Methods and compositions for determining immunoglobulin chain mispairing

Abstract

The present disclosure provides methods for evaluating and characterizing immunoglobulin chain associate in compositions (e.g., pharmaceutical compositions) of nucleic acids encoding multiple antibody agents, and compositions including antibody variants for use in the same.

Description

Attorney Docket No.: 2013237-1583 (BNT REF: P2042W001)METHODSAND COMPOSITIONS FOR DETERMINING IMMUNOGLOBULIN CHAIN MISPAIRING CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 754,548, filed February 5, 2025, which is herein incorporated by reference in its entirety.BACKGROUND

[0002] Utilizing more than one antibody or a bispecific antibody in a therapeutic composition has been a strategy for targeting a broader range of epitopes and increasing efficacy of immunotherapies. However, using multiple antibodies or bispecific antibodies adds complexity in generating antibodies that are correctly assembled, especially when the antibodies are delivered as nucleic acids. Mispairing of immunoglobulin chains can lead to formation of unintended antigen-binding domains, increasing the risk for off-target activity. There exists a need for improving the rate of mispairing of immunoglobulin chains in therapeutics containing multiple antibodies, and assessing risk of immunoglobulin chain mispairing in these compositions.SUMMARY

[0003] The present disclosure provides insights that address these challenges, making it possible to evaluate safety and efficacy of therapeutics including multiple antibody agents. For example, the present disclosure describes antibody agents or portions thereof (e.g., immunoglobulin chains) that are delivered to a subject via nucleic acids (e.g., polyribonucleotides). An antibody agent that is delivered as one or more polyribonucleotides that encode the antibody agent are referred to herein as "RiboMabs." After delivery of one or more nucleic acids (e.g., polyribonucleotides) that encode an antibody agent to a cell (e.g., in or from a subject), the antibody agent, e.g., the RiboMab" is expressed in the cell. Utilizing nucleic acids as a therapeutic agent (in contrast to administering an antibody agent itself) involves simpler and less expensive manufacturing processes. The less complex production of nucleic acid s (e.g., polyribonucleotides) encoding antibody agent(s) can streamline manufacturing {e.g., by circumventing the need for intensive glycan production and profiling), mitigating regulatory and production challenges associated with developing and using antibody agents themselves. Additionally, nucleic acids (e.g., polyribonucleotides) are effective at producing similar effects to recombinant proteins but tend to require much lower volumes be administered to a subject. In particular, with polyribonucleotides encoding antibody agent(s), they can be administered to a subject and the subject's body produces the antibody agent(s) itself. Using a lower volume can provide a patient with a more pleasant experience and increase patient compliance with a treatment regimen. The present disclosure also provides technologies that address certain limitations of recombinant antibody technologies, including for example, the short serum half-life of recombinant antibodies by utilizing nucleic acids (e.g., RNA technologies) as a modality to express antibody agents directly in the patient's cells (see FIG. 1).

[0004] RiboMab technology also allows for two or more antibody agents to be administered to a subject simultaneously. Typically, an antibody produced by, e.g., humans, comprises four polypeptide chains - two "heavy" chains and two "light" chains. Each polypeptide chain (whether heavy or light) includes (1) a "variable" domain, having a sequence that varies among antibodies and a structure that determines the antigen to which an antibody binds, and (2) a "constant" domain(s), having a sequence and a structure that generally remain unchanged across antibodies of a given class, thus imparting little effect on antigen binding. In humans, specialized white blood cells,Attorney Docket No.: 2013237-1583 (BNT REF: P2042W01)"B cells," produce antibodies. Heavy chains and light chains are assembled to form an antibody through two key pairings: (1) the fragment crystallizable (Fc) domains of the two heavy chains pair together, and (2) the two light chains each pair with a heavy chain via disulfide linkages. During normal antibody production in a human, a single antibody is produced by a single B cell. In that situation, the correct pairing of heavy and light chains is ensured because only one species of heavy chain and one species of light chain are present in each B cell. Antibody agents can be recombinantly produced and combined prior to their administration. While this does not face the problem of mispairing as each antibody is produced separately and only afterwards combined with one or more other antibodies, this recombinant antibody production however has several disadvantages, like e.g., challenges in protein manufacturing, administration and immunogenicity.

[0005] In vivo production of antibodies is an appealing way to leverage monoclonal antibodies uses in medical settings. In some instances, utilizing combinations of antibody agents in therapeutics is useful to target a broader range of epitopes and increase efficacy of interventions. This requires co-delivery of expression templates (i.e., DNA or RNA), regardless of the vector of delivery used (see FIG. 1). Co-expression of multiple antibody agents comes with a unique risk of mis-association, or mispairing, of the light chain of one antibody agent to the non-cognate heavy chain of another antibody agent (see FIG. 1 and FIG. 2). In some instances, a combination of antibody agents is directed against the same target making the evaluation of mispairing or the efficacy of mispairingpreventing strategies difficult to assess (see FIG. 5B). While strategies have been developed to reduce the risk of immunoglobulin heavy chain mispairing (see FIG. 3D) and mispairing between immunoglobulin heavy and light chains (see FIG. 2), complete prevention of mispairing is not guaranteed and can lead to serious off-target binding by a mispaired Fab when developing therapeutics.

[0006] The present disclosure provides, among other things, an in vitro methodology that circumvents hurdles of assessing mispairing for the development of interventions relying on in vivo co-expression of more than one antibody. In some embodiments, methods provided herein include generating antibody variants, including monovalent versions of an antibody agent to test, along with its cognate light chain and a "competing" light chain (or non-cognate light chain) to evaluate the loss of functional Fab fragments via mispairing (see FIG. 5B). Use of monovalent antibody agents reduces the risk of undetected "silent mispairing" in which an antibody agent contains a correctly assembled Fab, able to bind the target, while the other valency is mispaired (see e.g., FIG. 5A). In some embodiments, methods provided herein include evaluating mispairing among two or more bivalent antibody agents, e.g., when the bivalent antibody agents are co-delivered as nucleic acids.

[0007] In one aspect, the present disclosure provides an antibody variant comprising: (i) a first immunoglobulin chain comprising a first variable domain and a first constant domain; and (ii) a second immunoglobulin chain comprising a second variable domain and a second constant domain; wherein the first constant domain is a heavy chain constant domain that is unable to homodimerize; and wherein the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to a target antigen.

[0008] In some embodiments, the first variable domain comprises a heavy chain variable domain. In some embodiments, the first constant domain comprises a heavy chain constant domain. In some embodiments, the first constant domain comprises a CHI domain operably linked to a CH2 domain and a CH3 domain. In some- 2 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)embodiments, the first constant domain is unable to homodimerize because of one or more mutations. In some embodiments, the second variable domain comprises a light chain variable domain. In some embodiments, the second constant domain comprises a light chain constant domain. In some embodiments, the antibody variant further comprises a third immunoglobulin chain comprising a third constant domain. In some embodiments, the third constant domain comprises a heavy chain constant domain. In some embodiments, the third constant domain comprises a CH2 domain and a CH3 domain.

[0009] In some embodiments, the third constant domain is unable to homodimerize. In some embodiments, the third constant domain is unable to homodimerize because of one or more mutations. In some embodiments, the third immunoglobulin chain consists of the constant domain. In some embodiments, the third immunoglobulin chain does not comprise a functional variable domain.

[0010] In another aspect, the present disclosure provides a combination comprising: an antibody variant described herein, and a polypeptide comprising a fourth variable domain, wherein the first variable domain and the fourth variable domain are capable of associating to form a mispaired domain. In some embodiments, the fourth variable domain comprises an immunoglobulin light chain variable domain. In some embodiments, the first immunoglobulin chain and / or the second immunoglobulin chain comprise one or more mutations to: (i) increase association between the first immunoglobulin chain and the second immunoglobulin chain; (ii) decrease association between the first immunoglobulin chain and the third immunoglobulin chain; and / or (iii) decrease association between the first immunoglobulin chain and the fourth variable domain. In some embodiments, the third immunoglobulin chain comprises one or more mutations to: (i) increase association between the first immunoglobulin chain and the second immunoglobulin chain; (ii) decrease association between the first immunoglobulin chain and the third immunoglobulin chain; and / or (iii) decrease association between the first immunoglobulin chain and the fourth variable domain. In some embodiments, the fourth variable domain comprises one or more mutations to: (i) increase association between the first immunoglobulin chain and the second immunoglobulin chain; (ii) decrease association between the first immunoglobulin chain and the third immunoglobulin chain; and / or (iii) decrease association between the first immunoglobulin chain and the fourth variable domain.

[0011] In some embodiments, the second constant domain comprises one or more charge variant mutations. In some embodiments, the one or more charge variant mutations comprises an amino acid at position 123 (according to the EU numbering scheme) that is substituted by the amino acid K, R or H. In some embodiments, the one or more charge variant mutations comprises an amino acid at position 124 (according to the EU numbering scheme) that is substituted by the amino acid K, R or H. In some embodiments, the one or more charge variant mutations comprises any one of the following combinations of mutations according to the EU numbering scheme: (i) E123K and Q124R; (ii) E123K and Q124K; (iii) E123R and Q124R; and (iv) E123R and Q124K. In some embodiments, the CHI domain of the first constant domain comprises one or more charge variant mutations. In some embodiments, the one or more charge variant mutations in the CHI domain of the first constant domain comprises an amino acid at position 147 (EU numbering) that is substituted by the amino acid E or D, according to the EU numbering scheme. In some embodiments, the one or more charge variant mutations comprises a K213D mutation. In some embodiments, the one or more charge variant mutations in the CHI domain of the first constant domain comprises one of the following combinations of mutations: (i) K147E and K213D; or (ii) K147D and K213D. In some embodiments, the- 3 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)polypeptide comprises an immunoglobulin light chain variable domain operably linked to an immunoglobulin heavy chain CHI domain. In some embodiments, the CHI domain of the polypeptide comprises a Q124E mutation, according to the EU numbering scheme.

[0012] In some embodiments, the first variable domain comprises a heavy chain variable domain and the first constant domain comprises a light chain constant domain. In some embodiments, the second immunoglobulin chain comprises a light chain variable domain and the second constant domain comprises a CHI domain. In some embodiments, the first constant domain comprises a Q124E mutation, according to the EU numbering scheme.

[0013] In another aspect, the present disclosure provides a composition for use in a mispairing assay comprising a combination described herein.

[0014] In another aspect, the present disclosure provides a composition comprising one or more nucleic acids (e.g., polyribonucleotides) encoding a combination described herein.

[0015] In another aspect, the present disclosure provides a kit comprising the antibody variant, a combination or a composition described herein.

[0016] In another aspect, the present disclosure provides a method comprising: (i) measuring the binding affinity of one or more antibody agents to a target antigen, wherein the one or more antibody agents are in a composition comprising: (A) a first immunoglobulin chain comprising a first variable domain and a first constant domain; (B) a second immunoglobulin chain comprising a second variable domain and a second constant domain; and (C) a third immunoglobulin chain comprising a third constant domain; (D) a polypeptide comprising a third variable domain; wherein the first constant domain is a heavy chain constant domain that is unable to homodimerize; and wherein the first variable domain and the second variable domain are capable of associating to form an antigenbinding domain that specifically binds to the target antigen and wherein the first variable domain and the third variable domain are capable of associating to form a mispaired antigen-binding domain; (ii) comparing the binding affinity of the one or more antibody agents to the binding affinity measured in a control composition, wherein the control composition does not comprise the polypeptide; wherein the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of mispairing between the first and third variable domains, and wherein a decrease in binding affinity measured in the composition compared to the control composition indicates the detection of mispairing between the first and third variable domains.

[0017] In some embodiments, the first, second, and third immunoglobulin chains and the polypeptide are present in the composition as first, second, third, and fourth nucleic acid sequences encoding the first, second, and third immunoglobulin chains and polypeptide, respectively.

[0018] In some embodiments, the method further comprises the step of: expressing the first, second, and third immunoglobulin chains and polypeptide from the first, second, third, and fourth nucleic acid sequences, respectively, thereby producing the composition. In some embodiments, expressing the first, second, and third, immunoglobulin chains and the polypeptide from the first, second, third, and fourth nucleic acid sequences, respectively, comprises transfecting a host cell with the first, second, third and fourth nucleic acid sequences and culturing the host cell under conditions such that the first, second, and third immunoglobulin chains and polypeptide are expressed.- 4 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0019] In some embodiments, the third constant domain comprises a heavy chain constant domain. In some embodiments, the third constant domain comprises a CH2 domain and a CH3 domain. In some embodiments, the third constant domain is unable to homodimerize. In some embodiments, the third constant domain is unable to homodimerize because of one or more mutations. In some embodiments, third immunoglobulin chain consists of the heavy chain constant domain. In some embodiments, the third immunoglobulin chain does not comprise a functional variable domain. In some embodiments, the third immunoglobulin chain comprises a third variable domain that does not associate with the first or second variable domains. In some embodiments, the third immunoglobulin chain comprises a VHH domain. In some embodiments, the third immunoglobulin chain comprises a single-chain variable fragment (scFv). In some embodiments, the third immunoglobulin chain comprises fluorescent protein and / or a Tag. In some embodiments, the fluorescent protein comprises a green fluorescent protein (GFP).

[0020] In some embodiments, the first immunoglobulin chain and the third immunoglobulin chain heterodimerize to form an Fc domain. In some embodiments, the first immunoglobulin chain and the third immunoglobulin chain heterodimerize as a result of a knob-in-hole (KIH) mutation.

[0021] In another aspect, the present disclosure provides a method comprising: (i) measuring the binding affinity of one or more antibody agents to a target antigen, wherein the one or more antibody agents are in a composition comprising: (A) a first immunoglobulin chain comprising a first variable domain and a first constant domain; (B) a second immunoglobulin chain comprising a second variable domain and a second constant domain; and (C) a third immunoglobulin chain comprising a third variable domain and a third constant domain; and wherein the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to the target antigen; wherein the first variable domain and the third variable domain associate to a mispaired antigen-binding domain; (ii) comparing the binding affinity of the one or more antibody agents to the binding affinity measured in a control composition, wherein the control composition does not comprise the third immunoglobulin chain, wherein the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of mispairing between the first and third variable domains, and wherein a decrease in binding affinity measured in the composition compared to the control composition indicates detection of mispairing between the first and third variable domains.

[0022] In some embodiments, the composition further comprises: (D) a fourth immunoglobulin chain comprising a fourth variable domain and a fourth constant domain. In some embodiments, the third variable domain and the fourth variable domain associate to form an antigen-binding domain to a second target antigen. In some embodiments, the first, second, third, and fourth immunoglobulin chains are present in the composition as first, second, third, and fourth nucleic acid sequences encoding the first, second, third, and fourth immunoglobulin chains, respectively.

[0023] In some embodiments, the method further comprises the step of: expressing the first, second, third, and fourth immunoglobulin chains from the first, second, third, and fourth nucleic acid sequences, respectively, thereby producing the composition. In some embodiments, expressing the first, second, third, and fourth immunoglobulin chains from the first, second, third, and fourth nucleic acid sequences, respectively, comprises transfecting a host cell with the first, second, third and fourth nucleic acid sequences and culturing the host cell under conditions such that the first, second, third, and fourth immunoglobulin chains are expressed. In some embodiments, the first, second, third and / or fourth nucleic acids are polyribonucleotides. In some embodiments, the- 5 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)polyribonucleotides comprise a polyribonucleotide encoding each of the first, second, third, and fourth immunoglobulin chains.

[0024] In some embodiments, the polyribonucleotides are fully or partially encapsulated within lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes. In some embodiments, the first, second, third, and / or fourth nucleic acids comprise a DNA template. In some embodiments, the DNA template is encoded in a vector (e.g., viral vectors, plasmid vectors, bacteriophage vectors, cosmids, phagemids, and / or artificial chromosomes). In some embodiments, the vector comprises a viral vector. In some embodiments, the viral vector comprises an adeno-associated viral (AAV) vector. In some embodiments, the first, second, third, and fourth nucleic acid sequences are comprised in a single multicistronic expression cassette.

[0025] In some embodiments, the method comprises a method of characterizing the risk of mispairing of immunoglobulin chains. In some embodiments, the composition comprises two or more antibody agents and the method characterizes the risk of mispairing between antibody variable domains of the antibody agents.

[0026] In some embodiments, the method comprises a method of determining if immunoglobulin chains in a composition mispair. In some embodiments, the composition comprises two or more antibody agents and the method determines if antibody variable domains of two different antibody agents in a composition mispair.

[0027] In some embodiments, the method comprises a method of determining the suitability of one or more antibody agents for manufacture, administration, use as a therapeutic. In some embodiments, the therapeutic comprises two or more antibody agents. In some embodiments, the therapeutic comprises two or more antibody agents delivered as nucleic acids encoding the antibody agents. In some embodiments, the therapeutic comprises a bispecific antibody. In some embodiments, the bispecific antibody is delivered as a nucleic acid encoding the bispecific antibody.

[0028] In some embodiments, the composition comprises a relative amount of first nucleic acid compared to the third nucleic acid is between about 1:0.1 to about 1:100. In some embodiments, measuring binding affinity comprises performing an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the ELISA comprises a plate pre-coated with target antigen. In some embodiments, the ELISA further comprises a detection antibody. In some embodiments, the detection antibody comprises an anti-human Fc antibody linked to an enzyme. In some embodiments, the enzyme comprises horseradish peroxidase (HRP). In some embodiments, measuring binding affinity comprises measuring color change produced from a reaction between the enzyme and a substrate added to the sample.BRIEF DESCRIPTION OF THE DRAWING

[0029] FIG. 1 shows a schematic of formulation and delivery of exemplary antibody agents encoded by polyribonucleotides (e.g., RiboMabs) and potential paired and mispaired products.

[0030] FIG. 2 shows a schematic of possible antibody chain mispairing combinations.

[0031] FIG. 3 shows exemplary Fc modifications utilized in antibody agents as described herein. Exemplary antibody agent formats may include an unmodified Fc domain (FIG. 3A), or modifications shown in FIGS. 3B-D, including GAALIE / GAIE / GA / IE (FIG. 3B), L / S (FIG. 3C), and / or knob-into-holes (FIG. 3D).- 6 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0032] FIG. 4 shows exemplary antibody agent formats described herein. Exemplary formats may include IgG (FIG. 4A), CrossMabCH1'CLxCH1 / CL domain swap (FIG. 4B), CrossMabCH1-CLcvcharge variants (FIG. 4C), or various orientations / linkers of scFv-Fc (FIGS. 4D and 4E).

[0033] FIGs. 5A-5B show exemplary schematics of antibody heavy chain / light chain mispairing and detection of the same. FIG. 5A shows an exemplary schematic of an antibody with one mispaired light chain in a bivalent antibody, where mispairing may still be undetected in conventional ELISAs as the antibody still binds to its target through the correctly paired arm. FIG. 5B shows an exemplary schematic of antibody variants described herein utilized to reduce valencies (from 2 to 1) to completely disrupt binding when mispairing between a heavy chain / light chain occurs.

[0034] FIGs. 6A-6C show exemplary schematics of immunoglobulin heavy chain "null" domains that can be utilized in methods described herein, e.g., including a VHH-Fc or vNAR-Fc fusion (FIG. 6A), a fluorescent protein or protein tag fused to an Fc domain (FIG. 6B), and a random amino acid sequence (that does not affect Ig protein folding) or a "blunt" null domain containing only a constant domain of an antibody agent (FIG. 6C).

[0035] FIG. 7 shows a schematic of the experimental conditions in Example 1 using antibody variants described herein to measure antibody heavy chain and light chain mispairing. The exemplary experiment includes transfecting cells with polyribonucleotides encoding the exemplary antibody variants (a Ab2 light chain, a Abl light chain, a Abl-LS heavy chain with knob KiH mutations, and a VHH-Fc fusion with hole KiH mutations) such that the antibody variants are expressed in a composition. Mispairing of the antibody light chain and heavy chain is then measured by ELISA detect Abl antibody binding in conditions with increasing amounts of Ab2-LS antibody light chain.

[0036] FIG. 8 shows a schematic of immunoglobulin chains and compositions including the same utilized in a mispairing assay described in Example 1. Specifically, exemplary compositions include a "null" domain (e.g., a VHH-Fc L / S chain) for one arm of the formed antibody variant. The schematic illustrates the antibody variants (both correctly paired HC / LC combinations and mispaired HC / LC combinations) formed when immunoglobulin chains encoded by the polyribonucleotides included in Groups A-D associate, as assayed in Example 1.

[0037] FIGs. 9A-9B show schematics related to exemplary methods and assays described herein for detecting mispairing between an antibody LC / HC. FIG. 9A shows the principle of an exemplary mispairing assay utilizing an anti-human Fc detection antibody [HRP], an exemplary antibody variant, and a ligand coating on a streptavidin-coated polystyrene plate. FIG. 9B shows a series of steps in an exemplary mispairing assay described herein, where (1) pre-coated wells are coated with biotinylated ligand (target antigen); (2) an antibody variant binds to the target antigen; (3) the detection antibody binds to the bound antibody variant; and (4) HRP catalyzes an enzymatic color reaction.

