthy individuals and can cause repeated infections throughout life, leading to loss of productivity and transmission to vulnerable patients. Moreover, HPIV2 and HPIV4 infections can be fatal in children, adults with underlying lung disease, and immunocompromised individuals. There are currently no vaccines or drugs to prevent or treat infections caused by these viruses.

[0268] Worldwide, 13% of acute lower respiratory tract infections and 4% of deaths due to acute lower respiratory tract infections in children under 5 are attributable to the parainfluenza viruses. In the US, parainfluenza viruses account for 7% of hospitalizations in children due to pneumonia. The annual incidence of hospitalization due to parainfluenza viruses is 1 per 1,000 children.

[0269] Parainfluenza viruses account for 3% of hospitalizations due to pneumonia in adults. Age over 65 is a significant risk factor for developing severe disease. Outbreaks in long-term care facilities have occurred with up to 50% of patients developing lower respiratory tract disease.

[0270] Parainfluenza viruses also account for 8% of COPD exacerbations, which is associated with increased risk of hospitalization, rehospitalization, and death. In lung transplant recipients, the annual incidence of infection with parainfluenza viruses is 5%. Parainfluenza viral infection in a transplanted lung can lead to respiratory failure, bronchiolitis obliterans, and chronic allograft dysfunction. HPIV2 and HPIV4 also cause 2-5% of respiratory viral infections in HCT recipients and are frequently associated with difficult to treat bacterial and fungal super-infections. In the US alone, there are 10,000 new HCT recipients and 2,700 new lung transplants every year.

[0271] Zoonotic rubulaviruses cause infections in livestock, pets, and bats and have spilled over into humans. HPIV2 and HPIV4 were likely once zoonotic viruses that jumped out of animal reservoirs to become endemic in the human population. Outbreaks of emerging zoonotic rubulaviruses could have a significant global impact, like a pandemic.

[0272] There are currently no effective strategies for the prevention or treatment of HPIV2 or HPIV4. As a result, there remains an unmet need for safe and effective drugs for prophylaxis and therapy. Infants receive the mAb nirsevimab prophylactically to protect against RSV lower respiratory tract disease, but nothing is available for HPIV2 or HPIV4.

[0273] There are also no vaccines or antivirals available for any other of the rubulaviruses, except for a live attenuated vaccine for mumps virus. Notably, in the past 13 years, multiple mumps outbreaks have occurred. This, coupled with an increase in genetic diversity among circulating mumps virus strains, has led some to suggest an update to the currently available vaccine Zoonotic rubulaviruses, including new strains of mumps from bats and viruses that afflict canines and swine, threaten to spill over into the human population. The ability to infect numerous cell types due to the use of sialic acid, the fact that novel viruses have been found in animal reservoirs, and the fact that zoonoticF053-0199PCT / 25-069-WO-PCTtransmission has already been observed all raise concerns about an emerging rubulavirus. Currently, the world is unprepared for an outbreak caused by an emerging rubulavirus.

[0274] This example describes the development of a broadly protective mAb to prevent and treat HPIV2 and HPIV4 infections in vulnerable patients. This class of antibodies is called 2x4, because they can be used to simultaneously protect against HPIV2 and HPIV4. The 2x4 antibodies could be used as immunoprophylaxis in children, adults with underlying lung disease, and immunocompromised individuals, including hematopoietic stem cell transplant recipients who can be vulnerable to infections for months to years post-transplant. The 2x4 antibodies can also be used as therapy. A secondary goal is to determine if these antibodies protect against zoonotic rubulaviruses with pandemic potential. A cocktail of 2x4 and 3x1 (a mAb that neutralizes HPIV1 and HPIV3) would provide protection against all known parainfluenza viruses.