[0038] FIGs. 10A-10B shows an exemplary readout from an assay described herein where mispairing is indicated by a shifted binding profile (FIG. 10B) compared to a regular binding profile (positive, correctly paired control) (FIG. 10A).

[0039] FIGs. 11A-11D show antibody concentrations measured by ELISA in Example 1 from samples in Group A (FIG. 11A), Group B (FIG. 11B), Group C (FIG. 11C), and Group D (FIG. 11D).- 7 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0040] FIG. 12 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 12A) and reducing conditions (FIG. 12B) and stained for Goat anti-human Kappa Light Chain Antibody, HRP.

[0041] FIG. 13 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 13A) and reducing conditions (FIG. 13B) and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0042] FIG. 14 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 14A) and reducing conditions (FIG. 14B) and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0043] FIG. 15 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 15A) and reducing conditions (FIG. 15B) and stained for Goat IgG anti-Alpaka IgG (VHH).

[0044] FIG. 16 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 16A) and reducing conditions (FIG. 16B) and stained for Goat IgG anti-Alpaka IgG (VHH).

[0045] FIG. 17 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 17A) and reducing conditions (FIG. 17B) and stained for anti-idiotype Abl.

[0046] FIG. 18 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 18A) and reducing conditions (FIG. 18B) and stained for anti-idiotype Abl.

[0047] FIGs. 19A-19B show binding profiles of positive and negative controls used in Example 1 (FIG. 19A) and schematic of antibody variants used in Example 1 (FIG. 19B). FIG. 19A shows binding of the positive control antibodies and Abl to target antigen and negative controls show no binding to target antigen. Negative controls include either Abl HC used in Example 1 tested in the absence of a Abl LC and a negative control antibody that does not bind the target antigen.

[0048] FIGs. 20A-20E show results from the target antigen ELISA described in Example 1. FIG. 20 shows titration curves (binding profiles measuring Abl binding) in Group A (FIG. 20A), Group B (FIG. 20B), Group C (FIG. 20C), Group D (FIG. 20D). FIG. 20E provides a description of each condition that includes increasing relative amounts of Ab2-LS LC (competing LC) a Abl LC for association with a Abl HC, resulting in increased light chain competition.

[0049] FIG. 21 shows binding profiles obtained from a target antigen ELISA used to assess mispairing as described in Example 1. FIG. 21 compares the amount of mispairing in Abllight chains containing a charge variant (Abl LS CrossmabCHl-cv) vs. a nonchanged Abllight chain (Abl LS).

[0050] FIGs. 22A-22E show results from a neutralization assay measuring neutralization of target antigen described in Example 1. FIG. 22 shows neutralization (IC50 fold change) of target antigen in Group A (FIG. 22A), Group B (FIG. 22B), Group C (FIG. 22C), Group D (FIG. 22D). FIG. 22E provides a description of each condition that includes increasing relative amounts of Ab2 LC (competing LC) a Abl LC for association with a Abl HC, resulting in increased light chain competition. ND indicates that neutralization was not detected.

[0051] FIG. 23 shows a schematic of the experimental conditions in Example 2 using antibody variants described herein to measure antibody heavy chain and light chain mispairing. The exemplary experiment includes - 8 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)transfecting cells with polyribonucleotides encoding exemplary antibody variants (a Ab2-LS antibody light chain, a 1 Abl-LS antibody light chain, and a Abl-LS heavy chain) such that the antibody variants are expressed in a composition.

[0052] FIG. 24 shows a schematic of antibody variants and compositions including the same utilized in a mispairing assay described in Example 2. The schematic illustrates immunoglobulin heavy and light chains encoded by polyribonucleotides included in Groups A-D, as assayed in Example 2, as well as the resulting antibody variants from pairing and mispairing of the various HC / LC combinations.

[0053] FIGs. 25A-D show antibody concentrations measured by ELISA in Example 2 from samples in Group A (FIG. 25A), Group B (FIG. 25B), Group C (FIG. 25C), and Group D (FIG. 25D).

[0054] FIGs. 26A-26B show an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 26A) and reducing conditions (FIG. 26B) and stained for Goat anti-human Kappa Light Chain Antibody, HRP.

[0055] FIG. 27 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0056] FIG. 28 shows an exemplary Western Blot analysis of antibody agent mispairing under reducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0057] FIG. 29 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0058] FIG. 30 shows an exemplary Western Blot analysis of antibody agent mispairing under reducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0059] FIG. 31 shows exemplary binding curves obtained from the target antigen ELISA in Example 2 of positive control samples including a AblLS antibody or control LS antibody (fully assembled) and negative control samples including only a Abl-LS heavy chain, a Abl-LS heavy chain in CrossMabCHl-CLcv format, or negative control antibody (antibody that does not bind to the target antigen).

[0060] FIG. 32 shows exemplary binding curves obtained from a target antigen ELISA in Example 2 of Abl-LS antibody agents with and without a charge variant modification challenged with Ab2-LS light chain.

[0061] FIGs. 33A-33E show exemplary binding curves obtained from the target antigen ELISA in Example 2 for each of the Groups A, B, C, and D (FIG. 33A, FIG. 33B, FIG. 33C, and FIG. 33D, respectively) and exemplary ratios of the two light chains tested in each sample (FIG. 33E).

[0062] FIGs. 34A-34E show concentrations of antibody measured in the target antigen ELISA at 50% signal in Example 2 for each of the Groups A, B, C, and D (FIG. 34A, FIG. 34B, FIG. 34C, and FIG. 34D, respectively) and exemplary ratios of the two light chains tested in each sample (FIG. 34E).

[0063] FIGs. 35A-35C show exemplary components (FIG. 35A) and strategy for modifying a Ab2 light chain (FIGs. 35B-C) utilized in Example 3. FIG. 35A shows antibody agents used in Example 3, which are expressed in a cell after mRNA transfection of polyribonucleotides encoding a Ab2 light chain in CrossMabCH1'CLxformat "Ab2-LSxLc"),- 9 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)a Abl light chain (e.g., Abl-LSic and Abl-LSic in CrossMabCH1-CLcvformat "Abl-LScv c"), and a Ab2 heavy chain (e.g. a Ab2-LS heavy chain). FIG. 35B shows a Ab2-LS antibody in CrossMabCH1'CLx(domain swap) format to prevent mispairing. FIG. 35C shows a Abl-LS antibody in CrossMabCH1'CLcv(charge variant) format to prevent mispairing.

[0064] FIG. 36 shows a schematic of antibody variants and compositions including the same utilized in a mispairing assay described in Example 3. The schematic illustrates immunoglobulin heavy and light chains encoded by polyribonucleotides included in Groups A-D, as assayed in Example 3, as well as the resulting antibody variants from pairing and mispairing of the various HC / LC combinations.

[0065] FIGs. 37A-37E show results and sample conditions from the Gyros ELISA in Example 3 measuring intact antibody concentration (FIG. 37A shows results from Group A, FIG. 37B shows results from Group B, FIG.37C shows results from Group C, and FIG. 37D shows results from Group D) and exemplary ratios of the two light chains tested in each sample (FIG. 37E).

[0066] FIG. 38 shows exemplary Western Blot analysis of antibody agent mispairing under nonreducing conditions, stained for anti-human Fcg IgG + anti-human kappa light chain.

[0067] FIG. 39 shows exemplary Western Blot analysis of antibody agent mispairing under reducing conditions, stained for anti-human Fcg IgG + anti-human kappa light chain.

[0068] FIG. 40 shows an exemplary binding curve obtained from the target antigen ELISA of positive control samples including a AblLS antibody or control LS antibody (fully assembled) and negative control samples including only a Ab2-LSx (in domain swap or "CrossMabCH1'CLx" format) heavy chain, a Ab2-LS heavy chain, or a negative control antibody (antibody that does not bind to the target antigen).

[0069] FIG. 41A-41E show exemplary binding curves obtained from the target antigen ELISA in Example 3 for each of the Groups A, B, C, and D (FIG. 41A, FIG. 41B, FIG. 41C, and FIG. 41D, respectively) and exemplary ratios of the two light chains tested in each sample (FIG. 41E).DEFINITIONS

[0070] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0071] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents- 10 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0072] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.

[0073] About. The term "about", when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0074] Agent, s used herein, the term "agent," may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term "therapeutic agent" refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.

[0075] Amino acid-. In its broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a nonnatural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0076] Antibody agent. As used herein, the term "antibody agent" refers to any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding to a particular antigen. Exemplary antibody agents include but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of - 11 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In some embodiments, the term "antibody agent" is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody agent utilized in accordance with the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); CrossMabs (e.g., CrossMabCH1'CL; CrossMabCH1 Lcv; bispecific CrossMabCH1-CLwith knob-in-hole); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or sets thereof; single chain Fvs (scFvs); scFv-Fc fusions; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins® ; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®;MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, chains and / or fragments of such antibodies and fragments may be used in combination, e.g., a scFv-Fc immunoglobulin chain is used with a conventional antibody (e.g., IgG) immunoglobulin chain. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.)).

[0077] Antibody Variant: An "antibody variant" as used herein refers to an antibody agent that includes one or more modifications that alter association of one of more of its immunoglobulin chains. For example, in some embodiments, an antibody variant includes an immunoglobulin chain (e.g., an immunoglobulin heavy chain) that includes one or more modification that prevents association between the immunoglobulin chain and another identical immunoglobulin chain, or prevents homodimerization. In some embodiments, an antibody variant refers to an antibody agent that includes in immunoglobulin chain (e.g., an immunoglobulin heavy chain variable domain) that prevents or reduces risk of mispairing with a non-cognate immunoglobulin chain (e.g., an immunoglobulin light chain variable domain). Exemplary modifications of antibody variants described herein are shown in e.g., FIG. 2 and FIG.3. In some embodiments, an antibody variant includes an immunoglobulin chain that reduces valency of the antibody agent, such that silent mispairing of the antibody variant heavy chain and light chain is eliminated. In some embodiments, an antibody variant is a monovalent antibody agent. In some embodiments, such an immunoglobulin chain is an "null" domain (see e.g., FIG. 5B, where the "Fc-null domain" is a VHH-Fc fusion).

[0078] Antigen-binding domain: An "antigen-binding domain" refers to a portion of an antibody that binds the antigen to which the intact antibody binds. An antigen-binding domain of an antibody includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Exemplary antigen-binding domains include, but are not limited to, a Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, dsFv, SMIP, diabody, triabody, tetrabody, minibody, nanobody, maxibody, tandab, DVD, BiTe, TandAb, or the like, or any combination thereof. In some embodiments, the antigenbinding domain of the antibodies described herein are scFvs. In some embodiments, an scFv antigen-binding domain is part of an immunoglobulin chain that comprises an Fc domain (e.g., an scFv-Fc fusion). In some embodiments, an- 12 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)antigen-binding domain comprises an antigen-binding domain in CrossMab format (e.g., CrossMabCHl-CLx;CrossMabCHl-CLcv as described herein). As with full antibody molecules, antigen-binding domains may be mono-specific or multi-specific (e.g., bispecific).

[0079] Associated-. Two events or entities are "associated" with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0080] Co-administratiorr. s used herein, the term "co-administration" refers to administration of compositions described herein (e.g., a pharmaceutical composition) for delivery of more than one therapeutic agents. In some embodiments, a therapeutic agent comprises an antibody agent described herein. In some embodiments, co-administration refers to delivery of multiple antibody agents. In some embodiments, a therapeutic agent is a nucleic acid (e.g., a polyribonucleotide) encoding an antibody agent. In some embodiments, co-administration refers to co-delivery of nucleic acids encoding multiple antibody agents. In some embodiments, co-administration refers to delivery of multiple antibody agents (e.g., nucleic acids encoding multiple antibody agents) and an additional therapeutic agent. The combined use of a composition described herein (e.g., containing nucleic acids encoding multiple antibody agents) and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a composition described herein (e.g., containing nucleic acids encoding multiple antibody agents) and an additional therapeutic agent may be combined in one pharmaceutically-acceptable excipient, or they may be placed in separate excipient and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of "co-administration" or "combination," provided that nucleic acids encoding multiple antibody agents, optionally with an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0081] Combination therapy. As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be- 13 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition. In some embodiments, a combination therapy comprises nucleic acids (e.g., polyribonucleotides) encoding two or more antibody agents.

[0082] Comparable.- As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0083] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term "corresponding to" may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.- 14 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0084] Derived-. In the context of an amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide), it refers to a structural analogue of a designated amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, antibody agents utilized according to the present disclosure may include amino acid sequences (e.g., CDRs, variable domains, constant domains, etc.) derived from other antibodies, e.g., naturally produced antibodies.

[0085] Detecting.- The term "detecting" is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, "detecting" may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term "quantifying" when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards {e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0086] Encoder. As used herein, the term "encode" or "encoding" refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding an antibody agent refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such an antibody agent. In some embodiments, a coding region of a polyribonucleotide encoding an antibody agent refers to a non-coding strand of such an antibody agent, which may be used as a template for transcription of a gene or cDNA.

[0087] Engineered-. In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.- 15 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0088] Epitope.- As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous portion of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0089] Expression.- As used herein, the term "expression" of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0090] Increased, Induced, or Reduced-. As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be "increased" relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, an assessed value achieved (e.g., antibody agent binding) with a provided composition (e.g., binding of an antibody agent) may be "increased" or "decreased" relative to a control composition (e.g., a composition that does not contain a competing light chain). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term "reduced" or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term "reduced" or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term "increased" or "induced" refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at- 16 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0091] In order: As used herein with reference to a polynucleotide or polyribonucleotide, "in order" refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, "in order" refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. "In order" does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being "in order, Feature A, Feature B, and Feature C," this description does not exclude, e.g., Feature D being located between Features A and B.

[0092] Lipid, s used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0093] RNA iipid nanopartider. s used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as described herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size {e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0094] Mispairing-, s used herein, the term "mispairing" refers to association of an immunoglobulin chain (e.g., an immunoglobulin heavy chain) with a non-cognate immunoglobulin chain (e.g., a non-cognate immunoglobulin light chain). In some embodiments, mispairing occurs when an immunoglobulin heavy chain associates with a non-cognate light chain. Cognate immunoglobulin chains refer to immunoglobulin chains that form an antigen-binding domain that binds to, e.g., a target antigen. A non-cognate immunoglobulin chain refers to an immunoglobulin chain that does not form an intended antigen-binding domain. Mispairing may occur in mixtures of immunoglobulin chains from different antibody agents (e.g., antibody agents that bind to different target antigens or epitopes) when an immunoglobulin chain of one antibody agent associates with an immunoglobulin chain of another antibody agent. In some embodiments, mispairing between an immunoglobulin chain of one antibody agent and an immunoglobulin chain of another antibody agent abrogates binding to the target antigen of both antibody agents. In some embodiments, the immunoglobulin chains are one immunoglobulin heavy chain and one immunoglobulin light chain, that when associated, form and antigen-binding domain (e.g., a Fab). In some embodiments, a "mispaired"- 17 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)antigen-binding domain as used herein, refers to an antigen-binding domain that includes mispaired immunoglobulin chains, or paired immunoglobulin chains where one of the immunoglobulin chains is from a first antibody agent recognizing a target antigen or epitope and one of the immunoglobulin chains is from a second antibody agent recognizing a different target antigen or epitope.

[0095] Neutralization,- is used herein, the term "neutralization" refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term "neutralization" refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0096] Nucleic acid / Polynucleotide.- As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and doublestranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphoroth ioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a "peptide nucleic acid". In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.- 18 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0097] Polypeptide.- As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0098] Prevent, s used herein, the term "prevent" or "prevention" when used in connection with the occurrence of immunoglobulin chain mispairing, refers to reducing the risk of immunoglobulin mispairing between e.g., an immunoglobulin heavy chain and a non-cognate immunoglobulin light chain. Prevention may be considered complete when there is no difference in binding affinity in a composition that contains an immunoglobulin chains encoding an antibody agent and a competing light chain of a second antibody agent and a control composition that does not contain the competing light chain.- 19 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0099] Reference.- As used herein, the term "reference" describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, a reference composition or a control composition used in methods described herein refers to a composition that does not contain a competing light chain. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0100] Ribonucleic acid (RNA) or Polyribonucleotide-, s used herein, the term "ribonucleic acid, ""RNA," or "polyribonucleotide" refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of "Nucleic acid / Polynucleotide" above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is an mRNA. In some embodiments, where an RNA is a mRNA, a RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0101] Ribonucleotide.- As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 21position or 41position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0102] Risk-, is will be understood from context, "risk" of mispairing between immunoglobulin chains as described herein refers to a likelihood that a particular immunoglobulin heavy chain will associate with its noncognate light chain. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference composition or control composition as described herein. In some embodiments, a reference composition or control composition described herein is known to not have a risk of immunoglobulin chain mispairing. In some embodiments, a reference composition or control composition- 20 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)described herein does not contain a competing light chain such that there is no risk of mispairing with a non-cognate or competing light chain.

[0103] Selective or specific. The term "selective" or "specific," when used herein in reference to an agent (e.g., antibody agent) having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind "specifically" to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0104] Subject. As used herein, the term "subject" refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0105] Suffering from-. An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0106] Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0107] Therapy. The term "therapy" refers to an administration or delivery of an agent (e.g., one or more antibody agents) or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to - 21 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.DETAILED DESCRIPTION

[0108] The present disclosure provides, among other things, methods of identifying and / or characterizing mispairing between immunoglobulin chains. In some embodiments, the present disclosure provides methods for identifying mispairing of immunoglobulin chains expressed from one or more nucleic acids. Methods described herein are particularly useful in determining immunoglobulin chain mispairing in compositions that include immunoglobulin chains that form multiple antibody agents.

[0109] In some embodiments, compositions include nucleic acids that encode multiple immunoglobulin chains that are co-delivered, and encode for more than one antibody agent (see FIG. 1). The present disclosure recognizes, among other things, that such compositions of nucleic acids represent an alternative delivery modality for recombinant therapeutic proteins, e.g., therapeutic antibody agents, and that delivery of nucleic acids (e.g., polyribonucleotides) presents unique challenges including the expressed immunoglobulin chains form undesired "mispaired" combinations (see e.g., FIG. 1 and FIG. 2). Such challenges are not normally present when multiple antibody agents are delivered as recombinant proteins, where the antibody agents have been generated separately and associate correctly, and then combined. When nucleic acids encoding immunoglobulin chains of different antibody agents are co-delivered to and expressed in a cell, mispairing events may arise.

[0110] The present disclosure also recognizes that mispairing of immunoglobulin chains in compositions described herein may be difficult to detect. When immunoglobulin chains of multiple antibody agents are utilized or delivered together, desired combinations of those immunoglobulin chains can form, but undesired "mispaired" combinations of the immunoglobulin chains can also form (see, e.g., FIG. 1 and FIG. 2). For example, mispairing can occur on one or both arms of an antibody agent. Detection of mispairing at both Fabs of antibody agents within a population (e.g., in a composition) can be detected using binding assays (e.g., an ELISA as described herein). For example, if both Fabs of an antibody agent are mispaired or the heavy chain of one antibody agent is mispaired with a light chain of another antibody agent, the antibody agent will not form the intended epitope binding site and binding of the antibody agent to the epitope or antigen of interest will be reduced. While mispairing on both Fabs of an antibody agent can occur, a more likely occurrence is mispairing on only one Fab of an antibody agent. If only one Fab of an antibody agent pairs correctly and the other Fab mispairs, mispairing becomes challenging to detect because the one correctly paired Fab can still bind to its target antigen and some binding will still occur (also referred to herein as "silent mispairing" as shown in e.g., FIG. 5A). The challenge arises because the correctly paired Fab can still bind to its cognate epitope or antigen of interest and thus the antibody agent will be observed as properly binding. This makes traditional binding assays insufficient to definitively confirm that immunoglobulin chains are not mispairing in a composition of multiple antibody agents, e.g., multiple antibody agents expressed from nucleic acids.

[0111] To address this challenge, the present disclosure provides methods for identifying mispairing among combinations of antibody agents, and antibody variants for use in identifying mispairing. In some embodiments,- 22 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)antibody agent mispairing is characterized in a composition by utilizing a control polypeptide, e.g., a "null" domain e.g., as shown in Fig. 5B and FIGs. 6A-6C. A null domain is utilized to reduce valency of an antibody agent so reduced binding to a target antigen can attributed to mispairing of immunoglobulin chains forming the single antigenbinding domain.

[0112] In some embodiments, the present disclosure provides methods (e.g., methods of characterizing mispairing between immunoglobulin chains) that include (i) measuring the binding affinity of one or more antibody agents to a target antigen, (ii) comparing the binding affinity of the one or more antibody agents to the target antigen to the binding affinity measured in a control composition, wherein the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of mispairing, and wherein a decrease in binding affinity measured in the composition compared to the control composition indicates the occurrence of mispairing.