[0275] Innovation. There are no vaccines or antiviral agents currently available for the prevention or treatment of HPIV2, HPIV4, or any of the other related rubulaviruses, except for a live attenuated mumps vaccine. Clinicians have nothing to offer beyond supportive care to patients with severe lower respiratory tract disease caused by HPIV2 or HPIV4. This proposal builds upon the discovery of a novel class of cross-neutralizing mAbs that target both HPIV2 and HPIV4. This was unexpected given the relatively low amino acid homology between HPIV2 and HPIV4 (35%). This Example describes the development of mAbs for prophylaxis and / or therapy of respiratory infections caused by HPIV2, HPIV4, and potentially other related human and zoonotic rubulaviruses with spillover potential. This fills an important gap in the ability to care for vulnerable patients and, at the same time, prepare for the next pandemic.

[0276] Results. HPIV2 and HPIV4 have two surface glycoproteins, hemagglutinin-neuraminidase (HN) and the fusion (F) protein. Each is essential for the virus to infect host cells. The HN protein attaches to host cell receptors and cleaves these receptors for viral release, whereas the F protein mediates viral fusion and entry into the host cell. Initially, the focus was on F because the development of neutralizing antibodies to F is clinically correlated with decreased disease severity. A discovery campaign was started for anti-F antibodies to HPIV2 and HPIV4, because anti-F antibodies were demonstrated to suppress replication of other parainfluenza viruses, including HPIV3 and HPIV1, in animal models.

[0277] The F proteins of the parainfluenza viruses exist in a metastable pre-fusion (preF) conformation on the infectious virion and irreversibly flips into a post-fusion (postF) conformation after viral entry. Since most of the neutralizing activity is conferred by antibodies targeting preF, preF was used, rather than postF, as bait to identify parainfluenza virus-binding B cells. Because HPIV2 preF was unstable, the bait for HPIV4 preF was used. Therefore, the novel approach for isolating cross-neutralizing antibodies called "bait and switch” was leveraged. Single HPIV4-binding B cells were stimulated in vitro and screened for the ability to secrete antibodies that cross-neutralized HPIV2. From this, two novel HPI V2 / HPIV4 cross-neutralizing mAbs were identified, which were named 2x4-KaF (or 2x4-Ka) and 2x4-LaF (or 2x4-La). 2x4-KaF and 2x4-LaF demonstrated high binding affinity for HPIV4 with a Ko = 3 x 10’10M and KD< 10'12M, respectively (FIGs. 1A, 1B). Despite utilizing different immunoglobulin heavy and light chain variable genes, 2x4 KaF and 2x4 LaF competed with each other for binding to HPIV4 preF. Therefore, both mAbs target aF053-0199PCT / 25-069-WO-PCTsimilar conserved epitope present on HPIV2, HPIV4, and possibly other related human and zoonotic rubulaviruses.2x4 KaF and 2x4 LaF could block HPIV2 infection in vitro, with a neutralization titer of 0.2 pig / mL and 0.4 g / mL, respectively (FIG. 2). The in vitro neutralization potency of 2x4-KaF and 2x4-LaF against HPIV4 was 0.05|jg / mL. For comparison, the mAb palivizumab that is (U.S. Food and Drug Administration (FDA) approved for RSV prophylaxis in infants has an I C50 of 0.5 g / mL The 2x4 antibodies are the first ever mAbs isolated that are capable of neutralizing both HPIV2 and HPIV4.

[0278] Prophetic Example 2. The main objective of this example is to accelerate the preclinical characterization and development of novel cross-neutralizing monoclonal antibodies (mAbs) to Human parainfluenza virus types 2 (HPIV2), type 4 (HPIV4), and other rubulaviruses.

[0279] Characterize 2x4-KaF and 2x4-LaF in vitro and in vivo, a) Cryo-electron microscopy will be performed to visualize the molecular interactions between the neutralizing mAbs and HPIV4 preF. These results will provide a mechanistic understanding of the molecular basis of neutralization and the potential for viral escape; b) HPIV2 will be passaged in the presence of serial dilutions of mAb to generate escape mutations that confer mAb resistance; c) In vivo efficacy (EC50 and ECgo)will be determined by administering the 2x4 mAbs i) prophylactically and ii) therapeutically in a dose response hamster challenge model of HPIV2; and d) Other rubulaviruses, including mammalian orthorubulavirus 5 and mumps virus, which have 38-48% amino acid sequence homology to HPIV2, will be cultured and whether the 2x4 mAbs can neutralize these homologous viruses will be tested. Together, these results will provide critical preclinical information on neutralization breadth, neutralization mechanism, neutralization escape, and in vivo efficacy.