[0113] In some embodiments, one or more antibody agents are in a composition comprising: a first immunoglobulin chain comprising a first variable domain and a first constant domain (e.g., an immunoglobulin heavy chain of an antibody agent), a second immunoglobulin chain comprising a second variable domain and a second constant domain (e.g., a cognate light chain) and a third immunoglobulin chain comprising a third constant domain (e.g., a null domain) and a polypeptide comprising a third variable domain (e.g., a non-cognate immunoglobulin light chain) (see e.g., FIGs. 6A-6C). In some embodiments, the first constant domain is a heavy chain constant domain that is unable to homodimerize and / or may heterodimerize with third immunoglobulin chain. In some embodiments, such association with the first immunoglobulin chain and the third immunoglobulin chain produces an antibody agent with reduced valency (see e.g., FIG. 5B). In some embodiments, the first variable domain and the second variable domain are capable of associating to form an antigen-binding domain that specifically binds to the target antigen and where the first variable domain and the third variable domain are capable of associating to form a mispaired antigenbinding domain. In some embodiments, immunoglobulin chains and a polypeptide are present in the composition as nucleic acid sequences encoding the first, second, and third immunoglobulin chains and the polypeptide, respectively, and methods described herein include expressing the nucleic acids thereby producing the composition (e.g., by transfecting a host cell and culturing the host cell under conditions such that the immunoglobulin chains and polypeptide are expressed).

[0114] In some embodiments, mispairing is assessed in compositions that include immunoglobulin chains of two or more antibody agents, where valency is not reduced or there is no Fc null domain included in the composition. In some embodiments, compositions include a first immunoglobulin chain comprising a first variable domain and a first constant domain, a second immunoglobulin chain comprising a second variable domain and a second constant domain, a third immunoglobulin chain comprising a third variable domain and a third constant domain. In some embodiments, the first variable domain and the second variable domain associate to form an antigen-binding domain of a first antibody agent that specifically binds to a target antigen and the first variable domain and the third variable domain associate to a mispaired antigen-binding domain. In some embodiments, such compositions further comprise a fourth immunoglobulin chain that includes e.g., an immunoglobulin heavy chain that is capable of associating with the third immunoglobulin chain to form an antigen-binding domain of a second antibody agent.

[0115] Methods described herein, in some embodiments, include methods of characterizing mispairing of immunoglobulin chains. In some embodiments, compositions for use in such methods comprise two or more antibody- 23 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)agents and the method characterizes the risk of mispairing between antibody variable domains of the antibody agents.

[0116] Methods described herein, in some embodiments, include a method of determining if immunoglobulin chains in a composition mispair. In some embodiments, compositions for use in such methods comprise two or more antibody agents and the method determines if antibody variable domains of two different antibody agents in a composition mispair.

[0117] In some embodiments, methods described herein are useful in determining the suitability of one or more antibody agents for manufacture, administration, use as a therapeutic.Methods of Identifying and / or Characterizing Immunoglobulin Chain Mispairing

[0118] The present disclosure provides methods and compositions for identifying and / or characterizing immunoglobulin chain mispairing. In some embodiments, compositions provided include immunoglobulin chains from multiple antibody agents. In some embodiments, immunoglobulin chains are formulated in a composition as nucleic acids (e.g., polyribonucleotides) encoding the immunoglobulin chains. Methods described herein are particularly useful in identifying immunoglobulin chain mispairing in compositions that include immunoglobulin chains that form multiple antibody agents and / or antibody agents that contain two or more different antigen-binding domains (e.g., a bispecific antibodies).

[0119] In some embodiments, the present disclosure provides methods (e.g., methods of identifying and / or characterizing mispairing between immunoglobulin chains) that include (i) measuring the binding affinity of one or more antibody agents to a target antigen, (ii) comparing the binding affinity of the one or more antibody agents to the binding affinity measured in a control composition, wherein the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of mispairing, and wherein a decrease in binding affinity measured in the composition compared to the control composition indicates the detection of mispairing.Monovalent Antibody Variants

[0120] In some embodiments, methods described herein include measuring mispairing in a composition of immunoglobulin chains that, when associated, form a monovalent antibody variant (see FIG. 5B). In some embodiments, a composition comprises an antibody variant that includes a first immunoglobulin chain comprising a first variable domain and a first constant domain, a second immunoglobulin chain comprising a second variable domain and a second constant domain. In some embodiments, a composition further comprises a polypeptide comprising a third variable domain (e.g., a competing light chain). In some embodiments, an antibody variant further comprises a third immunoglobulin chain comprising a third constant domain (e.g., an "null domain"). In some embodiments, the first constant domain is a heavy chain constant domain that is unable to homodimerize. In some embodiments, the first variable domain and the second variable domain are capable of associating to form an antigen-binding domain that specifically binds to a target antigen. In some embodiments, the first variable domain and the third variable domain are capable of associating to form a mispaired antigen-binding domain. In some embodiments, methods described herein identify and / or characterize mispairing between the third variable domain and the first variable domain. In some embodiments, mispairing is measured by measuring binding affinity of the antigen-binding domain to the target antigen when the third variable domain is added to a composition.- 24 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0121] In some embodiments, first, second, and third immunoglobulin chains and the polypeptide are present in a composition as first, second, third, and fourth nucleic acid sequences encoding the first, second, and third immunoglobulin chains and polypeptide, respectively (see FIG. 7 and FIG. 8). In some embodiments, methods described herein further comprise the step of expressing the first, second, and third immunoglobulin chains and polypeptide from the first, second, third, and fourth nucleic acid sequences, respectively, thereby producing the composition. In some embodiments, expressing the nucleic acid sequences include transfecting a host cell with the nucleic acids and culturing the host cell under conditions such that the nucleic acids are expressed. In some embodiments, a host cell may be in vitro (e.g., a cell line) - for example a cell or cell line (e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for expressing antibody agents and / or nucleic acids encoding antibody agents described herein.

[0122] In some embodiments, the third constant domain comprises a heavy chain constant domain. In some embodiments, the third constant domain is null domain), e.g., as shown in Fig. 5B (an exemplary embodiment that uses a VHH-Fc domain fusion) and FIGs. 6A-6C. A null domain as described is utilized in order to reduce valency of an antibody agent so that any impact of mispairing on a Fab not bound to an epitope or antigen of interest can be eliminated. In some embodiments, the nucleic acids are polyribonucleotides. In some embodiments, the polyribonucleotides are fully or partially encapsulated within lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0123] As described herein, a null domain may include an immunoglobulin heavy chain includes a CH2 domain and a CH3 domain. In some embodiments, a null immunoglobulin heavy chain is unable to homodimerize, e.g., to one or more mutations. In some embodiments, a null immunoglobulin heavy chain includes only the heavy chain constant domain. In some embodiments, a null immunoglobulin heavy chain does not include a functional variable domain (so that it cannot bind to a target antigen). In some embodiments, a null immunoglobulin heavy chain, does not comprise a functional variable domain (e.g., may include a variable domain that is not functional, e.g., to bind to a target antigen). In some embodiments, a null immunoglobulin heavy chain includes a variable domain that does not associate with the other variable domains in compositions described herein. In some embodiments, a null immunoglobulin heavy chain comprises a VHH domain or a single-chain variable fragment (scFv). In some embodiments, a null immunoglobulin heavy chain includes fluorescent protein and / or a Tag (e.g., a green fluorescent protein). In some embodiments, a null domain including an immunoglobulin heavy chain constant domain heterodimerizes with a constant domain of an antibody variant described herein to form an Fc domain, e.g., as a result of a knob-in-hole (KIH) mutation.

[0124] In some embodiments, methods described herein include measuring the binding affinity of the antigenbinding domain to a target antigen in a composition described herein and comparing the binding affinity of the antigen-binding domain in the composition to the binding affinity measured in a control composition. In some embodiments, a control composition is a composition that does not contain the third variable domain (e.g., a competing light chain). In some embodiments, the same or similar binding affinity is measured in the composition compared to the control composition indicates no detection of mispairing. In some embodiments, a decrease in binding affinity measured in the composition compared to the control composition indicates the detection of mispairing.Bivalent Antibody Agents- 25 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0125] In some embodiments, methods described herein include measuring mispairing in a composition of immunoglobulin chains, that, when associated, form multiple bivalent antibody agents, where each antibody agent binds to a different target antigen (see FIG. 24). In some embodiments, such compositions include a first immunoglobulin chain comprising a first variable domain and a first constant domain, a second immunoglobulin chain comprising a second variable domain and a second constant domain, and a third immunoglobulin chain comprising a third variable domain and a third constant domain (see FIG. 23). In some embodiments, the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to the target antigen. In some embodiments, the first variable domain and the third variable domain associate to a mispaired antigen-binding domain that does not bind to the target antigen. In some embodiments, a composition further includes a fourth immunoglobulin chain comprising a fourth variable domain and a fourth constant domain, where the third variable domain and the fourth variable domain associate to form an antigen-binding domain to a second target antigen. In some embodiments, the first, second, third, and fourth immunoglobulin chains are present in the composition as first, second, third, and fourth nucleic acid sequences encoding the first, second, third, and fourth immunoglobulin chains, respectively. In some embodiments, the first, second, third, and fourth immunoglobulin chains are encoded by the same nucleic acid. In some embodiments, the first, second, third, and fourth immunoglobulin chains are encoded by separate nucleic acids (e.g., a first, second, third, and fourth nucleic acid, respectively).

[0126] In some embodiments, the method comprises the step of expressing the first, second, third, and fourth immunoglobulin chains from the first, second, third, and fourth nucleic acid sequences, respectively, thereby producing the composition. In some embodiments, expressing the first, second, third, and fourth immunoglobulin chains from the first, second, third, and fourth nucleic acid sequences, respectively, comprises transfecting a host cell with the first, second, third and fourth nucleic acid sequences and culturing the host cell under conditions such that the first, second, third, and fourth immunoglobulin chains are expressed. In some embodiments, the nucleic acids are polyribonucleotides. In some embodiments, the polyribonucleotides are fully or partially encapsulated within lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0127] In some embodiments, methods described herein include measuring the binding affinity of the antigenbinding domain to a target antigen in a composition described herein and comparing the binding affinity of the antigen-binding domain in the composition to the binding affinity measured in a control composition. In some embodiments, a control composition is a composition that does not contain the third variable domain (e.g., a competing light chain). In some embodiments, the same or similar binding affinity is measured in the composition compared to the control composition indicates no detection of mispairing. In some embodiments, a decrease in binding affinity measured in the composition compared to the control composition indicates the detection of mispairing.Methods of Measuring Binding Affinity

[0128] In some embodiments, methods described herein include measuring the binding affinity of one or more antibody agents to a target antigen. Those skilled in the art will be familiar with a variety of technologies for measuring binding affinity and / or dissociation constants in accordance with the present disclosure, including, e.g., but not limited to Enzyme Linked Immunosorbent Assay (ELISA), gel-shift assays, pull-down assays, equilibrium- 26 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), bio-layer interferometry, grating-coupled interferometry, flow cytometry and spectroscopic assays.

[0129] Binding of an antigen-binding domain of an antibody agent to a target antigen described herein can be measured by methods known in the art, e.g., one of the following methods: BIACORE analysis, ELISA, x-ray crystallography, sequence analysis and scanning mutagenesis. The binding interaction of an antibody agent and a target antigen can be analyzed using surface plasmon resonance (SPR). SPR or Biomolecular Interaction Analysis (BIA) detects bio-specific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface. The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No.5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345 (1991); Szabo et al., Curr. Opin. Struct. Biol. 5:699-705 (1995) and on-line resources provided by BIAcore (Cytiva, USA). Additionally, a KinExA (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho), and / or an Octet BLI (Bio-Layer Interferometry) from Sartorius (Goettingen, Germany) can also be used.

[0130] Information from SPR or from similar BIA methods can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (KD), and kinetic parameters, including Kon and Koff, for the binding of an antigen-binding domain to a target antigen. Such data can be used to compare different molecules. Information from SPR can also be used to develop structure-activity relationships (SAR).

[0131] In some embodiments, measuring the binding affinity of one or more antibody agents to a target antigen is measuring binding affinity by performing an ELISA. In some embodiments, an ELISA comprises a plate pre-coated with target antigen. In some embodiments, an ELISA further comprises a detection antibody. In some embodiments, a detection antibody comprises an anti-human Fc antibody linked to an enzyme. In some embodiments, an enzyme comprises horseradish peroxidase (HRP). In some embodiments, measuring binding affinity comprises measuring color change produced from a reaction between the enzyme and a substrate added to the sample. An exemplary schematic of an assay used to measure binding affinity in methods described herein is shown in FIG. 8.

[0132] In some embodiments, binding is measured using flow cytometry. In some embodiments, cells are transfected with a target antigen and the target antigen is displayed on the cell surface, and cells are subsequently sorted based on antibody agent binding in a sample. Without wishing to be bound by any theory, such methods would allow for target antigens, including transmembrane proteins, to be expressed in their native conformation (and folding), rather than being denatured or not properly folded as they may be when other assays are used. Using flow cytometry to measure binding provides a measurement of functionality of the tested antibody agent. Additionally, utilizing flow cytometry to measure antibody agent binding to assess mispairing in a composition allows use of methods described herein to a broader range of target antigens (e.g., transmembrane proteins).Identifying and / or Characterizing Immunoglobulin Chain M isoairing

[0133] In some embodiments, methods described herein include comparing binding affinity of one or more antibody agents to the binding affinity measured in a control composition. In some embodiments, the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of- 27 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)mispairing. In some embodiments, a decrease in binding affinity measured in the composition compared to the control composition indicates the detection of mispairing.

[0134] In some embodiments, an antigen-binding domain in control compositions described herein (e.g., without the presence of a competing light chain) exhibits high affinity for a target antigen. In various embodiments, KD of an antigen-binding domain in a control composition as described herein for a target antigen is less than about 10'4, IO’5, 10'6, IO’7, 10'8, 10'9, IO0, IO41, IO42, IO43, IO44, or IO45M or any range there between. In certain instances, KD of an antigen-binding domain in control compositions as described herein for a target antigen is between 0.001 and 1 nM, e.g., 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM or any range there between.

[0135] In some embodiments, when a competing light chain is added to compositions described herein, binding affinity of the antigen-binding domain to a target antigen decreases in the composition. In some embodiments, a decreased binding affinity is measured by a shift in the binding curves in a composition with increased amounts of competing light chain compared to the binding curve of a control composition. For example, FIG. 9 shows an exemplary regular binding curve (e.g., in a control composition) (FIG. 9A) and a shifted binding profile (FIG. 9B), where the binding affinity decreases compared to a control composition (e.g., when a competing light chain is added to a composition described herein). In some embodiments, mispairing is identified when a binding curve is shift by at least about at least about 10%, about 20%, about 30%, about 50%, about 60%, about 70%, about 80%, about 90% or more, compared to the binding cure of in a control composition described herein.

[0136] In some embodiments, comparing the binding affinity of the one or more antibody agents to the binding affinity measured in a control composition includes comparing the antibody concentrations at the midpoint of a binding curve (e.g., 50% signal), e.g., as shown in FIG. 34. In some embodiments, mispairing is identified when concentration of antibody required to reach 50% signal in a binding curve in a composition is increased compared to a control composition. In some embodiments, concentration is increased by at least about 10%, about 20%, about 30%, about 50%, about 60%, about 70%, about 80%, about 90% or more compared to a control composition.

[0137] In some embodiments, a decrease in binding affinity to a target antigen compared to a control composition indicates presence of mispairing. In some embodiments, binding affinity is decreased by at least about 10%, about 20%, about 30%, about 50%, about 60%, about 70%, about 80%, about 90% or more compared to a control composition.

[0138] In some embodiments, binding affinity decreases with increasing concentrations of competing light chain, indicating presence of immunoglobulin chain mispairing. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain (or competing chain) is between about 1:0.1 to about 1:100. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain (or mispaired light chain) is about 1:0.1. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain (or mispaired light chain) is about 1:0.5. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:1. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:2. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:4. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid - 28 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)encoding a non-cognate light chain is about 1:8. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:10. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:20. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:30. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:40. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:50. In some embodiments, a relative amount of a nucleic acid encoding a cognate light chain to amount of nucleic acid encoding a non-cognate light chain is about 1:100.

[0139] Methods described herein may be used, e.g., with compositions that comprise two or more antibody agents and the method characterizes the risk of mispairing between antibody variable domains of the antibody agents. In some embodiments, methods described herein comprise determining if immunoglobulin chains in a composition mispair.

[0140] Methods described herein may be used, e.g., with compositions that comprise two or more antibody agents and the method determines if antibody variable domains of two different antibody agents in a composition mispair.

[0141] Methods described herein may be used, e.g., to determine the suitability of one or more antibody agents for manufacture, administration, use as a therapeutic. In some embodiments, a therapeutic comprises two or more antibody agents. In some embodiments, a therapeutic comprises two or more antibody agents delivered as nucleic acids encoding the antibody agents. In some embodiments, a therapeutic comprises a bispecific antibody. In some embodiments, a bispecific antibody is delivered as a nucleic acid encoding the bispecific antibody.Compositions

[0142] In some embodiments, the present disclosure provides compositions utilized in methods described herein to characterize and / or identify immunoglobulin chain mispairing in a composition.

[0143] In some embodiments, compositions described herein are formulated for use in a mispairing assay. In some embodiments, a mispairing assay described herein identifies and / or characterizes mispairing between immunoglobulin chains of multiple antibody agents in a composition. In some embodiments, a mispairing assay described herein detects and / or characterizes mispairing between immunoglobulin chains of an antibody variant and a polypeptide in a composition. In some embodiments, a composition described herein includes one or more recombinant antibody agents. In some embodiments, compositions described herein include one or more nucleic acids encoding a combination described herein (e.g., including an antibody variant and a polypeptide, e.g., competing light chain, and / or multiple antibody agents).

[0144] In some embodiments, a composition for use in a mispairing assay described herein comprises one or more nucleic acids that encode an antibody variant, where the antibody variant comprises a first immunoglobulin chain comprising a first variable domain and a first constant domain and a second immunoglobulin chain comprising a second variable domain and a second constant domain. In some embodiments, an antibody variant is characterized in that the first constant domain is a heavy chain constant domain that is unable to homodimerize. In some embodiments, antibody variant is characterized in that the first variable domain and the second variable domain- 29 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)associate to form an antigen-binding domain that specifically binds to a target antigen. In some embodiments, nucleic acids of a composition further encode a third immunoglobulin chain comprising a third constant domain (e.g., an "null" domain). In some embodiments, nucleic acids of a composition described herein further encode a combination that includes the antibody variant and a further polypeptide comprising a third variable domain (e.g., a light chain variable domain, e.g., a "competing light chain"). In some embodiments, each immunoglobulin chain is encoded by a separate nucleic acid. In some embodiments, the first, second, and third immunoglobulin chains and the polypeptide are present in a composition described herein as a first, second, third, and fourth nucleic acid sequence, encoding the first, second, and third immunoglobulin chains and polypeptide, respectively. In some embodiments, two or more immunoglobulin chains are encoded by the same nucleic acid. In some embodiments, each immunoglobulin chain in a composition is encoded by the same nucleic acid. In some embodiments, the nucleic acids are polyribonucleotides.

[0145] In some embodiments, a composition for use in a mispairing assay described herein comprises one or more nucleic acids that encode a first immunoglobulin chain comprising a first variable domain and a first constant domain, a second immunoglobulin chain comprising a second variable domain and a second constant domain, and a third immunoglobulin chain comprising a third variable domain and a third constant domain. In some embodiments, the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to the target antigen. In some embodiments, the first variable domain and the third variable domain associate to a mispaired antigen-binding domain. In some embodiments nucleic acids in a composition further encode a fourth immunoglobulin chain comprising a fourth variable domain and a fourth constant domain. In some embodiments, each immunoglobulin chain is encoded by a separate nucleic acid. In some embodiments, the first, second, third, and fourth immunoglobulin chains are present in a composition described herein as a first, second, third, and fourth nucleic acid sequence, encoding the first, second, third, and fourth immunoglobulin chains, respectively. In some embodiments, two or more immunoglobulin chains are encoded by the same nucleic acid. In some embodiments, each immunoglobulin chain in a composition is encoded by the same nucleic acid. In some embodiments, the nucleic acids are polyribonucleotides.

[0146] Compositions including provided nucleic acids (e.g., polyribonucleotides) encoding one or more antibody agents (e.g., antibody variants) may be delivered to a cell in methods described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs or DNAs, or delivery mediated by viral and / or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, efc), and / or peptide-based vectors. See, e.g., Wadhwa etal. "Opportunities and Challenges in the Delivery of mRNA-Based Vaccines" Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference.

[0147] In some embodiments, provided nucleic acids are polyribonucleotides. In some embodiments, polyribonucleotides in compositions described herein are fully or partially encapsulated within lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes. In some embodiments, one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration). In some embodiments, lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and / or engineered for systemic delivery of the RNA to target cells (e.g., liver cells). Polyribonucleotides provided herein can be delivered by particles. In the context of the present disclosure, the term "particle" relates to a structured entity formed by- 30 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)molecules or molecule complexes. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising a polyribonucleotide.

[0148] A "nucleic acid particle" (e.g., a ribonucleic acid particle) are particles that encompass or contain a nucleic acid, and are used to deliver nucleic acid (e.g., a polyribonucleotide) to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle (e.g., a ribonucleic acid particle) may be formed from (i) at least one cationic or cationically ionizable lipid or lipid-like material, (ii) at least one cationic polymer such as protamine, or a mixture of (i) and (ii), and (iii) nucleic acid (e.g., a polyribonucleotide). Nucleic acid particles (e.g., a ribonucleic acid particle) include lipid nanoparticles (lipid nanoparticle) and lipoplexes (LPX).

[0149] In some embodiments, nucleic acid particles (e.g., ribonucleic acid particles) comprise more than one type of nucleic acid molecules (e.g., polyribonucleotides), where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

[0150] In some embodiments, provided nucleic acid particles (e.g., ribonucleic acid particles) can comprise lipid nanoparticles. As used in the present disclosure, "nanoparticle" refers to a particle having an average diameter suitable for parenteral administration. In various embodiments, lipid nanoparticles can have an average size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm.

[0151] Nucleic acid particles (e.g., ribonucleic acid particles) described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0152] Different types of nucleic acid particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60, which is herein incorporated by reference). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0153] The present disclosure describes particles comprising nucleic acid (e.g., a polyribonucleotide), at least one cationic or cationically ionizable lipid or lipid-like material, and / or at least one cationic polymer which associate with the nucleic acid (e.g., a polyribonucleotide) to form nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) and compositions comprising such particles. The nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) may comprise nucleic acid (e.g., a polyribonucleotide) which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular, they are not infectious viral particles, i.e., they are not able to virally infect cells.

[0154] Some embodiments described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species (e.g., polyribonucleotide species).- 31 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0155] In a nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation, it is possible that each nucleic acid species (e.g., polyribonucleotide species) is separately formulated as an individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. In that case, each individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation will comprise one nucleic acid species (e.g., polyribonucleotide species). The individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species (e.g., polyribonucleotide species) separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species (e.g., polyribonucleotide species) that is being provided when the particles are formed (individual particulate formulations).

[0156] In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation.Respective pharmaceutical compositions are referred to as "mixed particulate formulations." Mixed particulate formulations according to the invention are obtainable by forming, separately, individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations, as described above, followed by a step of mixing of the individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) populations may be together in one container, comprising a mixed population of individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanopartide) formulations.

[0157] Alternatively, it is possible that different nucleic acid species (e.g., polyribonucleotide species) are formulated together as a "combined particulate formulation." Such formulations are obtainable by providing a combined formulation (typically combined solution) of different nucleic acid species (e.g., polyribonucleotide species) species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a "mixed particulate formulation," a "combined particulate formulation" will typically comprise particles that comprise more than one nucleic acid species (e.g., polyribonucleotide species) species. In a combined particulate composition different nucleic acid species (e.g., polyribonucleotide species) are typically present together in a single particle.

[0158] In certain embodiments, nucleic acids (e.g., polyribonucleotides), when present in provided nucleic acid particles (e.g., ribonucleic acid particles, e.g., lipid nanoparticles) are resistant in aqueous solution to degradation with a nuclease.

[0159] In some embodiments, nucleic acid particles (e.g., ribonucleic acid particles) are lipid nanoparticles. In some embodiments, lipid nanoparticles are liver-targeting lipid nanoparticles. In some embodiments, lipid nanoparticles are cationic lipid nanoparticles comprising one or more cationic lipids {e.g., ones described herein). In some embodiments, cationic lipid nanoparticles may comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid {e.g., at least one neutral lipid).

[0160] In some embodiments, the present disclosure provides a kit comprising compositions or combinations described herein (e.g., including antibody agents, e.g., antibody variants and / or nucleic acids encoding the same).- 32 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Applications

[0161] Methods described herein may be used, e.g., to determine mispairing of immunoglobulin chains in compositions that include more than one antibody agent. In some embodiments, methods described herein may be used to determine suitability of multiple antibody agents for co-delivery as nucleic acids encoding the antibody agents. Methods described herein may be used to characterize antibody agents for therapeutic use, prophylactic use, tool antibodies, and / or diagnostic antibodies.

[0162] Methods described herein may be used, e.g., to determine the suitability of one or more antibody agents for use as a therapeutic. Therapeutic antibody agents described herein, especially those that can be used in compositions including multiple antibody agents, may target a wide range of target antigens, for use in therapeutics treating a range of diseases and disorders, e.g., cancer, autoimmune disease, rare diseases, metabolic diseases, inflammatory diseases, infectious diseases, among others.

[0163] Target antigens targeted by antibody agents (e.g., antibody variants) used in methods described herein include antigens and potential antigens, e.g., autoantigens, tumor antigens, antigens identified from infectious agents / pathogens, and tissue-specific or non-specific antigens.Infectious Disease

[0164] Target antigens targeted by antibody agents (e.g., antibody variants) as used in methods described herein include antigens to pathogens and infectious agents. Exemplary pathogens include viruses, bacteria, fungi, protozoa, or helminths that infect humans.

[0165] In some embodiments, an antigen is identified from a viruses such as immunodeficiency virus {e.g., a human immunodeficiency virus (HIV), e.g., HIV-1, HIV-2), a hepatitis virus e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus, non-A and non-B hepatitis virus), a herpes virus {e.g., herpes simplex virus type I (HSV-1), HSV-2, Varicella-zoster virus, Epstein Barr virus, human cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8), a poxvirus {e.g., variola, vaccinia, monkeypox, Molluscum contagiosum virus), an influenza virus, a human papilloma virus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus, rabies virus, coxsackie virus, human T-cell leukemia virus (types I, II and III), parainfluenza virus, paramyxovirus, poliovirus, rotavirus, rhinovirus, rubella virus, measles virus, mumps virus, adenovirus, yellow fever virus, Norwalk virus, West Nile virus, a Dengue virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), bunyavirus, Ebola virus, Marburg virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Junin virus, Lassa virus, and Lymphocytic choriomeningitis virus.

[0166] In some embodiments, an antigen is identified from a bacteria {e.g., from a bacterial pathogen). In some embodiments, the bacterial pathogen is an intracellular pathogen. In some embodiments, the bacterial pathogen is an extracellular pathogen. Examples of bacterial pathogens include bacteria from the following genera and species: Chlamydia {e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Legionella {e.g., Legionella pneumophila), Listeria {e.g., Listeria monocytogenes), Rickettsia {e.g., R. australis, R. rickettsii, R. akari, R. conorii, R. sibirica, R. japonica, R. africae, R. typhi, R. prowazekii), Actinobacter {e.g., Actinobacter baumannii), Bordetella {e.g., Bordetella pertussis), Bacillus {e.g., Bacillus anthracis, Bacillus cereus), Bacteroides {e.g., Bacteroides fragilis), Bartonella {e.g., Bartonella henselae), Borrelia {e.g., Borrelia burgdorferi), Brucella {e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter {e.g., Campylobacter jejuni),- 33 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Clostridium {e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium {e.g., Corynebacterium diphtheriae, Corynebacterium amycolatum), Enterococcus {e.g., Enterococcus faecalis, Enterococcus faecium), Escherichia {e.g., Escherichia coli), Francisella {e.g., Francisella tularensis), Haemophilus {e.g., Haemophilus influenzae), Helicobacter {e.g., Helicobacter pylori), Klebsiella {e.g., Klebsiella pneumoniae), Leptospira {e.g., Leptospira interrogans), Mycobacteria {e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma {e.g., Mycoplasma pneumoniae), Neisseria {e.g., Neisseria gonorrhoeae, Neisseria meningitidis), Pseudomonas {e.g., Pseudomonas aeruginosa), Salmonella {e.g., Salmonella typhi, Salmonella typhimurium, Salmonella enterica), Shigella {e.g., Shigella dysenteriae, Shigella sonnei), Staphylococcus {e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Streptococcus {e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes), Treponoma {e.g., Treponoma pallidum), Vibrio {e.g., Vibrio cholerae, Vibrio vulnificus), and Yersinia {e.g., Yersinia pestis). Libraries for other bacteria can also be produced and used according to methods described herein.

[0167] In some embodiments, an antigen is identified from a protozoan. Examples of protozoal pathogens include the following organisms: Cryptosporidium parvum, Entamoeba {e.g., Entamoeba histolytica), Giardia {e.g., Giardia lambila), Leishmania {e.g., Leishmania donovani), Plasmodium spp. {e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae), Toxoplasma {e.g., Toxoplasma gondii), Trichomonas {e.g., Trichomonas vaginalis), and Trypanosoma {e.g., Trypanosoma brucei, Trypanosoma cruzi). Libraries for other protozoa can also be produced and used according to methods described herein.

[0168] In some embodiments, an antigen is identified from a fungus. Examples of fungal pathogens include the following: Aspergillus, Candida {e.g., Candida albicans), Coccidiodes {e.g., Coccidiodes immitis), Cryptococcus {e.g., Cryptococcus neoformans), Histoplasma {e.g., Histoplasma capsulatum), and Pneumocystis {e.g., Pneumocystis carinii). Libraries for other fungi can also be produced and used according to methods described herein.

[0169] In some embodiments, an antigen is identified from a helminth. Examples of helminthic pathogens include Ascaris lumbricoides, Ancylostoma, Clonorchis sinensis, Dracuncula medinensis, Enterobius vermicularis, Filaria, Onchocerca volvulus, Loa loa, Schistosoma, Strongyloides, Trichuris trichura, and Trich inella spiralis. Libraries for other helminths can also be produced and used according to methods described herein.

[0170] Sequence information for genomes and ORFs for infectious agents is publicly available. See, e.g., the Entrez Genome Database (URL: ncbi.nlm.nih.gov / sites / entrez?db=Genome&itool=toolbar) and the ERGO™ Database (URL: igweb.integratedgenomics.com / ERGO_supplement / genomes.html), the Genomes Online Database (GOLD) (URL: genomesonline.org) (Liolios et al., Nucleic Acids Res. 1; 34(Database issue):D332-4, 2006).HIV

[0171] In some embodiments, a target antigen described herein is an epitope of HIV. In some embodiments an epitope of HIV is targeted by an antibody agent that comprises or be derived from a broadly neutralizing antibody (bNAb). In some embodiments, a target HIV antigen described herein may targeted by any one of the HIV-targeting antibodies described in Barouch, eta!., Nature 503: 7475 224-228, 2013, Shingai, eta!., Nature 503: 7475 277-280, 2013, Caskey, eta!., Nature 522.7557: 487-491, 2015, Caskey eta!., Nature Medicine 23.2: 185-191, 2017, Baret al., New England Journal of Medicine 375.21: 2037-2050, 2016, Mendoza, eta / ., Nature 561.7724: 479-484, 2018, Gautam, Rajeev, eta / ., Nature Medicine 24.5: 610-616, 2018, the contents of each of which are incorporated herein by reference in their entirety for the purposes described herein.- 34 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0172] In some embodiments, an HIV target antigen may be targeted by e.g., 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, PGDM1400, fragments thereof, or combinations thereof. Exemplary anti-HIV antibodies that can be used in compositions described herein include, but are not limited to, 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, PGDM1400, fragments thereof, or combinations thereof. For example, in some embodiments, a polyribonucleotide as described herein can comprise one or more heavy chain complementarity determining regions (HCDRs) {e.g., HCDR1, HCDR2, and / or HCDR3) from 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400. In some embodiments, a polyribonucleotide as described herein can comprise HCDR1, HCDR2, and HCDR3 from 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400. In some embodiments, a polyribonucleotide as described herein can comprise a heavy chain variable domain from 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400. In some embodiments, a polyribonucleotide as described herein can comprise one or more light chain complementarity determining regions (LCDRs) {e.g., LCDR1, LCDR2, and / or LCDR3) from 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400. In some embodiments, a polyribonucleotide as described herein can comprise LCDR1, LCDR2, and LCDR3 from 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400. In some embodiments, a polyribonucleotide as described herein can comprise a light chain variable domain from 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400.

[0173] In some embodiments, a plurality of nucleic acids (e.g., polyribonucleotides) that each encode an immunoglobulin chain of an antibody agent can be used to deliver two or more antibody agents {e.g., 10E8v4-5R-lOOcF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400, or fragments or variants thereof). In some embodiments, a plurality of nucleic acids (e.g., polyribonucleotides) that each encode an immunoglobulin chain of an antibody agent can be used to deliver three or more antibody agents {e.g., 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400, or fragments or variants thereof). In some embodiments, a plurality of nucleic acids (e.g., polyribonucleotides) that each encode an immunoglobulin chain of an antibody agent can be used to deliver {e.g., by administration to a subject) four or more antibody agents {e.g., 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400, or fragments or variants thereof). In some embodiments, a plurality of nucleic acids (e.g., polyribonucleotides) that each encode an immunoglobulin chain of an antibody agent can be used to deliver {e.g., by administration to a subject) two, three, four, five or six antibody agents {e.g., 10E8v4-5R-100cF, 1-18, PGT121, 3BNC117, bl2, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523, VRC07, N6, PG9, 4E10, PGT151, PGT128, 8ANC195, 2F5, IOMA, or PGDM1400, or fragments or variants thereof).- 35 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Cancer

[0174] Target antigens targeted by antibody agents (e.g., antibody variants) used in methods described herein include tumor antigens and antigens associated with certain cancers, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), and / or cancer / testis antigens (CTAs). In some embodiments, antibody agents described herein may be included in compositions where multiple antibody agents target different cancer antigens (e.g., associated with the same cancer) and / or different epitopes on the same cancer antigen.

[0175] Exemplary tumor antigens and antigens associated with certain cancers include, e.g., MART-l / MelanA (MART-I or MLANA), gplOO (Pmel 17 or SILV), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3 (also known as HIP8), BAGE, GAGE-1, GAGE-2, pl5, Calcitonin, Calretinin, Carcinoembryonic antigen (CEA), Chromogranin, Cytokeratin, Desmin, Epithelial membrane protein (EMA), Factor VIII, Glial fibrillary acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45, Human chorionic gonadotropin (hCG), inhibin, lymphocyte marker, MART-1 (Melan-A), Myo DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, PTPRC (CD45), prostate-specific membrane antigen (PMSA), Cryptic, EGF-CFC Family Member 1 (CFC-1), S100 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-1, Tumor M2-PK, vimentin, p53, Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens (e.g., EBNA1), human papillomavirus (HPV) antigen E6 or E7 (HPV_E6 or HPV_E7), TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1 (also known as CTAG1B), erbB, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein (AFP), beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, MUC16, IL13RO2, FRa, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, F0LR1, endothelial receptor, STEAP1, SLC44A4, Nectin-4, AGS-16, guanalyl cyclase C, MUC-1, CFC1B, integrin alpha 3 chain (of a3bl, a laminin receptor chain), TPS, CD19, CD20, CD22, CD30, CD31, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CD34, CD99, CD117, CD80, CD28, CD13, CD15, CD25, CD10, CLL-1 / CLEC12A, ROR1, Glypican 3 (GPC3), Mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, Glycolipid F77, EGFRvIII, BCMA, GD-2, PSAP, prostein (also known as P501S), PSMA, Survivin (also known as BIRC5), and MAGE-A3, MAGEA2, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, BIRC5, CDH3, CEACAM3, CGB_isoform2, ELK4, ERBB2, HPSE1, HPSE2, KRASJsoforml, KRAS_isoform2, MUC1, SMAD4, TERT, 2. TERT.3, TGFBR2, EGAG9_isoforml, TP53, CGBJsoforml, IMPDH2, LCK, angiopoietin-1 (Angl) (also known as ANGPT1), XIAP (also known as BIRC4), galectin-3 (also known as LGALS3), VEGF-A (also known as VEGF), ATP6S1 (also known as ATP6AP1), MAGE-A1, cIAP-1 (also known as BIRC2), macrophage migration inhibitory factor (MIF), galectin-9 (also known as LGALS9), progranulin PGRN (also known as granulin), OGFR, MLIAP (also known as BIRC7), TBX4 (also known as ICPPS, SPS orT-Box4), secretory leukocyte protein inhibitor (Slpi) (also known as antileu koproteinase), Ang2 (also known as ANGPT2), galectin-1 (also known as LGALS1), TRP-2 (also known as DCT), hTERT (telomerase reverse transcriptase) tyrosinase-related protein 1 (TRP-1, TYRP1), NOR-90 / UBF-2 (also known as UBTF), LGMN, SPA17, PRTN3, TRRAP_1, TRRAP_2, TRRAP_3, TRRAP_4, MAGEC2, PRAME, 50X10, RAC1, HRAS, GAGE4, AR, CYP1B1, MMP8, MMP9, 7YR, PDGFRB, KLK3, PAX3, PAX5, ST3GAL5, PLAC1, RhoC, MYCN, REG3A, CSAG2, CTAG2-la, CTAG2-lb, PAGE4, BRAF, GRM3, ERBB4, KIT, MAPK1, MFI2, SART3, ST8SIA1, WDR46, AKAP-4, RGS5, F0SL1, PRM2,- 36 - 13276319V1Attorney Docket No.: 2013237-1583 (BNT REF: P2042W01)ACRBP, CTCFL, CSPG4, CCNB1, MSLN, WT1, SSX2, KDR, ANKRD30A, MAGED1, MAP3K9, XAGE1B, PREX2, CD276, TEK, AIM1, ALK, F0LH1, GRIN2A MAP3K5 and one or more isoforms of any preceding tumor antigens.

[0176] Tumor associated antigens (TAAs) include proteins encoded in a normal genome (see, e.g., Ward eta / ., Adv. Immunol. 130:25-74 (2016)). In some embodiments, TAAs are either normal differentiation antigens or aberrantly expressed normal proteins. Overexpressed normal proteins that possess growth / survival-promoting functions, such as Wilms tumor 1 (WT1) (Ohminami eta / ., Blood 95:286-293 (2000)) or Her2 / neu (Kawashima eta / ., Cancer Res. 59:431-435 (1999)), are TAAs that directly participate in the oncogenic process. Post-translational modifications, such as phosphorylation, of proteins may also lead to formation of TAAs (Doyle, J. Biol. Chem.281:32676-32683 (2006); Cobbold, Sci. Transl. Med. 5:203ral25 (2013)). TAAs are generally shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10%, 1-20%, or more of subjects suffering from a cancer. In some embodiments, TAAs are known or pre-selected tumor antigens. In some embodiments, with respect to an individual subject, TAAs are potential or putative tumor antigens. Cancer / testis antigens (CTAs) are expressed by various tumor types and by reproductive tissues (for example, testes, fetal ovaries and trophoblasts) but have limited or no detectable expression in other normal tissues in the adult and are generally not presented on normal reproductive cells, because these tissues do not express MHC class I molecules (see, e.g., Coulie etaL, Nat. Rev. Cancer 14:135-146 (2014); Simpson eta!., Nat. Rev. Cancer 5:615-625 (2005); Scanlan eta!., Immunol. Rev. 188:22-32 (2002)).

[0177] Tumor associated antigens (TAAs) include proteins encoded in a normal genome (see, e.g., Ward eta / ., Adv. Immunol. 130:25-74 (2016)). In some embodiments, TAAs are either normal differentiation antigens or aberrantly expressed normal proteins. Overexpressed normal proteins that possess growth / survival-promoting functions, such as Wilms tumor 1 (WT1) (Ohminami eta / ., Blood 95:286-293 (2000)) or Her2 / neu (Kawashima eta / ., Cancer Res. 59:431-435 (1999)), are TAAs that directly participate in the oncogenic process. Post-translational modifications, such as phosphorylation.Autoimmune Disease

[0178] Target antigens targeted by antibody agents (e.g., antibody variants) used in methods described herein include antigens associated with an autoimmune disease. In some embodiments, an autoimmune disease can be any known autoimmune disease or condition associated with an autoimmune disease. In some embodiments, an autoimmune disease is one of achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal & neuronal neuropathy (AMAN), Balo disease, asthma, Behcet's disease, aenign mucosal pemphigoid, aullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressier's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-- 37 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Barre syndrome, transplant rejection / graft versus host disease (GVHD), Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), hidradenitis S\suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (UP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sderosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, lyme disease chronic, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, Nneonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes type I, II, III, polymyalgia rheumatica, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, Type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, or Vogt-Koyanagi-Harada disease (autoimmune disease list according to the AARDA https : / / w ww.aa rda . org / d isease I ist / ) .