[0280] Discovery campaign for additional cross-neutralizing antibodies targeting other rubulavirus epitopes or antigens. While the “bait-and-switch” approach allowed for circumventing the unavailability of bait for HPIV2, this approach constrains the identification of cross-neutralizing B cells to only those that bind to the recombinant version of the HPIV4 preF protein. Recombinant proteins may not recapitulate all epitopes present on a live virion. Furthermore, cross-neutralizing B cells targeting HN are missed.

[0281] An antigen-agnostic approach was developed for neutralizing antibody discovery that does not rely on recombinant bait. In this approach, memory B cells are enriched from human peripheral blood, spleen, or tonsils. Lentiviral transduction of the transcription factors BCL-6 and BCL-xL will be used to imbue B cells with high proliferative capacity and antibody secretion. These B cells are sorted at a frequency of 200 cells / well. The B cells from wells with supernatant that neutralize HPIV2 will be sorted into new wells at a frequency of 1 cell / well. Culture supernatant will be screened for HPIV4 neutralization to identify HPIV2 and HPIV4 cross-neutralizing B cells. In vitro binding assys and neutralization assays can then be performed with the individual mAbs and in combination with 2x4-KaF and -LaF to determine potency and synergy, respectively.

[0282] A cross-protective mAb against HPIV2, HPIV4, and zoonotic rubulaviruses would have important medical implications for several vulnerable populations. One of the 2x4 mAbs could also be combined with other cross-F053-0199PCT / 25-069-WO-PCTneutralizing antibodies that 1) target other epitopes to provide synergy or 2) target other respiratory viruses to increase the breadth of protection

[0283] Advantages and impact on standard of care. The current stand of care for patients with parainfluenza viral infections is supportive care. Steroids are associated with increased mortality and are no longer used. A mAb could be used to protect vulnerable populations from infection and could also provide a therapeutic option for clinicians who otherwise have little to nothing to offer beyond supportive care. By leveraging cross-neutralizing mAbs that target a conserved region on multiple viruses, the risk of developing mAb resistance through viral escape is also likely decreased.

[0284] (XII) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0285] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programswell known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0286] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non-polar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0287] In making such changes, the hydropathic index of amino acids may be considered. The importance of theF053-0199PCT / 25-069-WO-PCThydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0288] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0289] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1 5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0290] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid sidechain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistical ly-significant degree.

[0291] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0292] “ % sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods toF053-0199PCT / 25-069-WO-PCTdetermine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the PASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0293] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pig / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pig / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0294] "Specifically binds" refers to an association of a binding domain (of, for example, an antibody (e.g., neutralizing antibody) to a virus) to its cognate binding molecule with an affinity or Ka( / .e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M'1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains withF053-0199PCT / 25-069-WO-PCTa Kaof at least 107M-1, at least 108M’1, at least 109M-1, at least 1010M'1, at least 1011M’1, at least 1012M-1, or at least 1013M'1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Kaof up to 107M'1, up to 106M’1, up to 105M'1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 105M to 10'13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd(dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (KOff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0295] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I Freshney, ed. Animal Cell Culture (1987).

[0296] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms "include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of' limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant increase in Rubulavirus (e.g., HPIV2 and HPIV4) as described herein.

[0297] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of theF053-0199PCT / 25-069-WO-PCTdoctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0298] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0299] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0300] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0301] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claimsF053-0199PCT / 25-069-WO-PCTappended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0302] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0303] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0304] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0305] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

F053-0199PCT / 25-069-WO-PCTCLAIMSWhat is claimed is:

1. A binding domain that binds a Rubulavirus, wherein the binding domain comprises a variable heavy chain comprising a complementarity determining region (CDR) heavy (H)1 , a CDRH2, and a CDRH3 and a variable light chain comprising a CDR light (L)1 , CDRL2, and CDRL3; wherein:the CDRH1 comprises the sequence as set forth in SEQ ID NO: 4, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 5, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 6, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 7, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 8, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 9;the CDRH1 comprises the sequence as set forth in SEQ ID NO: 10, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 11, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 12, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 13, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 14, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 9;the CDRH1 comprises the sequence as set forth in SEQ ID NO: 15, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 16, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 12, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 13, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 14, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 9;the CDRH1 comprises the sequence as set forth in SEQ ID NO: 17, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 18, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 6, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 13, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 19, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 9; orthe CDRH1 comprises the sequence as set forth in SEQ ID NO: 20, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 21, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 22, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 23, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 24, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 25.