[0179] In some embodiments, an autoimmune disease is an inflammatory autoimmune condition. Examples of inflammatory autoimmune conditions include autoimmune (Hashimoto's) thyroiditis, hyperthyroidism (Grave's disease), autoimmune adrenal insufficiency (Addison's disease), autoimmune oophoritis, autoimmune orchitis, autoimmune hepatitis, autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria, autoimmune thrombocytopenia, autoimmune neutropenia, pernicius anemia, pure red cell anemia, autoimmune coagulopathies, myasthenia gravis, autoimmune polyneuritis, multiple sclerosis, pemphigus and other bullous diseases, rheumatic carditis, Goodpasture's syndrome, postcardiotomy syndrome, systemic lupus erythematosus, Sjorgen's syndrome, polymyositis, dermatomyositis, scleroderma, inflammatory bowel diseases: Crohn's disease, ulcerative colitis; chronic obstructive pulmonary diseases, chronic inflammatory diseases, celiac disease, vasculitis, Wegener's disease, Churg- Strauss syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, cardiovascular disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis juvenile idiopathic arthritis, polydermatomyositis, septic shock, host versus graft disease, graft versus host disease, asthma, rhinitis, psoriasis, cachexia associated with cancer, eczema, vitiligo, Reiter's syndrome, Kawasaki's disease, idiopathic thrombocytopenic purpura, Guillain-Barre syndrome, antiphosphoiipid antibody syndrome (APS), atherosclerosis, and narcolepsy.

[0180] In some embodiments, an autoimmune disease is systemic lupus erythematosus (SLE). SLE is associated with excessive complement activation, which can cause tissue damage. SLE can affect the joints, skin,- 38 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)kidneys, blood cells, brain, heart, and lungs. SLE symptoms vary but can include fatigue, joint pain, rash, and fever, which can periodically get worse (flare-up).

[0181] In some embodiments, an autoimmune disease is Henoch Schonlein purpura (HSP) nephritis.

[0182] In some embodiments, an autoimmune disease is antiphospholipid antibody syndrome (APS). APS is a clinical entity that encompasses thrombosis, recurrent miscarriages, and pregnancy-related complications, mediated by anti-phospholipid antibodies (APLA). In some embodiments, an autoimmune disease is a vasculitis, e.g., vasculitis affecting large vessels including, Polymyalgia rheumatic, Takayasu's arteritis, temporal arteritis (and giant cell arteritis); vasculitis affecting medium vessels including, Buerger's disease, cutaneous vasculitis, Kawasaki disease, polyarteritis nodosa; vasculitis affecting small vessels including, Behget's syndrome, Churg-Strauss syndrome, cutaneous vasculitis, Henoch-Schonlein purpura, microscopic polyangiitis, Granulomatosis with polyangiitis (GPA), Golfer's vasculitis, and cryoglobulinemia.

[0183] In some embodiments, an autoimmune disease is rheumatoid arthritis (RA).

[0184] In some embodiments, a target antigen is a human protein target. In some embodiments, a human protein target is CD3.Combinations

[0185] In some embodiments, antibody variants encoded by nucleic acids described herein may be part of a combination described herein, e.g., with one or more immunoglobulin chains (e.g., variable domains). In some embodiments, such combinations are used in methods described herein, to characterize and / or identify immunoglobulin mispairing of immunoglobulin domains.

[0186] For example, a combination described herein includes an antibody agent (e.g., an antibody variant) and a polypeptide. In some embodiments, an antibody variant may include a first immunoglobulin chain comprising a first variable domain and a first constant domain and a second immunoglobulin chain comprising a second variable domain and a second constant domain; wherein the first constant domain is a heavy chain constant domain that is unable to homodimerize; and wherein the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to a target antigen. In some embodiments, an antibody variant further comprises a third immunoglobulin chain comprising, e.g., a null domain including an immunoglobulin heavy chain constant domain. In some embodiments, an antibody variant is monovalent. In some embodiments, a combination described herein may include an antibody variant and a polypeptide comprising a third immunoglobulin variable domain, where the third immunoglobulin variable domain is capable of associating with the first immunoglobulin variable domain to form a mispaired antigen-binding domain. Methods described herein may identify and / or characterize mispairing between the first variable domain the third variable domain in a combination. In some embodiments, the first variable domain and the second variable domain form an antigen-binding domain to a target antigen. In some embodiments, the first variable domain and the third variable domain form a mispaired antigenbinding domain that does not bind the target antigen. In some embodiments, such a combination is useful in identifying and / or characterizing mispairing between immunoglobulin chains of a monovalent antibody variant.

[0187] In some embodiments, combinations described herein may include a first immunoglobulin chain comprising a first variable domain and a first constant domain, a second immunoglobulin chain comprising a second variable domain and a second constant domain, and a third immunoglobulin chain comprising a third variable domain and a third constant domain, where the first variable domain and the second variable domain associate to form an- 39 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)antigen-binding domain that specifically binds to the target antigen and the first variable domain and the third variable domain associate to a mispaired antigen-binding domain. In some embodiments, a combination described herein includes a fourth immunoglobulin chain, wherein the fourth immunoglobulin chain includes a fourth variable domain and a fourth constant domain. In some embodiments, the fourth immunoglobulin chain comprises a heavy chain variable domain and a heavy chain constant domain. In some embodiments, the third immunoglobulin chain and the fourth immunoglobulin chain associate to form an antigen-binding domain that binds to a second target antigen. In some embodiments, the first target antigen and the second target antigen are different antigens. In some embodiments, the first target antigen and the second target antigen are different epitopes on the same target antigen. In some embodiments, such a combination is useful in identifying and / or characterizing mispairing between two different bivalent antibodies.Antibody Agents

[0188] In some embodiments, methods described herein are used to characterize immunoglobulin chain mispairing in compositions comprising two or more antibody agents. In some embodiments, antibody agents are present in a composition described herein as nucleic acids (e.g., polyribonucleotides) encoding immunoglobulin chains of the antibody agents. In some embodiments, an antibody agent is present in a combination described herein.

[0189] Antibody agents, as described herein, may be characterized by the amino acid sequence of one or more domains within their antibody structure. For example, an antibody agent can comprise at least one heavy (H) chain and at least one light (L) chain interconnected, e.g., by disulfide bonds. Each H chain comprises a heavy chain variable domain (abbreviated herein as VH), encoded by a nucleic acid comprising a heavy chain variable region and a heavy chain constant domain (abbreviated herein as CH), encoded by a nucleic acid comprising a heavy chain constant region. Each light chain comprises a light chain variable domain (abbreviated herein as VL), encoded by a nucleic acid comprising a light chain variable region, and a light chain constant domain (abbreviated herein as CL), encoded by a nucleic acid comprising a light chain constant region. The variable domains of each lig ht / heavy chain (VL / VH) pair or associate to form an antigen-binding domain. As used herein, an immunoglobulin heavy chain and immunoglobulin light chain that pair or associate to form an antigen-binding domain are considered each's "cognate" immunoglobulin chain.

[0190] Within each light or heavy chain variable domain, there are three short segments called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three in the light chain variable domain and three in the heavy chain variable domain) fold up together in 3-dimensional space to form the actual antibody binding site. The terms "LCDR1," "LCDR2" and "LCDR3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody agent. In some embodiments, the light chain variable domain provided herein includes in N-terminal to C-terminal direction a LCDR1, a LCDR2 and a LCDR3. Likewise, the terms "HCDR1,""HCDR2" and "HCDR3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody agent. In certain embodiments, the heavy chain variable domain provided herein includes in N-terminal to C-terminal direction a HCDR1, a HCDR2 and a HCDR3.

[0191] Also within the variable region, but not contained within the CDRs, are regions called the framework regions ("FR"). Thus, the complementarity determining regions (CDRs) are interspersed with the framework regions.- 40 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Accordingly, each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Generally, framework regions are more conserved than variable regions across naturally produced antibodies.

[0192] The positions of the CDRs and framework regions within the VH and VL domains of an antibody agent described herein can be determined using various numbering systems known in the art, e.g., Kabat, Chothia, AbM and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering; Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), each of which is incorporated herein by reference). Accordingly, CDRs within 10E8 antibody agents within the same VH or VL domain can be determined by different numbering systems.

[0193] In some embodiments, an antibody agent is formed by one, two, three, or four immunoglobulin chains.

[0194] In some embodiments, a nucleic acids (e.g., polyribonucleotide), as described herein, encode a single immunoglobulin chain. In some embodiments, a first nucleic acid encodes a first immunoglobulin chain of an antibody agent. In some embodiments, a first nucleic acid encodes a first immunoglobulin chain of an antibody agent and a second nucleic acid encodes a second immunoglobulin chain of the antibody agent. In some embodiments, a first nucleic acid encodes a first immunoglobulin chain of an antibody agent, a second nucleic acid encodes a second immunoglobulin chain of the antibody agent, and a third nucleic acid encodes a third immunoglobulin chain of the antibody agent. In some embodiments, a first nucleic acid encodes a first immunoglobulin chain of an antibody agent, a second nucleic acid encodes a second immunoglobulin chain of the antibody agent, a third nucleic acid encodes a third immunoglobulin chain of the antibody agent, and a fourth nucleic acid encodes a fourth immunoglobulin chain of the antibody agent.

[0195] In some embodiments, a nucleic acid as described herein encodes two immunoglobulin chains. In some embodiments, a single nucleic acid (e.g., a polyribonucleotide) can include a first coding region that encodes a first immunoglobulin chain of an antibody and a second coding region that encodes a second immunoglobulin chain of the antibody. In some embodiments, the first coding region and the second coding region are separated by an internal ribosome entry sides (IRES), an internal promoter, or a peptide sequence, such as "self-cleaving" 2A or 2A-like sequences (see, e.g., Szymczak etal. Nat Biotechno / ll'. ^, May 2004; ePub April 42004, which is herein incorporated by reference) to yield the first immunoglobulin chain and the second immunoglobulin chain from the single nucleic acid.

[0196] Antibody agents encoded by one or more nucleic acid (e.g., polyribonucleotides) described herein may be in various formats described herein. Exemplary types of antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, chimeric, etc., as is known in the art. An antibody agent utilized in accordance with the present disclosure, in some embodiments, is in a format selected from, but not limited to, intact IgG, IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); CrossMabs (e.g., CrossMabCH1'CLx; CrossMabCH1-CLcv; bispecific CrossMabCH1'CLxwith knob-in-holes); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or sets thereof; single chain Fvs (scFvs); scFv-Fc fusions; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies;- 41 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®;MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, immunoglobulin chains and / or fragments of such antibodies may be used in combination, e.g., an scFv-Fc arm with a conventional antibody arm.

[0197] Exemplary formats that may be used in accordance with the present disclosure are described further below.

[0198] In some embodiments, an antibody agent may be a conventional antibody agent. For clarity, the term "conventional antibody" is not intended to imply that an antibody described herein is routine and conventional (e.g., to make or use). Rather, as used herein, a "conventional antibody" refers to an antibody agent that includes two heavy chains and two light chains (see e.g., Fig. 2A and Fig. 3A). Each heavy chain includes a heavy chain variable domain operably linked to one or more heavy chain constant domains. In some embodiments, one or more heavy chain constant domains comprise a CHI domain, a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. In some instances, one or more heavy chain constant domains comprise a CHI domain, a hinge domain, a CH2 domain, a CH3 domain, a CH4 domain, or a combination thereof. Each light chain includes a light chain variable domain operably linked to a light chain constant domain.

[0199] The Fc region of an antibody agent described herein binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, conventional antibody agents produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. In some embodiments, conventional antibody agents are naturally produced {e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, a conventional antibody agent is polyclonal; in some embodiments, a conventional antibody agent is monoclonal. In some embodiments, a conventional antibody agent has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, conventional antibody agent sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0200] In some embodiments, an immunoglobulin chain of an antibody agent described herein includes a VH domain operably linked to one or more constant domains.

[0201] In some embodiments, an antibody agent as described herein comprises a CHI domain. In some embodiments, an antibody agent as described herein comprises a CHI domain that comprises a Glm3 allotype. In some embodiments, an antibody agent as described herein comprises a CHI domain that comprises a Glml7 allotype. In some embodiments, an antibody agent comprises a CHI domain that comprises one or more mutations. In some embodiments, a CHI domain comprises the addition of one or more serine residues. In some embodiments, a CHI domain comprises addition of two additional serine residues (referred to herein as"SS").

[0202] In some embodiments, one or more heavy chain constant domains comprise a CH3 domain. In some embodiments, a conventional antibody agent as described herein comprises a CH3 domain that comprises a Glm3, Glml7, or a Glml7,l allotype.

[0203] Antibody agents described herein may comprise one or more heavy chain constant domains comprising an amino acid modification {e.g., a substitution or deletion) at one or more amino acid positions. For example, an - 42 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)antibody agent as described herein may include an L / S mutation within a CH3 region (for enhanced FcRn binding) (see Zalevsky J et al. Nat Biotechnol. 2010, which is herein incorporated by reference). Such mutations are noted as M428L and N434S according to EU numbering and referred to herein as "LS" or "L / S" (see e.g., FIG. 3C). In some embodiments, an antibody agent encoded by one or more polyribonucleotides as described herein comprises an E294 deletion (for Fc hypersialylation) (see Bas M eta / . J Immunol 2019, which is herein incorporated by reference.

[0204] The present disclosure also provides technologies that can be used to express an antibody agent, e.g., as illustrated in FIG. 1 or described in Stadler et al. (2016) Oncoimmunology 5(3): el091555; and / or in Stadler et al. (2017) Nature Medicine 23(7): 815-817.

[0205] In some embodiments, an antibody agent as described herein comprises a hinge domain.

[0206] In some embodiments, an antibody agent as described herein comprises a CH2 domain. In some embodiments, an antibody agent described herein comprises a CH2 domain having one or more mutations. For example, in some embodiments, an antibody agent as described herein comprises one or more of the following mutations: G236A, A330L, and I332E (according to EU numbering). In some embodiments, an antibody agent as described herein comprises the following mutations: G236A, A330L, and I332E (according to EU numbering), referred to herein as"GAALIE". Such mutations in the CH2 domain have been associated with increased affinity to Fc receptors FcgRIIA and FcgRIII for enhanced antibody effector function (see FIG. 3B).

[0207] In some embodiments, an antibody agent as described herein comprises one or more mutations selected from: G236A and I332E (according to EU numbering). In some embodiments, an antibody agent as described herein comprises the mutations selected from: G236A and I332E (according to EU numbering), referred to herein as "GAIE".

[0208] In some embodiments, an antibody agent as described herein comprises a mutation: G236A (according to EU numbering), referred to herein as "GA".

[0209] In some embodiments, a conventional antibody encoded by one or more polyribonucleotides as described herein comprises a mutation: I332E (according to EU numbering), referred to herein as "IE".

[0210] In some embodiments, a conventional antibody encoded by one or more polyribonucleotides as described herein comprises a signal peptide comprising a human signal peptide.

[0211] In some embodiments, a conventional antibody encoded by one or more polyribonucleotides as described herein comprises a light chain constant domain, where the light chain constant domain comprises a kappa light chain constant domain. In some embodiments, a conventional antibody encoded by one or more polyribonucleotides as described herein comprises a light chain constant domain, where the light chain constant domain comprises a lambda light chain constant domain. In some embodiments, a conventional antibody encoded by one or more polyribonucleotides as described herein comprises a lambda chain variable domain.Modifications to Reduce Misoairing

[0212] Challenges exist in producing multiple antibody agents from a single composition {e.g., a composition comprising nucleic acids encoding multiple antibody agents), particularly because the random pairing of different antibody heavy and light chains can yield undesired antibody species. Due to the presence of mispaired byproducts, and significantly reduced production yields, sophisticated purification procedures are required to isolate the desired antibody agent in those situations (see, e.g., Morrison, S.L., Nature Biotech. 25, 1233-1234, 2007, which is herein incorporated by reference). The present disclosure also provides antibody agents in formats that reduce mispairing- 43 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)between an immunoglobulin heavy chain and a non-cognate light chain, that can be used to deliver and express multiple antibody agents.CrossMabCH1 CLx

[0213] In some embodiments, such a format comprises an antibody agent in "CrossMab" format (see e.g., W02015 / 101588 Al, WO 2009 / 080253A1, and Schaefer, W. et al, PNAS, 108, 11187-1191, 2011, which are herein incorporated by reference in their entirety). In some embodiments, antibody agents in CrossMab format contain a CL-CH1 crossover in one or both binding arms (referred to herein as"CrossMabCH1'CLx"or"CHl-CLx"). Such a modification reduces the byproduct formation caused by a mismatch of a light chain of a first antibody that specifically binds to a first antigen with the wrong heavy chain of a second antibody that specifically binds to a second antigen (when compared to approaches without such domain exchanges).

[0214] In some embodiments, an antibody agent encoded by one or more nucleic acids provided herein comprises a first immunoglobulin chain and a second immunoglobulin chain. In some embodiments, a nucleic acid may encode a first immunoglobulin chain and a second immunoglobulin chain of a CrossMabCH1'CLxantibody agent as described herein. In some embodiments, a nucleic acid encoding a first immunoglobulin chain comprises a nucleic acid sequence encoding a VH domain, a CL domain, a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, a nucleic acid encoding a second immunoglobulin chain comprises a nucleic acid sequence encoding a light chain variable (VL) domain and a CHI domain (see e.g., Fig. 4B).

[0215] In some embodiments, a CrossMabCH1'Cb<antibody agent may be encoded by two separate nucleic acids (e.g., polyribonucleotides): a first nucleic acid comprising a coding region that encodes (in 5' to 3' order): a heavy chain variable domain (VH), a light chain constant region (CL), a hinge region, a CH2 domain, and a CH3 domain; and a second nucleic acid comprising a coding region that encodes (in 5' to 3' order): a light chain variable domain (VL) and a CHI domain.CrossMabCH1 CLcv

[0216] In some embodiments, antibody agents as described herein are in a format where one or more charge variants (cv) are introduced into a domain (e.g., a constant domain, e.g., a CHI domain, a CL domain, or a combination thereof). Such formats are referred to herein as"CrossMabCH1‘CLcv" or"CHl-CLcv". In some embodiments, such antibody agents include charge variants in both arms of the antibody (see e.g., Fig. 4C).Exemplary charge variants are described e.g., in WO2017055539 Al, which is herein incorporated by reference in its entirety. In some embodiments, in contrast to the formats described in WO2017055539 Al, CHl-CLcv antibody agents described do not include a swap of the antibody CHI and CL domains in either arm of the antibody agent.

[0217] In some embodiments, a charge variant (cv) is introduced into a CHI and / or CL domain of an antibody agent in order to prevent mispairing of immunoglobulin chains of an antibody agent. Such charge variants may include, for example, introducing one or more positively charged amino acid residues in a CHI domain and one or more negatively charged amino acid residues in the CL, or vice versa, at specific positions in the CHI and CL interface.

[0218] In some embodiments, a nucleic acid (e.g., a polyribonucleotide) may encode a heavy chain and / or light chain of a CrossMabCH1‘CLcvantibody agent as described herein.- 44 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0219] In some embodiments, a CrossMabCH1'CLcvantibody agent may be encoded by two separate nucleic acids (e.g., polyribonucleotides): a first nucleic acid comprising a coding region that encodes (in 5' to 3' order): a heavy chain variable domain (VH), a CHI domain that includes one or more charge variants as described herein, a hinge region, a CH2 domain, and a CH3 domain; and a second nucleic acid comprising a coding region that encodes (in 5' to 3' order): a light chain variable domain (VL) and a light chain constant domain (CL) that includes one or more charge variants as described herein.

[0220] In some embodiments, a CrossMabCH1-CLcvantibody agent comprises a first immunoglobulin chain, wherein the first immunoglobulin chain comprises a CL domain and the CL domain an amino acid at position 123 (EL) numbering) that is substituted by an amino acid selected from K, R and H. In some embodiments, a CrossMabCH1-CLcvantibody agent comprises a first immunoglobulin chain, wherein the first immunoglobulin chain comprises a CL domain and the CL domain an amino acid at position 124 (EL) numbering) that is substituted by an amino acid selected from K, R and H. In some embodiments, a CrossMabCH1‘CLcvantibody agent comprises a second immunoglobulin chain, wherein the second immunoglobulin chain comprises a CHI domain having an amino acid at position 147 (EL) numbering) that is substituted by an amino acid selected from E or D (seeWO2017055539Al, which is herein incorporated by reference in its entirety).

[0221] In some embodiments, a CrossMabCH1-CLcvantibody agent encoded by one or more polyribonucleotides as described herein comprises a CHI domain that comprises one or more charge variant mutations. In some embodiments, a CrossMabCH1-CLcvantibody agent comprises a CHI domain, wherein the CHI domain comprises one or more substitutions including K147E, K213D, or a combination thereof. In some embodiments, a CrossMabCH1-CLcvantibody agent comprises a CHI domain that comprises a Glm3 allotype. In some embodiments, a CrossMabCH1 Lcvantibody agent comprises a CHI domain that comprises a Glml7 allotype.