2. The binding domain of claim 1, wherein the variable heavy chain comprises a sequence as set forth in SEQ ID NO: 48 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 48; and the variable light chain comprises a sequence as set forth in SEQ ID NO: 49 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 49.

3. The binding domain of claim 1 , wherein the variable heavy chain is encoded by a sequence as set forth in SEQ ID NO: 50 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 50; and the variable light chain is encoded by a sequence as set forth in SEQ ID NO: 51 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 51.

4. A binding domain that binds a Rubulavirus, wherein the binding domain comprises a variable heavy chainF053-0199PCT / 25-069-WO-PCTcomprising a complementarity determining region (CDR) heavy (H)1 , a CDRH2, and a CDRH3 and a variable light chain comprising a CDR light (L)1 , CDRL2, and CDRL3; wherein:the CDRH1 comprises the sequence as set forth in SEQ ID NO: 26, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 27, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 28, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 29, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 30, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 31;the CDRH1 comprises the sequence as set forth in SEQ ID NO: 32, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 33, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 34, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 35, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 36, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 31;the CDRH1 comprises the sequence as set forth in SEQ ID NO: 37, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 38, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 34, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 35, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 36, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 31;the CDRH1 comprises the sequence as set forth in SEQ ID NO: 39, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 40, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 28, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 35, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 41, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 31; orthe CDRH1 comprises the sequence as set forth in SEQ ID NO: 42, the CDRH2 comprises the sequence as set forth in SEQ ID NO: 43, and the CDRH3 comprises the sequence as set forth in SEQ ID NO: 44, and the CDRL1 comprises the sequence as set forth in SEQ ID NO: 45, the CDRL2 comprises the sequence as set forth in SEQ ID NO: 46, and the CDRL3 comprises the sequence as set forth in SEQ ID NO: 47.

5. The binding domain of claim 4, wherein the variable heavy chain comprises a sequence as set forth in SEQ ID NO: 52 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 52; and the variable light chain comprises a sequence as set forth in SEQ ID NO: 53 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 53.

6. The binding domain of claim 4, wherein the variable heavy chain is encoded by a sequence as set forth in SEQ ID NO: 54 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 54; and the variable light chain is encoded by a sequence as set forth in SEQ ID NO: 55 or a sequence having at least 95% sequence identity to the sequence as set forth in SEQ ID NO: 55.

7. A binding molecule comprising the binding domain of claim 1 or claim 4.

8. The binding molecule of claim 7, wherein the binding molecule further comprises a human constant region.

9. The binding molecule of claim 8, wherein the human constant region comprises a human light chain constant region and / or a human heavy chain constant region.F053-0199PCT / 25-069-WO-PCT10. The binding molecule of claim 9, wherein the human light chain constant region comprises a human IgK light chain constant region or a human IgA light chain constant region.

11. The binding molecule of claim 10, wherein the human IgK light chain constant region comprises the sequence as set forth in SEQ ID NO: 68.

12. The binding molecule of claim 10, wherein the human IgA light chain constant region comprises the sequence as set forth in SEQ ID NO: 69.

13. The binding molecule of claim 7, wherein the binding molecule is an IgG antibody, an IgA antibody, an IgM antibody, an IgD antibody, or an IgE antibody.

14. The binding molecule of claim 13, wherein the IgG antibody comprises an lgG1 antibody, an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody.

15. The binding molecule of claim 7, wherein the binding molecule is a neutralizing antibody.

16. The binding molecule of claim 7, wherein the binding molecule is an scFV or a Fab.

17. The binding molecule of claim 7, comprising one or more modified amino acids.18 The binding molecule of claim 17, wherein the one or more modified amino acids comprise a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, or an amino acid conjugated to an organic derivatizing agent.