[0222] In some embodiments, an antibody agent described herein may be a combination of a CrossMabCH1‘CLxand CrossMabCH1-CLcvantibody agent. For example, an antibody agent described herein can include one arm of the antibody that is in a CrossMabCH1‘CLxformat and a second arm of the antibody in a CrossMabCH1-CLcvformat. In some embodiments, a composition comprising multiple antibody agents includes one antibody agent that is a CrossMabCH1‘Cb<antibody agent and one antibody agent that is a CrossMabCH1‘CLcvantibody agent.

[0223] In some embodiments, antibody agents described herein can be designed to include one or more mutations to promote heterodimization (e.g., knob-into-holes or "KIH" technology). For example, in some embodiments, a CrossMabCH1‘Cb<antibody agent as described herein can be designed to further include mutations to utilize KIH technology. In some embodiments, a CrossMabCH1‘CLcvantibody agent as described herein can be designed to further include mutations to utilize KIH technology. In some embodiments, KIH technology may be applied to promote pairing of two different heavy chains, for example, to produce a bispecific and / or bivalent antibody agent. In some embodiments, KIH technology may be applied to promote pairing of two different heavy chains, for example, to produce a bispecific and / or bivalent antibody comprising one arm of the antibody that comprises an immunoglobulin chain of a CrossMabCH1‘CLxantibody agent and a second arm that comprises an immunoglobulin chain of a CrossMabCH1-CLcvantibody agent.

[0224] In some embodiments, CrossMab formats are applied to multiple antibody agents delivered together in a compositions. In some embodiments one antibody agent in a composition is a CrossMabCH1‘Cb<antibody agent and another antibody agent in the composition is a CrossMabCH1‘CLcvantibody agent.- 45 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Antibody Variants

[0225] Antibody variants described herein include one or more immunoglobulin chains, where one or more of the immunoglobulin chains includes a modification that alters association of the immunoglobulin chains. In some embodiments, an antibody variant comprises two immunoglobulin chains, a first immunoglobulin chain comprising a first variable domain and a first constant domain and a second immunoglobulin chain comprising a second variable domain and a second constant domain. In some embodiments, an antibody variant is a monovalent antibody or comprises only one antigen-binding domain. In some embodiments, the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to a target antigen. In some embodiments, the first constant domain is a heavy chain constant domain that is unable to homodimerize.

[0226] In some embodiments, an antibody variant described herein contains one or more modifications to one or more of its immunoglobulin chains to prevent homodimerization and / or to promote heterodimerization. In some embodiments, an antibody variant is utilized in methods described herein to detect and / or characterize mispairing between immunoglobulin chains of antibody agents. In some embodiments, an antibody variant includes an immunoglobulin chain that reduces valency of the antibody variant (e.g., is a monovalent antibody agent), such that silent mispairing of the antibody variant heavy chain and light chain is eliminated.

[0227] In some embodiments, an antibody variant includes an immunoglobulin chain that (e.g., an immunoglobulin heavy chain) that includes one or more mutations to prevent homodimerization and / or to promote heterodimerization. In some embodiments, an antibody variant comprises an immunoglobulin heavy chain constant domain. In some embodiments, an immunoglobulin heavy chain constant domain comprises a CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin heavy chain constant domain of an antibody variant is unable to homodimerize. In some embodiments, homodimerization is prevented by introducing one or more mutations.

[0228] In some embodiments, mutations known as "knob-into-holes" (KIH) mutations are utilized to prevent homodimerization. KIH technology, which aims to force the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids were replaced by amino acids with short side chains to create a "hole" and amino acids with large side chains were introduced into the other CH3 domain, to create a "knob". By co-expressing these two heavy chains with two light chains, high yields of heterodimer formation versus homodimer was observed (see Ridgway, J.B., etai, Protein Eng.9, 617-621, 1996; and WO 96 / 027011, which are herein incorporated by reference). In some embodiments, antibody variants described herein utilize KIH technology as described in, e.g., WO 1998 / 050431, which is herein incorporated by reference in its entirety. As described herein, an antibody variant may comprise certain mutations that utilize KIH technology that include, but are not limited to, a CH3 modification. In some embodiments, an antibody variant comprises a CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU numbering). In some embodiments, an antibody variant comprises a CH3 domain, wherein the CH3 domain comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU numbering). Such a combination of mutations is referred to herein as "cah". In some embodiments, an antibody variant comprises a CH3 domain comprising one or more mutations selected from: S354C and T366W (according to EU numbering). In some embodiments, an antibody agent comprises a CH3 domain comprising each of the following mutations: S354C and T366W (according to EU numbering). Such a combination of CH3 mutations is referred to herein as "cak".- 46 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0229] Accordingly, in some embodiments, an antibody variant encoded by one or more nucleic acids (e.g., polyribonucleotides) as described herein comprises a CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU numbering). In some embodiments, an antibody variant encoded by one or more nucleic acids (e.g., polyribonucleotides) as described herein comprises a CH3 domain comprising one or more mutations selected from: S354C and T366W (according to EU numbering).

[0230] In addition to preventing homodimerization, KIH mutations also promote heterodimerization with a different immunoglobulin heavy chain. Additionally, providing an antibody variant including a mutation to prevent homodimerization and / or promote homodimerization is useful in methods described here to detect and / or characterize mispairing of immunoglobulin chains of antibody agents. For example, in some embodiments, an immunoglobulin chain containing a mutation that prevents homodimerization (e.g., a "KIH" mutation) is included in a composition, where the composition does not include other immunoglobulin chains with which it can heterodimerize with. Because there can be no formation of a bivalent antibody agent, the "silent mispairing" effect (e.g., as shown in FIG. 5A) is eliminated. Such antibody variants are useful in methods described herein to detect and / or characterize immunoglobulin chain mispairing with certainty.Null Domains

[0231] In some embodiments, an antibody variant, including an immunoglobulin heavy chain modified to prevent homodimerization and promote heterodimerization, is included in a composition that includes another immunoglobulin chain modified to promote heterodimerization of the immunoglobulin chains. In some embodiments, heterodimerization of the immunoglobulin heavy chains (e.g., via KIH mutations) produces a monovalent antibody agent (see e.g., FIG. 5B, FIGs. 6A-6C, and FIG. 7). Such embodiments include an antibody variant that includes an immunoglobulin heavy chain that is modified such that it does not form an antigen-binding domain with another light chain (e.g., a "null domain"). In some embodiments, heterodimerization of the immunoglobulin heavy chains (e.g., via KIH mutations) produces a monovalent antibody agent (see e.g., FIG. 5B and FIG. 7).

[0232] In some embodiments, a null domain comprises a KIH mutation. In some embodiments, n null domain heterodimerizes with another immunoglobulin heavy chain of the antibody variant as a result of the KIH mutation. In some embodiments, a null domain of an antibody variant comprises the Y349T mutation and the other immunoglobulin heavy chain of the antibody variant comprises the T394F mutation. In some embodiments, a null domain of an antibody variant (immunoglobulin heavy chain constant domain) comprises the T394F mutation and the other immunoglobulin heavy chain of the antibody variant comprises the Y349T mutation.

[0233] In some embodiments, a KIH mutation comprises T366W, S354C, T366S, L368A, Y407V, and Y349C, according to the EU numbering scheme. In some embodiments, an null domain of an antibody variant (immunoglobulin heavy chain constant domain) comprises the T366W and S354C mutations and the other immunoglobulin heavy chain of the antibody variant comprises the T366S, L368A, Y407V, and Y349C mutations, according to the EU numbering scheme. In some embodiments, an null domain of an antibody variant (immunoglobulin heavy chain constant domain) comprises the T366S, L368A, Y407V, and Y349C mutations and the other immunoglobulin heavy chain of the antibody variant comprises the T366W and S354C mutations, according to the EU numbering scheme.

[0234] In some embodiments, a Fc-null domain included in antibody variants described herein includes only an immunoglobulin heavy chain constant domain. In such embodiments, when the null domain associates with another- 47 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)immunoglobulin heavy chain constant domain, the resulting antibody variant is a monovalent (the null domain cannot form an antigen-binding domain).

[0235] In some embodiments, a null domain may not be limited to only having an immunoglobulin heavy chain constant domain, but still does not include a functional immunoglobulin variable domain. In some embodiments, a null domain comprises another domain that does not function as an antigen-binding domain. In some embodiments, a null domain includes another antibody variable domain that does not bind to the target antigen targeted by the functional arm of an antibody variant. In some embodiments, an Fc-null domain comprises a variable domain that does not associate with other immunoglobulin domains (e.g., variable domains) of an antibody variant.

[0236] An antibody variable domain included in null domains described herein may include, e.g., a VHH domain or a single VH domain (see e.g., FIG. 6A) . In some embodiments, a variable domain included in null domains described herein may include a single-chain variable fragment (scFv). Various domains may be included in Fc null domains described herein, where the null domain does not form an antigen-binding domain that binds to the target antigen of the antibody variant. FIGs. 6A-6C show exemplary schematics of immunoglobulin heavy chain "null" domains that can be utilized in methods described herein, e.g., including a VHH-Fc or vNAR-Fc fusion (FIG. 6A), a fluorescent protein or protein tag fused to an Fc domain (FIG. 6B), and a random amino acid sequence (that does not affect Ig protein folding) or a "blunt" null domain containing only a constant domain of an antibody agent (FIG.6C). A null domain described herein may include any immunoglobulin heavy chain constant domain that does not bind to a target antigen. In some embodiments, a null domain may include any immunoglobulin heavy chain constant domain with a protein sequence that does not bind to the antigen target and does not affect folding of the antibody variant.

[0237] In some embodiments, an null domain includes a domain does not bind to the target antigen, but instead may be used as a detection agent, e.g., in binding assays to detect binding of the antibody variant to the target antigen. Exemplary detection agents include, for example, various ligands, radionuclides (e.g., 3H, 14C, 18F, 19F, 32P, 35S, 1351, 1251, 1231, 64Cu, 187Re, Ulin, 90Y, 99mTc, 177Lu, 89Zr etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available. Methods of measuring a detectable entity include, but are not limited to, visible detection, fluorescence, chemiluminescence, radioactivity, colorimetry, gravimetry, X-ray diffraction, X-ray absorption, magnetism, and enzymatic activity.

[0238] In some embodiments, a null domain of an antibody variant includes a domain such as a fluorescent protein or Tag. In some embodiments, a fluorescent protein comprises a green fluorescent protein. In some embodiments, a fluorescent protein is a luciferase or green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), a green fluorescent protein (eGFP), a His tag, GUS uidA lacz, or a gene expressing a beta glucuronidase or uidA gene (GUS) for which various chromogenic substrates, or any combination thereof. In some embodiments, a detection agent comprises a His tag comprising the sequence HIS-Tag: CATCACCATCACCATCAC-TAA, CATCATCATCATCATCAT, CATCACCATCACCATCAC, or a fragment or variant thereof. In some embodiments, a detection agent can be or include one or more of the sequences encoding yellow fluorescent protein (YFP, GenBank: GQ221700.1), red fluorescent protein (DsRED, GenBank:- 48 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)KY426960.1 or SEQ ID NO: 7), a green fluorescent protein (eGFP, GenBank: AAB02572.1), or cyan fluorescent protein (CFP, GenBank: HQ993060.1).Nucleic Acids Encoding Antibody Agents

[0239] Among other things, the present disclosure provides compositions for co-delivery of multiple antibody agents encoded by nucleic acid sequences, and methods of identifying and characterizing mispairing between immunoglobulin chains of the expressed antibody agents. Nucleic acids encoding multiple antibody agents may be delivered to a cell together, and the immunoglobulin chains of the antibody agents are expressed within the cell and associate to form the multiple antibody agents. Nucleic acids encoding antibody agents may include DNA or RNA, and antibody agents may be delivered and expressed by various methods.DNA Constructs

[0240] Among other things, the present disclosure provides DNA constructs, for example that may encode one or more antibody agents as described herein, or components thereof. In some embodiments, DNA constructs provided by and / or utilized in accordance with the present disclosure are comprised in a vector. In some embodiments, compositions comprising multiple antibody agents as described herein are delivered to a cell using in a vector.

[0241] Non-limiting examples of an vectors include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Non-limiting examples of viral vectors include: retrovirus (e.g., Moloney murine leukemia virus (MMLV), Harvey murine sarcoma virus, murine mammary tumor virus, Rous sarcoma virus), adenovirus, adeno-associated virus, SV40-type virus, polyomavirus, Epstein-Barr virus, papilloma virus, herpes virus, vaccinia virus, and polio virus.

[0242] Retroviruses are enveloped viruses that belong to the viral family Retroviridae. Once in a host's cell, the virus replicates by using a viral reverse transcriptase enzyme to transcribe its RNA into DNA. The retroviral DNA replicates as part of the host genome, and is referred to as a provirus. A selected nucleic acid (e.g., encoding multiple antibody agents) can be inserted into a vector and packaged in retroviral particles using techniques known in the art. Protocols for the production of replication-deficient retroviruses are known in the art (see, e.g., Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991)). The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art, for example See U.S. Pat Nos. 5,994,136, 6,165,782, and 6,428,953. Retroviruses include the genus of Alpharetrovirus (e.g., avian leukosis virus), the genus of Betaretrovirus; (e.g., mouse mammary tumor virus) the genus of Deltaretrovirus (e.g., bovine leukemia virus and human T-lymphotropic virus), the genus of Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), and the genus of Lentivirus.

[0243] In some embodiments, a retrovirus is a lentivirus of the Retroviridae family. Lentiviral vectors can transduce non-proliferating cells and show low immunogenicity. In some examples, the lentivirus is, but is not limited to, human immunodeficiency viruses (HIV-1 and HIV-2), simian immunodeficiency virus (S1V), feline immunodeficiency virus (FIV), equine infections anemia (EIA), and visna virus. Vectors derived from lentiviruses can achieve significant levels of nucleic acid transfer in vivo.- 49 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0244] In some embodiments, a vector is an adenovirus vector. Adenoviruses are a large family of viruses containing double stranded DNA. They replicate within the nucleus of a host cell, using the host's cell machinery to synthesize viral RNA, DNA and proteins. Adenoviruses are known in the art to affect both replicating and nonreplicating cells, to accommodate large transgenes, and to code for proteins without integrating into the host cell genome.

[0245] In some embodiments, a viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6):1110-17 (1994); Cotten et al., P.N.A.S. U.S.A., 89(13): 6094-98 (1992); Curiel, Nat Immun, 13(2-3):141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Then, 20(4):699-708 (2012)). Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368.

[0246] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof. Generally, any AAV serotype may be used to deliver one or more antibody agents described herein. However, the serotypes have different tropisms, e.g., they preferentially infect different tissues. In one embodiment, an AAV serotype is selected based on a liver tropism, found in at least serotypes AAV2, AAV3 (e.g., AAV3B), AAV5, AAV7, AAV8, and AAV9 (see, e.g., Shaoyong et al., Mol. Ther. 23:1867-1876 (2015)).

[0247] The AAV sequences of a rAAV vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are about 145 bp in length. In some embodiments, substantially the entire sequences encoding the ITRs are used in an rAAV vector, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of an rAAV vector of the present disclosure is a "cis-acting" plasmid containing a transgene (e.g., nucleic acid encoding one or more antibody agents described herein), in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.

[0248] In some embodiments, a vector is an expression vector. In some embodiments, a vector is a cloning vector. In general, a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.).

[0249] Expression vectors, which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest. In some embodiments, a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).

[0250] Cloning vectors are generally used to modify, engineer, and / or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process). In some embodiments, a cloning vector may lack expression signals.- 50 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0251] In many embodiments, a vector may include replication elements such as primer binding site(s) and / or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and / or guide RNA binding sites, etc.

[0252] In some embodiments, a vector is a viral vector {e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.

[0253] In some embodiments, polynudeotide(s) of the present disclosure are included in a DNA construct (e.g., a vector) amenable to transcription and / or translation.

[0254] In some embodiments, an expression vector comprises a polynucleotide that encodes proteins and / or polypeptides of the present disclosure operatively linked to a sequence or sequences that control expression {e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression {e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression {e.g., promoters) are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality of proteins and / or polypeptides. In some embodiments, a plurality of recombinant proteins and / or polypeptides are expressed from the same vector {e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic). In some embodiments, a plurality of polypeptides are expressed, each of which is expressed from a separate vector.

[0255] In some embodiments, an expression vector comprising a polynucleotide of the present disclosure is used to produce an RNA and / or protein and / or polypeptide in a host cell. In some embodiments, a host cell may be in vitro {e.g., a cell line) - for example a cell or cell line {e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for producing polynucleotides of the present disclosure and proteins and / or polypeptides encoded by said polynucleotides.

[0256] In some embodiments, an expression vector is an RNA expression vector. In some embodiments, an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix. In some embodiments, an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, a polynucleotide template is generated through PCR as a linear polynucleotide template. In some embodiments, a linearized polynucleotide is mixed with enzymes suitable for RNA synthesis, RNA capping and / or purification. In some embodiments, the resulting RNA is suitable for producing proteins encoded by the RNA.

[0257] A variety of methods are known in the art to introduce an expression vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction.

[0258] In some embodiments, transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art.- 51 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0259] In some embodiments, a vector has a copy number that is more than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In accordance with various embodiments, useful vectors for expressing antibody agents described herein include viral vectors or plasmids. Examples, without limitation include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAVs), pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEX vectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pBAD vectors (Invitrogen), pLEX vectors (Invitrogen), pMAL™ vectors (New England BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen). Vector systems for producing phage libraries are known and include Novagen T7Select® vectors, pMX vector plasmid (Invitrogen's GeneArt Gene Synthesis), and New England Biolabs Ph.D.™ Peptide Display Cloning System.Polyribonucleotides

[0260] Nucleic acids encoding antibody agents described herein may be polyribonucleotides.Polyribonucleotides described herein may encode an immunoglobulin chain of an antibody agent as described herein. Additionally, polyribonucleotides described herein, in some embodiments, include other elements such as a secretion signal-encoding region. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5'UTR of interest and / or a 3' UTR of interest. In some embodiments, polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail. In some embodiments, polyribonucleotides described herein may comprise a 5' cap, which may be incorporated during transcription, or joined to a polyribonucleotide post-transcription.Secretion signal-encoding region

[0261] According to certain embodiments, a signal peptide (or signal sequence) is fused, either directly or through a linker, to an encoded immunoglobulin chain of an antibody agent described herein.

[0262] In some embodiments, an open reading frame of the RNA described herein encodes an immunoglobulin chain of an antibody agent described herein with a signal sequence, e.g., that is functional in mammalian cells. In some embodiments, a utilized signal sequence is "intrinsic" in that it is, in nature, associated with (e.g., linked to) an immunoglobulin chain of an antibody agent or portion thereof.

[0263] In some embodiments, a utilized signal sequence is heterologous to an immunoglobulin chain of an antibody agent or portion thereof - e.g., is not naturally part of an immunoglobulin chain of an antibody agent or portion thereof.

[0264] In some embodiments, signal peptides are sequences, which are typically characterized by a length of about 15 to 30 amino acids.

[0265] In many embodiments, signal peptides are positioned at the N-terminus of an immunoglobulin chain of an antibody agent or portion thereof, without being limited thereto. In some embodiments, signal peptides preferably allow the transport of an immunoglobulin chain of an antibody agent or portion thereof encoded by RNAs of the present disclosure with which they are associated into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[0266] In some embodiments, a polyribonucleotide, as provided herein, that encodes an immunoglobulin chain of an antibody agent may comprise a ribonucleic acid sequence encoding a secretion signal. In some embodiments, a ribonucleic acid sequence encoding a secretion signal allows an immunoglobulin chain of an antibody agent encoded- 52 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)by the polyribonucleotide to be secreted upon translation by cells, e.g., present in a subject, thus yielding a plasma concentration of a biologically active antibody agent.5' Cap

[0267] A structural feature of mRNAs is cap structure at five-prime end (5'). Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5 ' to 5 '-triphosphate bridge resulting in capO structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2'-hydroxy-group (2'-OH) {e.g., the 2'-hydroxyl group may be methylated to form 2'-O-Me) of the first and subsequent nucleotides producing "capl" and "cap2" five-prime ends, respectively). Diamond, et al., (2014) Cytokine & growth Factor Reviews, 25:543-550 reported that capO-mRNA cannot be translated as efficiently as capl-mRNA in which the role of 2'-O-Me in the penultimate position at the mRNA 5' end is determinant. Lack of the 2'-O-met has been shown to trigger innate immunity and activate IFN response. Daffis, et al. (2010) Nature, 468:452-456; and Zlist et al. (2011) Nature Immunology, 12:137-143.

[0268] RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5'-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nudeobase, e.g. m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA ("anti-reverse cap analogue"), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1 -methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0269] The term "5'-cap" as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions {e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'OMeG). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3' O methylation at a ribose (m7(3'OMeG)). It will be understood that the notation used in the above paragraph, e.g., "(m27'3''°)G" or "m7(3'OMeG)", applies to other structures described herein.

[0270] In some embodiments, providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and / or translation rate of an RNA, and / or increase expression of an encoded polypeptide. In some embodiments, alterations- 53 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.

[0271] In some embodiments, a utilized 5' caps is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.Cap Proximal Sequences

[0272] In some embodiments, a utilized 5' caps is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.

[0273] In some embodiments, polyribonucleotides encoding antibody agents described herein include a 5' UTR utilized comprising a cap proximal sequence. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5' cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0274] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +1 (Ni) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1 and +2 (Ni and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1, +2, and +3 (Ni, N2, and N3) of an RNA polynucleotide.

[0275] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence {e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in an RNA by virtue of having been included in a cap entity {e.g., a capl or cap2 structure, etc); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added {e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m27'3''0Gppp(mi2'-°)ApG cap is utilized, +1 (i.e., Ni) and +2 (i.e. N2) are the (mi2'-°)A and G residues of the cap, and +3, +4, and +5 are added by polymerase {e.g., T7 polymerase).

[0276] In some embodiments, the 5' cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises Ni of the 5' cap, where Ni is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5' cap is a trinucleotide cap structure {e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni and N2 of the 5' cap, wherein Ni and N2 are independently any nucleotide, e.g., , C, G or U. In some embodiments, the 5' cap is a tetranucleotide cap structure {e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni, N2, and N3 of the 5' cap, wherein Ni, N2, and N3 are any nucleotide, e.g., , C, G or U.

[0277] In some embodiments, e.g., where the 5' cap is a dinucleotide cap structure, a cap proximal sequence comprises Ni of a the 5' cap, and N2, N3, N4and N5, wherein Ni to N5correspond to positions +1, +2, +3, +4, and / or- 54 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)+5 of an RNA polynucleotide. In some embodiments, e.g., where the 5' cap is a trinucleotide cap structure, a cap proximal sequence comprises Ni and N2 of a the 5' cap, and N3, N4and N5, wherein Ni to N5correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5' cap is a tetranucleotide cap structure, a cap proximal sequence comprises Ni, N2, and N3 of a the 5' cap, and N4and N5, wherein Ni to N5correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0278] In some embodiments, Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is G. In some embodiments, Ni is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4is A. In some embodiments, N4is C. In some embodiments, N4is G. In some embodiments, N4is U. In some embodiments, N5is A. In some embodiments, N5is C. In some embodiments, N5is G. In some embodiments, N5is U. It will be understood that, each of the embodiments described above and herein (e.g., for Ni through N5) may be taken singly or in combination and / or may be combined with other embodiments of variables described above and herein (e.g., 5' caps).5' UTR

[0279] In some embodiments, a nucleic acid {e.g., DNA, RNA) utilized in accordance with the present disclosure is a 5'-UTR. In some embodiments, a 5'-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements {e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments a 5' UTR comprises multiple different sequence elements.

[0280] The term "untranslated region" or "UTR" is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). As used herein, the terms "five prime untranslated region" or "5' UTR" refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, "5' UTR" refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. In some embodiments, a 5' UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5' cap.

[0281] Exemplary 5' UTRs include a human alpha globin (hAg) 5'UTR or a fragment thereof, a TEV 5' UTR or a fragment thereof, a HSP70 5' UTR or a fragment thereof, or a c-Jun 5' UTR or a fragment thereof.

[0282] In some embodiments, an RNA disclosed herein comprises a hAg 5' UTR or a fragment thereof.PolyA Tail

[0283] In some embodiments, a polynucleotide {e.g., DNA, RNA) disclosed herein comprises a polyadenylate (polyA) sequence, eg., as described herein. In some embodiments, a polyA sequence is situated downstream of a 3‘-UTR, e.g., adjacent to a 3'-UTR.- 55 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0284] As used herein, the term "poly(A) sequence" or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted Poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a templateindependent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0285] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).

[0286] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0287] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

[0288] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 Al may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coiiand is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.- 56 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0289] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e. , the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.

[0290] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0291] In some embodiments, a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.3' UTR

[0292] In some embodiments, an RNA utilized in accordance with the present disclosure comprises a 3'-UTR. As used herein, the terms "three prime untranslated region, ""3' untranslated region," or "3' UTR" refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context. The term "3‘-UTR"does preferably not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.

[0293] In some embodiments, an RNA disclosed herein comprises a 3' UTR comprising an F element and / or an I element. In some embodiments, a 3' UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHIs\ . In some embodiments, a restriction site is a XhoIs\e.

[0294] In some embodiments, an RNA construct comprises an F element. In some embodiments, a F element sequence is a 3'-UTR of amino-terminal enhancer of split (AES). In some embodiments, a 3'UTR is an FI element as described in W02017 / 060314, which is herein incorporated by reference in its entirety.EXEMPLIFICATIONExample 1: Assessment of Immunoglobulin Chain Misoairing in Antibody Variants

[0295] The present example demonstrates use of antibody variants described herein and compositions including the same in identifying and quantifying mispairing between immunoglobulin chains, e.g., that are to be utilized in a bispecific antibody or in multiple antibody agents to be delivered in the same composition.- 57 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0296] The present example measures mispairing between immunoglobulin chains encoded by polyribonucleotides, where the polyribonucleotides are co-delivered, and encode for more than one antibody agent (see FIG. 1). The present example provides antibody variants that target multiple epitopes on the same antigen. Specifically, the present example utilizes a first antibody agent (Abl) and a second antibody agent (Ab2) that bind to different epitopes of the same target antigen.

[0297] When immunoglobulin chains of multiple antibody agents are utilized or delivered together, desired combinations of those immunoglobulin chains can form, but undesired "mispaired" combinations of the immunoglobulin chains can also form (see, e.g., FIG. 1 and FIG. 2). Mispairing can occur on one or both arms of an antibody agent. Detection of mispairing at both Fabs of antibody agents within a population (e.g., in a composition) can be detected using binding assays (e.g., an ELISA as described herein). If both Fabs of an antibody agent are mispaired (e.g., a Abl heavy chain variable domain is paired with a Ab2 light chain variable domain), the antibody agent will not form the intended epitope binding site and binding of the antibody agent to the epitope or antigen of interest will be reduced. While mispairing on both Fabs of an antibody agent can occur, the more likely scenario is mispairing on only one Fab of an antibody agent. If only one Fab of an antibody agent pairs correctly and the other Fab mispairs, the mispairing is more challenging to detect. The challenge arises because the correctly paired Fab can still bind to its cognate epitope or antigen of interest and thus the antibody agent will be observed as properly binding.

[0298] To address this challenge, combinations of antibody agents described herein were assayed together with a "null" domain, as shown in FIG. 5 (an exemplary embodiment that uses a VHH-Fc domain fusion) and FIGs.6A-6C. By utilizing a null domain that does not bind to the epitope targets of the Abl or Ab2 antibody agent, any impact of mispairing on a Fab not bound to an epitope or antigen of interest can be eliminated.

[0299] Four Groups (A, B, C, and D) utilizing different combinations of polyribonucleotides encoding antibody agents, as shown in FIG. 8, were assayed. Various strategies were utilized in this example to minimize or prevent immunoglobulin chain mispairing of the Abl and Ab2 antibody agents, e.g., mispairing between immunoglobulin heavy chain constant domains (FIG. 3) and between immunoglobulin heavy and light chains (FIG. 4). The present example utilizes knob-into-holes mutations to prevent immunoglobulin heavy chain mispairing, as shown in FIG. 3D known as knob-into-holes (e.g., "cah" and "cak" mutations). Additionally, the present example identifies and quantifies mispairing between an immunoglobulin light chain variable domain and an immunoglobulin heavy chain variable domain of Abl that associate to form an antigen-binding domain or Fab (e.g., an epitope on a target antigen) and another light chain of Ab2 that is part of an antigen-binding domain to another epitope of the target antigen. Strategies shown in FIG. 4 were utilized in the present example to enhance correct pairing between the heavy and light chains of Abl, including antibody configurations: CrossMabCH1'CLx(FIG. 4B) and CrossMabCH1 Lcv(FIG. 4C). The specific groups of polyribonucleotides tested in the present example are as follows:

[0300] Group A, polyribonucleotides encoding a Ab2 light chain, a Abl light chain, a Abl heavy chain L / S (cak), and a VHH-Fc heavy chain L / S (cah) ("null domain") were used.

[0301] Group B, polyribonucleotides encoding a Ab2cm-CLx light chain, a Abl light chain, a Abl heavy chain L / S (cak), and a null domain (VHH-Fc) heavy chain L / S (cah) were used.- 58 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0302] Group C, polyribonucleotides encoding a Ab2 light chain, a Abiev light chain, a Abiev heavy chain L / S (cak), and a null domain (VHH-Fc) heavy chain L / S (cah) were used.

[0303] Group D, polyribonucleotides encoding a Ab2cm-cb< light chain, a Abiev light chain, a Abiev heavy chain L / S (cak), and a null domain (VHH-Fc) heavy chain L / S (cah) were used.

[0304] As shown in Table 1 below, seven samples were used within each group. Each of the samples used a different ratio of polyribonucleotides encoding the two light chains.Table 1:- 59 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Cell Transfection

[0305] The samples shown in Fig. 6 were delivered to a cell and were quantified by using Gyros xPand™ ELISA device (Gyros Protein Technologies AB, Uppsala, Sweden).

[0306] lpg of total RNA per well of polyribonucleotides encoding the antibody agents was delivered to HEK293T / 17 cells using RiboJuice™ RNA Transfection Kit (Merck, TR-1013). Briefly, transfection of cells was performed in duplicate in a 12-well culture cell vessel, lpg of polyribonucleotides of mixes were prepared according to the manufacturer protocol, by mixing lpg total RNA in lOOpL Optimem with 4pL Boost Reagent and 4pL RiboJuice transfection reagents. Mixes were added dropwise onto 4xl05cells in ImL DMEM+10% FCS. Cells were incubated for 48h at 37°C under humidified 5% CO2 atmosphere. Cell supernatants were then collected, centrifuged for 5 min at 4°C at 300xg and 900pL were collected for target antigen ELISA, Gyros ELISA and Western Blot analysis.Gyros ELISA

[0307] A sandwich immunoassay format was performed to determine intact (fully assembled) antibody agent content in cell culture supernatant samples from HEK293T17 cells transfected with RNA. The antibody agent (Abl antibody agent) concentration was analyzed using A) an anti-idiotype Abl Fab Biotin as capture antibody and Alexa Fluor 647 labeled F(ab')2 Rabbit Anti-Human IgG, Fey (Jackson ImmunoResearch Europe Ltd) as detection antibody, B) an Captu reSelect™ Human IgG-Fc PK Biotin as capture antibody and an Alexa Fluor 647 labeled F(ab')2 Rabbit Anti-Human IgG, Fey (Jackson ImmunoResearch Europe Ltd) as detection antibody. The assay was processed for low titer in a Gyrolab Bioaffy 1000 HC CD (Gyros Protein Technologies AB) with a dynamic range of 12.3 to 9,000 ng / mL in cell culture supernatant.

[0308] All samples, reference proteins and reagents were centrifuged for 4 minutes at 12,000xg to sediment any aggregates. Samples containing the respective antibody agents were diluted 10-fold in Reagent E buffer (Gyrolab huIgG Kit, Gyros Protein Technologies AB), respectively. The CD columns were washed with Reagent C and D (Gyrolab huIgG Kit, Gyros Protein Technologies AB).

[0309] Abl served as reference proteins to calculate the expressed antibody concentration in the assays.

[0310] For antibody agent quantitation, all materials prepared for the Gyros ELISA assay were loaded onto a 96-well plate (Gyros Protein Technologies AB) according to the Gyrolab loading list. Data were generated for low antibody agent titers with the huIgG Low Titer method (cell culture supernatant) Results were evaluated using the Gyrolab Evaluator software.Western Blot Analysis of Antibody Agents in Cell Culture Supernatants- 60 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0311] Western blots were performed on each of the groups to confirm antibody titers and to confirm antibody integrity (e.g., that there is no chain aggregation and all immunoglobulin chains expressed).

[0312] Briefly, a membrane was incubated with two HRP-conjugated detection antibodies: goat anti-human kappa LC at a 1:200 dilution (Thermo Fisher Scientific) and goat anti-human IgG, Fey-Fragment specific (Thermo Fisher Scientific) at a 1:500 dilution in 3% BSA Fraction V (Eurobio Scientific, Les Ulis, France). The membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 0.5 seconds and the data analyzed with Image Lab Software (Bio-Rad).

[0313] Further denatured samples were analyzed by western blots under non-reduced and reduced condition, by addition of 5% |3-mercaptoethanol. Briefly, the membrane was incubated with one HRP-conjugated detection antibody: Goat IgG anti-Alpaca IgG VHH HRP ( Thermo Fisher Scientific) at a 1:2000 dilution in 3% BSA Fraction V (Eurobio Scientific, Les Ulis, France). The membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 0.5 seconds and the data analyzed with Image Lab.

[0314] Further denatured samples were analyzed by western blots under non— reduced or reduced conditions, by addition of 5% |3-mercaptoethanol. Briefly, the membrane was incubated with a primary and HRP secondary detection antibodies: Anti-Idiotype Abl (Thermo Fisher Scientific) at a 1:250 dilution and Anti-6x His tag antibody HRP (Thermo Fisher Scientific) at a 1:10000 dilution in 3% BSA Fraction V (Eurobio Scientific, Les Ulis, France). The membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 0.5-3 seconds and the data analyzed with Image Lab.ELISA for Mispairing Determination

[0315] An ELISA was employed with a control antibody Fab to detect immunoglobulin chain mispairing.Antibody agents produced for each of Groups A, B, C, and D (FIG. 8) were subjected to ELISA to quantify antigenspecific binding titers. An exemplary schematic of the ELISA to measure mispairing is shown in FIG. 9.

[0316] For randomly biotinylated antigen ELISAs, target antigens were randomly biotinylated using the EZ-Link NHS-PEG4-Biotin kit (Thermo Fisher Scientific) according to the manufacturer's guidelines. The Pierce Biotin Quantitation kit (Thermo Fisher Scientific) was used to quantify the number of biotin molecules per target antigen, resulting in an average of 1-10 biotins attached to each target antigen.

[0317] Randomly biotinylated target antigen was added to ELISA plates (96 well Streptavidin plate, F-bottom) at 100 ng / 100 pL, diluted in lx Coating Buffer, and incubated overnight at 4 °C. Unbound target antigen was removed by washing with PBS-T (PBS with 0.01 % Tween20), and plates were blocked with lxBIocking Buffer (B6429-500ml) for one hour at 37°C, and then buffer was removed. Recombinant antibody agents or cell culture supernatants were serially diluted lxBIocking Buffer, and then incubated for one hour at 37°C. Recombinant antibody agents or cell culture supernatants were removed and plates were washed three times with PBS-T. Horseradish peroxidase (HRP) labeled secondary against the human IgG (Jackson Immuno Research, 109-035-098) was added at 1:5,000 dilution in PBS-T and incubated at 37°C for 45 min. Plates were then washed three times with PBS-T. Ultra TMB-ELISA Substrate Solution (ThermoFisher Scientific) was added at 100 pL / well for colorimetric detection and quenched with lOOpL / well 25% sulphuric acid (Merck, 1.007.161.000). Absorption was measured at 450 nm. Two independent biological replicates (n = 2) were performed for all assays.- 61 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Neutralization assay

[0318] Supernatants from HEK293T / 17 cells transfected with polyribonucleotides encoding the immunoglobulin chains shown in FIG. 8 or purified IgG samples were tested at indicated primary dilutions / concentrations and serially diluted 3-fold 7-times in duplicate wells. Diluted samples were mixed with an optimal titer of target antigen and coincubated at 37°C for 1 h. TZM-bl cells were then added at a final concentration of 104cells per well in a 96-well plate in medium supplemented with DEAE-dextran for 48hrs at 37°C and 5% CO2. 100 pl of culture supernatant was removed from each well and replaced with a Luc reporter gene assay system reagent (Brite-Glo, Promega, used as per manufacturer's recommendation). After a 2-min incubation at room temperature to allow cell lysis, 150 pl of cell lysate was transferred to 96-well black solid plates (Corning-Costar) for measurements of luminescence. After subtracting background relative luminescence units (RLUs) of non-infected TZM-bl cells, 50% and 80% inhibitory concentrations (IC50s and IC80s) were determined as the antibody / IgG concentrations resulting in a 50% / 80% RLU reduction compared to untreated antigen control wells.Results:Gyros ELISA

[0319] As shown in FIG. 11 and Table 1, Abl formed for all samples, except Samples 14 and 28. However, samples 14 and 28 were not expected to form antibody agents. Samples 14 and 28 included only a Ab2cni-cb< light chain. The Ab2cHi-cLx light chain includes a CHI domain, which is not expected to pair with the CHI domain of the Abl heavy chain L / S (cak) utilized with Group B or the Abiev heavy chain L / S (cak) utilized with Group D.Western Blot

[0320] Western blots performed on each of the groups demonstrated each of the Groups resulted in antibody titers and confirmation of the expressed antibody integrity as shown in FIGs. 12-18.

[0321] Fig. 12 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 12A) and reducing conditions (FIG. 12B) and stained for Goat anti-human Kappa Light Chain Antibody, HRP.

[0322] Fig. 13 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 13A) and reducing conditions (FIG. 13B) and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0323] Fig. 14 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 14A) and reducing conditions (FIG. 14B) and stained for anti-human Fcg IgG + anti-human kappa light chain.

[0324] Fig. 15 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 15A) and reducing conditions (FIG. 15B) and stained for Goat IgG anti-Alpaka IgG (VHH).

[0325] Fig. 16 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 16A) and reducing conditions (FIG. 16B) and stained for Goat IgG anti-Alpaka IgG (VHH).

[0326] Fig. 17 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 17A) and reducing conditions (FIG. 17B) and stained for anti-idiotype Abl.- 62 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0327] Fig. 18 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 18A) and reducing conditions (FIG. 18B) and stained for anti-idiotype Abl.Target Antigen ELISA

[0328] As shown in FIG. 20, antibody titration curves for different Groups A, B, C, and D were compared. With Group A, the Ab2 light chain, the Abl light chain, and the Abl heavy chain L / S (cak) did not include any modifications to prevent mispairing. Therefore, as the ratio of the Ab2 light chain to the Abllight chain increased, the amount of mispairing increased, an incremental increase in curve shift downward and to the right can be observed, indicated an incremental loss of target antigen binding titer at equal total antibody titer (FIG. 20A). With Group C, the Ab2 light chain did not include any modifications to prevent mispairing; however, the Abiev light chain and the Abiev heavy chain L / S (cak) were charge variants designed to promote binding to one another. As shown in Fig. 20C, the Group C titration curves shift less than observed with Group A. This demonstrates that the charge variants of Group C were able prevent some, but not all of the mispairing events observed with Group A. Group B included a Ab2cHi Lx light chain - a CrossMab including a CHI domain, which should prevent pairing with the Abl heavy chain L / S (cak). As shown in Fig. 20B, the CrossMab format was successful in significantly reducing the amount of mispairing that was observed. Finally, for Group D, a Ab2cm-CLx light chain in a CrossMab format was used.Additionally, the charge variants of the Abiev light chain and a Abiev heavy chain L / S (cak) were used. The combination of the two approaches for preventing mispairing eliminated any curve shift, indicating that mispairing had been abrogated (Fig. 20D). An overlay of selected curves from Figs. 20A and 20C is included in FIG. 21. FIG. 21 also shows that the AblCrossMabCHl-CLcv reduced mispairing compared to the Abl without the charge variant mutation.Neutralization Assay

[0329] FIG. 22 shows results from the neutralization assay measuring neutralization capacity of the antibody agents against target antigen (e.g., a viral antigen). If the immunoglobulin chains mispair (the Ab2 competing light chain pairs with the Abl heavy chain), then neutralization of the target antigen will decrease (IC50 / IC80 values will be higher). The results in FIG. 22 show neutralization was not detected in samples where no Abl light chain present in each of the four groups (A, B, C, and D). When no CrossMab modifications were included in the light chains or Abl heavy chain, neutralization capacity is lower or not detectable in more instances than Groups B, C, and D. Groups B, C, and D showed relatively similar IC50 values. These results show that the target antigen ELISA described in the present example for measuring mispairing is more sensitive to detecting antibody chain mispairing, and for identifying which immunoglobulin chain mutations are most effective in preventing mispairing.