19. The binding molecule of claim 7, wherein the binding molecule comprises one or more Fc modifications.

20. The binding molecule of claim 7, wherein the binding molecule is a part of a multi-domain binding molecule.

21. A multi-domain binding molecule comprising at least two binding domains wherein at least one binding domain comprises the binding domain of claims 1 or 4.

22. The multi-domain binding molecule of claim 21, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer.

23. The multi-domain binding molecule of claim 21, comprising an Fc region.

24. The multi-domain binding molecule of claim 23, wherein the Fc region is an IgA Fc region or an IgM Fc region.

25. The multi-domain binding molecule of claim 23, wherein the Fc region is an IgA Fc region having the sequence as set forth in SEQ ID NOs: 76 and 7726. The multi-domain binding molecule of claim 23, wherein the Fc region is an IgM Fc region having the sequence as set forth in SEQ ID NOs: 78, 79, or 111-119.

27. The multi-domain binding molecule of claim 23, wherein the Fc region comprises a multimerizing fragment of an IgA Fc region or a multimerizing fragment of an IgM Fc region.

28. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgA Fc region comprises an IgA tailpiece29. The multi-domain binding molecule of claim 28, wherein the IgA tailpiece has a sequence of residues 331-352 as set forth in SEQ ID NO: 76 or a sequence of residues 318-340 as set forth in SEQ ID NO: 77.F053-0199PCT / 25-069-WO-PCT30. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA3 domain and an IgA tailpiece.

31. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.

32. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgA Fc region comprises an IgA CA1 domain, an IgA CA2 domain, an IgA CA3 domain, and an IgA tailpiece.

33. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgM Fc region comprises an IgM tailpiece.

34. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgM Fc region comprises an Cpi4 domain and an IgM tailpiece.

35. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgM Fc region comprises a Cpi3 domain, a Cpi4 domain, and an IgM tailpiece.

36. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgM Fc region comprises a Cpi2 domain, a Cpi3 domain, a Cpi4 domain, and an IgM tailpiece.

37. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgM Fc region comprises a Cpi 1 domain, a Cpi2 domain, a Cpi3 domain, a Cpi4 domain, and an IgM tailpiece.

38. The multi-domain binding molecule of claim 27, wherein the multimerizing fragment of the IgM Fc region has the sequence as set forth in any of SEQ ID NOs: 111-119.

39. A single-chain variable fragment (scFv) comprising the binding domain of claims 1 or 4, wherein the binding domain comprises a humanized light chain variable region and / or a humanized heavy chain variable region and lacks a constant region.

40. A conjugate comprising the binding domain of claims 1 or 4, linked to a drug, a detectable label, or a particle.

41. A composition comprising the binding domain of claims 1 or 4, the binding molecule of claim 7, the multi-domain binding molecule of claim 21, the scFvofclaim 39, or the conjugate of claim 40; and a pharmaceutically acceptable carrier.42 The composition of claim 41 , further comprising one or more adjuvants.

43. The composition of claim 41, further comprising a second type of binding molecule.

44. A nucleic acid sequence encoding the binding domain of claims 1 or 4, the binding molecule of claim 7, or the scFv of claim 39.

45. A vector comprising the nucleic acid sequence of claim 44.

46. A cell genetically modified to express the binding molecule of claim 7.47 A method of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition of claim 41 thereby treating the subject in need thereof.

48. The method of claim 47, wherein the subject has been infected with a Rubulavirus.F053-0199PCT / 25-069-WO-PCT49. The method of claim 48, wherein the Rubulavirus comprises Human Parainfluenza Virus (HPIV) 2.50 The method of claim 48, wherein the Rubulavirus comprises HPIV4.

51. The method of claim 50, wherein the HPIV4 comprises HPIV4a or HPIV4b.

52. The method of claim 47, wherein the administering is prophylactical ly administering.

53. The method of claim 47, wherein the subject has previously been infected with a Rubulavirus.

54. The method of claim 47, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

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