[0330] In summary, the present example demonstrates the combination of successful RNA-construct design, RNA-co-transfection and ELISA to highlight the potential antibody chain association challenge.Example 2: Assessment of Immunoglobulin Chain Misoairing in mixtures of Bivalent Antibody Agents

[0331] The present example demonstrates an assay for identifying mispairing between immunoglobulin chains in a composition comprising nucleic acids (e.g., polyribonucleotides) encoding the immunoglobulin chains, and where the immunoglobulin chains, when expressed and correctly associated, form, e.g., one or more bispecific antibodies or one or more antibody agents.- 63 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0332] The present example measures mispairing between immunoglobulin chains encoded by polyribonucleotides, where the polyribonucleotides are co-delivered, and encode for more than one antibody agent (see FIG. 23). The present example provides antibody agents that target multiple epitopes on the same target antigen. Specifically, the present example utilizes a Abl antibody agent and a Ab2 antibody agent that bind to different epitopes of the same target antigen. The antibody agents are expressed in a cell after mRNA transfection of polyribonucleotides encoding a Ab2 light chain (e.g., Ab2 and Ab2in CrossMabCH1'CLxformat "Ab2xLc"), a Abl light chain (e.g., Abl-LSic and Abl-LSic in CrossMabCH1-CLcvformat "Abl-LScv c"), and a Abl heavy chain (e.g. a Abl LS heavy chain), as shown in FIG. 23 and FIG. 24. The present example examined mispairing between the Ab2 light chains and the Abl heavy chain when the three polyribonucleotides represented in FIG. 23 were delivered to a cell together with increasing amounts of polyribonucleotide encoding the Ab2 light chain (or the "competing light chain").

[0333] To screen mixtures of nucleic acids encoding antibody agents for mispairing, combinations of antibody agents described herein were assayed together as shown in Fig. 24.

[0334] Four Groups (A, B, C, and D) utilizing different combinations of polyribonucleotides encoding antibody agents, as shown, were assayed. Various strategies were utilized in this example to minimize or prevent immunoglobulin chain mispairing of the Abl and Ab2 antibody agents, e.g., mispairing between immunoglobulin heavy and light chains (FIG. 4). The present example identified and quantified mispairing between an immunoglobulin light chain variable and an immunoglobulin heavy chain (of a Abl antibody agent) that associate to form an antigen-binding domain or Fab to an epitope on the target antigen and another light chain (Ab2) that is part of an antigen-binding domain to another epitope on the target antigen. Strategies shown in FIG. 4 were utilized in the present example to enhance correct pairing between the heavy and light chains of Abl antibody agents, including antibody configurations: CrossMabCH1'CLx(FIG. 4B) and CrossMabCH1 Lcv(FIG. 4C). The specific groups of polyribonucleotides tested in the present example were as follows:

[0335] Group A, polyribonucleotides encoding a Ab2 light chain, a Abl light chain, a Abl heavy chain with an L / S mutation.

[0336] Group B, polyribonucleotides encoding a Ab2-LSxLc light chain (in CrossMabCHl-CLx format), a Abl light chain, and a Abl heavy chain L / S were used.

[0337] Group C, polyribonucleotides encoding a Ab2 light chain, a Abiev light chain (in CrossMabCHl-CLcv forma), and a Abiev heavy chain L / S were used.

[0338] Group D, polyribonucleotides encoding a Ab2cm-cb< light chain, a Abiev light chain, and a Abiev heavy chain L / S were used.

[0339] As shown in Fig. 25E, seven samples were used within each group. Each of the samples used a different ratio of the polyribonucleotides encoding the two light chains (Ablic: Ab2Lc was e.g., 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:8, and 0:1).Cell Transfection

[0340] The samples shown in FIG. 24 and Fig. 25 were delivered to a cell and were quantified by using Gyros xPand™ ELISA device (Gyros Protein Technologies AB, Uppsala, Sweden).- 64 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)

[0341] 2.5pg of total RNA per well of polyribonucleotides encoding the antibody agents was delivered to HEK293T / 17 cells using RiboJuice™ RNA Transfection Kit (Merck, TR-1013). Briefly, transfection of cells was performed in duplicate in a 6-well culture cell vessel. 2.5pg of polyribonucleotides of mixes were prepared according to the manufacturer protocol, by mixing 2.5pg total RNA in 250pL Optimem with 5pL Boost Reagent and 5pL RiboJuice transfection reagents. Mixes were added dropwise onto lxlO6cells in 2.5mL DMEM+10% FCS. Cells were incubated for 48h at 37°C under humidified 5% CO2 atmosphere. Cell supernatants were then collected, centrifuged for 5 min at 4°C at 300xg and 2400pL were collected for Gyros ELISA, Western Blot analysis, and target antigen ELISA.Gyros ELISA

[0342] A sandwich immunoassay format was performed to determine intact (fully assembled) antibody agent content in cell culture supernatant samples from HEK293T17 cells transfected with RNA. The antibody agent concentration was analyzed using a Captu reSelect™ Human IgG-Fc PK Biotin as capture antibody and an Alexa Fluor 647 labeled F(ab')2 Rabbit Anti-Human IgG, Fey (Jackson ImmunoResearch Europe Ltd) as detection antibody. The assay was processed for low titer in a Gyrolab Bioaffy 1000 HC CD (Gyros Protein Technologies AB) with a dynamic range of 12.3 to 9,000 ng / mL in cell culture supernatant.

[0343] All samples, reference proteins and reagents were centrifuged for 4 minutes at 12,000xg to sediment any aggregates. Samples containing the respective antibody agents were diluted 10-fold in Reagent E buffer (Gyrolab huIgG Kit, Gyros Protein Technologies AB), respectively. The CD columns were washed with Reagent C and D (Gyrolab huIgG Kit, Gyros Protein Technologies AB).

[0344] Abl served as reference proteins to calculate the antibody agent concentration in the assays.

[0345] For antibody agent quantitation, all materials prepared for the Gyros ELISA assay were loaded onto a 96-well plate (Gyros Protein Technologies AB) according to the Gyrolab loading list. Data were generated for low antibody agent titers with the huIgG Low Titer method (cell culture supernatant). Results were evaluated using the Gyrolab Evaluator software.Western Blot Analysis of Antibody Agents in Cell Culture Supernatants

[0346] Western blots were performed on each of the groups (A, B, C, and D in FIG. 24) to confirm antibody titers and correct assembly.

[0347] Briefly, a membrane was incubated with two HRP-conjugated detection antibodies: goat anti-human kappa LC at a 1:200 dilution (Thermo Fisher Scientific) and goat anti-human IgG, Fey-Fragment specific (Thermo Fisher Scientific) at a 1:500 dilution in 3% BSA Fraction V (Eurobio Scientific, Les Ulis, France). The membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 1 second and the data analyzed with Image Lab Software (Bio-Rad).

[0348] Further denatured samples were analyzed by western blots under non— reduced or reduced conditions, by addition of 5% |3-mercaptoethanol. Briefly, the membrane was incubated with a primary and HRP secondary detection antibodies: Anti-Idiotype Ab (Thermo Fisher Scientific) at a 1:250 dilution and Anti-6x His tag antibody HRP (Thermo Fisher Scientific) at a 1:10000 dilution in 3% BSA Fraction V (Eurobio Scientific, Les Ulis, France). The- 65 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 1 second and the data analyzed with Image Lab.Target Antigen ELISA for Mispairing Determination

[0349] An ELISA was employed with a control antibody Fab to detect immunoglobulin chain mispairing.Antibody agents produced for each of Groups A, B, C, and D were subjected to target antigen ELISA to quantify antigen-specific binding titers.

[0350] For randomly biotinylated antigen ELISAs, target antigen was randomly biotinylated using the EZ-Link NHS-PEG4-Biotin kit (Thermo Fisher Scientific) according to the manufacturer's guidelines. The Pierce Biotin Quantitation kit (Thermo Fisher Scientific) was used to quantify the number of biotin molecules per target antigen, resulting in an average of 1-10 biotins attached to each target antigen.

[0351] Randomly biotinylated target antigen was added to ELISA plates (96 well Streptavidin plate, F-bottom) at 100 ng / 100 pL, diluted in lx Coating Buffer, and incubated overnight at 4 °C. Unbound target antigen was removed by washing with PBS-T (PBS with 0.01 % Tween20), and plates were blocked with lxBIocking Buffer (B6429-500ml) for one hour at 37°C, and then buffer was removed. Recombinant antibody agents, or cell culture supernatants were serially diluted lxBIocking Buffer, and then incubated for one hour at 37°C. Recombinant antibody agents or cell culture supernatants were removed and plates were washed three times with PBS-T. Horseradish peroxidase (HRP) labeled secondary against the human IgG (Jackson Immuno Research, 109-035-098) was added at 1:5,000 dilution in PBS-T and incubated at 37°C for 45 min. Plates were then washed three times with PBS-T. Ultra TMB-ELISA Substrate Solution (ThermoFisher Scientific) was added at 100 pL / well for colorimetric detection and quenched with lOOpL / well 25% sulphuric acid (Merck, 1.007.161.000). Absorption was measured at 450 nm. Two independent biological replicates (n = 2) were performed for all assays.Results:Gyros ELISA

[0352] As shown in Figs. 25A-D, antibody agents formed for all samples (>600ng / mL) (FIG. 25A shows results from Group A, FIG. 25B shows results from Group B, FIG. 25C shows results from Group C, and FIG. 25D shows results from Group D), except those samples that included only a Ab2cni-cb< light chain, which showed low antibody production. The Ab2cni-cb< light chain includes a CHI domain, which is not expected to pair with the CHI domain of the Abl heavy chain L / S utilized with Group B or the Abiev heavy chain L / S utilized with Group D. These results indicate successful mismatch prevention when the CrossMabCH1‘CLxand CrossMabCH1‘CLcvstrategies were utilized.Western Blot

[0353] Western blots performed on each of the groups demonstrated each of the Groups resulted in antibody titers and with the correct assembly as shown in FIGs. 26-30.

[0354] Fig. 26 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing (FIG. 26A) and reducing conditions (FIG. 26B) and stained for Goat anti-human Kappa Light Chain Antibody, HRP.- 66 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Specifically, FIG. 26 shows that the LC:LC dimer formation for the CrossMAbCH1'CLxgroup (samples 8-14) is prevented if Abl LS CrossMabCH1'CLcv(charged variant) is used (samples 22-28).

[0355] Fig. 27 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain. Specifically, FIG. 27 shows no visible antibody agent aggregate formation, and successful Abl monomer formation, and no detectible mispairing between Ab2LC and AblHC in sample 14, where the CrossMabCH1'CLxformat is used, and where the sample contains no Abl LC.

[0356] Fig. 28 shows an exemplary Western Blot analysis of antibody agent mispairing under reducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain. Specifically, FIG. 28 shows no visible antibody agent aggregate formation and no detectible mispairing between Ab2LC and AblHC in sample 14, where the CrossMabCH1 Lxformat is used, and where the sample contains no Abl LC.

[0357] Fig. 29 shows an exemplary Western Blot analysis of antibody agent mispairing under nonreducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain. Specifically, FIG. 29 shows no visible antibody agent aggregate formation, and successful Abl monomer formation, and no detectible mispairing between Ab2 LC and AblHC in sample 28, where the CrossMabCH1'CLxformat is used in the Ab2 light chain and the CrossMabCHl-CLcv format is used in the Abl heavy chain and Abl light chain, and where the sample contains no Abl LC.

[0358] Fig. 30 shows an exemplary Western Blot analysis of antibody agent mispairing under reducing conditions and stained for anti-human Fcg IgG + anti-human kappa light chain. Specifically, FIG. 30 shows no visible antibody agent aggregate formation and no detectible mispairing between Ab2 LC and Ab 1HC in sample 28, where the CrossMabCH1 Lxformat is used in the Ab2 light chain and the CrossMabCHl-CLcv forma is used in the Abl heavy chain and Abl light chain, and where the sample contains no Ab 1LC.Target Antigen ELISA

[0359] FIG. 31 shows exemplary binding curves obtained from the target antigen ELISA of positive control samples including a Abl LS antibody or a control antibody LS (fully assembled) that binds a different epitope of the target antigen than Abl and Ab2 and negative control samples including only a Abl heavy chain, a Abl LS heavy chain in CrossMabCHl-CLcv format, or a negative control antibody (antibody that does not target an epitope of the target antigen). FIG. 31 shows successful binding of the Abl and control antibody positive control antibodies to the target antigen, and no binding of the negative controls (Abl HC without the Abl LC and the negative control antibody that does not bind to the target antigen). FIG. 32 shows exemplary binding curves obtained from the mispairing ELISA of Abl antibody agents with and without a charge variant modification challenged with Ab2 light chain. These results demonstrate that for each condition ("Abl baseline" or only Abl antibody agent, 1:1 Abl to Ab2 light chain, and 1:8 Abl to Ab2 light chain), the binding curves shifted when a charge variant modification was introduced into the Abl light chain, indicating a reduction in mispairing between the Ab2 LC and Abl heavy chain.FIG. 33 shows exemplary binding curves obtained from the target antigen ELISA for each of the Groups A, B, C, and D (FIG. 33A, FIG. 33B, FIG. 33C, and FIG. 33D, respectively) and conditions in each sample (FIG. 33E). Ratios of the two different light chains in each sample is shown in FIG. 33E. Overall, FIG. 33 shows a curve shift with- 67 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)increasing concentration of the competing Ab2 light chain when the light chains do not contain CrossMAb modifications (either charge variant "CHl-CLcv" or domain swap "CHl-CLx"). FIG. 33 also shows that the curve shift is particularly reduced when the CrossMabCH1'CLxmodifications are introduced into the Ab2 light chain (see FIG. 33B and FIG. 33D).

[0360] FIGs. 34A-34E shows concentrations of Abl antibody (ng / ml) at 50% signal from the binding curves shown in FIG. 33 for each of the Groups A, B, C, and D (FIG. 34A, FIG. 34B, FIG. 34C, and FIG. 34D, respectively) and the conditions in each sample (FIG. 34E). These results show that modifications utilized in the present example to reduce mispairing between the Abl and Ab2 light chains resulted in greater Abl binding to the target antigen (as indicated by lower concentrations of antibody required to reach 50% signal).

[0361] In summary, the present example demonstrates that mispairing can be detected in certain combinations of antibody agents even when the antibody agent valency is not reduced (e.g., without use of a "null" domain). Additionally, the target antigen ELISA was able to detect a decrease in target antigen binding with increased amounts of competing light chain. The present example also confirms successful RNA-construct design encoding the antibody agents to completely prevent HC / LC mispairing by engineering the Ab2 light chain to include a CrossMabCH1'CLxmodification and engineering the Abl light chain to include a CrossMabCH1-CLcvmodification.Example 3: Assessment of Immunoglobulin Chain Misoairing in mixtures of bivalent Antibody Agents

[0362] The present example demonstrates and assay for identification of mispairing between immunoglobulin chains in a composition comprising nucleic acids (e.g., polyribonucleotides) encoding the immunoglobulin chains, and where the immunoglobulin chains, when expressed and correctly associated, form, e.g., one or more bispecific antibodies or one or more antibody agents.

[0363] Similar to prior Examples 1 and 2, the present example measures mispairing between immunoglobulin chains encoded by polyribonucleotides, where the polyribonucleotides are co-delivered, and encode for more than one antibody agent (see FIG. 35A). The present example provides antibody agents that target multiple epitopes on the same target antigen. Specifically, the present example utilizes a Abl antibody agent and a Ab2 antibody agent that bind to different epitopes of a target antigen. The antibody agents are expressed in a cell after mRNA transfection of polyribonucleotides encoding a Ab2 light chain (e.g., Ab2-LS and Ab2-LS in CrossMabCH1'CLxformat "Ab2-LSx c"), a Abl light chain (e.g., Abl-LSic and Abl-LSic in CrossMabCH1-CLcvformat "Abl-LScv c"), and a Ab2 heavy chain (e.g. a Ab2-LS heavy chain), as shown in FIG. 35A and FIG. 36. The present example examined mispairing between the Abl light chain and the Ab2 heavy chain when the three polyribonucleotides represented in FIG. 35A were delivered to a cell together with increasing amounts of polyribonucleotide encoding the Abl light chain (or the "competing light chain").

[0364] To screen mixtures of nucleic acids encoding antibody agents for mispairing, combinations of antibody agents described herein were assayed together as shown in Fig. 36.

[0365] Four Groups (A, B, C, and D) utilizing different combinations of polyribonucleotides encoding antibody agents, as shown, were assayed. Various strategies were utilized in this example to minimize or prevent immunoglobulin chain mispairing of the Abl and Ab2 antibody agents, e.g., mispairing between immunoglobulin heavy and light chains as shown in FIG. 35B and FIG. 35C. The present example identified and quantified- 68 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)mispairing between an immunoglobulin light chain variable and an immunoglobulin heavy chain (of a Ab2 antibody agent) that associate to form an antigen-binding domain or Fab (e.g., an epitope on the target antigen) and another light chain (Abl) that is part of an antigen-binding domain to another epitope of the target antigen (e.g., a different epitope than the epitope targeted by Abl). Strategies shown in FIG. 35B and FIG. 35C were utilized in the present example to enhance correct pairing between the heavy and light chains of Ab2 antibody agent, including antibody configurations: CrossMabCH1'Cb<(FIG. 35B) and CrossMabCH1'CLcv(FIG. 35C). The specific groups of polyribonucleotides tested in the present example were as follows (as shown in FIG. 36):

[0366] Group A, polyribonucleotides encoding a Abl light chain, a Ab2 light chain, and a Ab2 heavy chain with an L / S mutation.

[0367] Group B, polyribonucleotides encoding a Abl light chain, a Ab2-LSx c light chain (in CrossMabCH1 Lxformat), and a Ab2-LSx heavy chain (Ab2 antibody agent heavy chain in CrossMabCH1'CLxformat) were used.

[0368] Group C, polyribonucleotides encoding a Abiev light chain (in CrossMabCH1 Lcvformat), a Ab2 light chain, and a Ab2-LS heavy chain were used.

[0369] Group D, polyribonucleotides encoding a Abiev light chain (in CrossMabCH1'CLcvformat), a Ab2-LSxLc light chain (in CrossMabCH1'CLxformat), and a Ab2-LSx heavy chain (in CrossMabCH1'CLxformat) were used.

[0370] As shown in Fig. 37E, seven samples were used within each group. Each of the samples used a different ratio of the two light chains (Ab2 c: Ablic was e.g., 1:0, 1:0.5, 1:1, 1:2, 1:4, 1:8, and 0:1).Cell Transfection

[0371] The samples shown in FIG. 35 and FIG. 36 were delivered to a cell and were quantified by using Gyros xPand™ ELISA device (Gyros Protein Technologies AB, Uppsala, Sweden).

[0372] 2.5pg of total RNA per well of polyribonucleotides encoding the antibody agents was delivered to HEK293T / 17 cells using RiboJuice™ RNA Transfection Kit (Merck, TR-1013). Briefly, transfection of cells was performed in duplicate in a 6-well culture cell vessel. 2.5pg of polyribonucleotides of mixes were prepared according to the manufacturer protocol, by mixing 2pg total RNA in 250pL Optimem with 5pL Boost Reagent and 5pL RiboJuice transfection reagents. Mixes were added dropwise onto 14xl06cells in 2.5mL DMEM+10% FCS. Cells were incubated for 48h at 37°C under humidified 5% CO2 atmosphere. Cell supernatants were then collected, centrifuged for 5 min at 4°C at 300xg and 2400pL were collected for Gyros ELISA, Western Blot analysis, and target antigen ELISA.Gyros ELISA

[0373] A sandwich immunoassay format was performed to determine intact (fully assembled) antibody agent content in cell culture supernatant samples from HEK293T17 cells transfected with RNA. The antibody agent concentration was analyzed using an Captu reSelect™ Human IgG-Fc PK Biotin as capture antibody and an Alexa Fluor 647 labeled F(ab')2 Rabbit Anti-Human IgG, Fey (Jackson ImmunoResearch Europe Ltd) as detection antibody. The assay was processed for low titer in a Gyrolab Bioaffy 1000 HC CD (Gyros Protein Technologies AB) with a dynamic range of 12.3 to 9,000 ng / mL in cell culture supernatant.

[0374] All samples, reference proteins and reagents were centrifuged for 4 minutes at 12,000xg to sediment any aggregates. Samples containing the respective antibody agents were diluted 10-fold in Reagent E buffer (Gyrolab - 69 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)huIgG Kit, Gyros Protein Technologies AB), respectively. The CD columns were washed with Reagent C and D (Gyrolab huIgG Kit, Gyros Protein Technologies AB).

[0375] Abl served as reference proteins to calculate the antibody agent concentration in the assays.

[0376] For antibody agent quantitation, all materials prepared for the Gyros ELISA assay were loaded onto a 96-well plate (Gyros Protein Technologies AB) according to the Gyrolab loading list. Data were generated for low antibody agent titers with the huIgG Low Titer method (cell culture supernatant). Results were evaluated using the Gyrolab Evaluator software.Western Blot Analysis of Antibody Agents in Cell Culture Supernatants

[0377] Western blots were performed on each of the groups (A, B, C, and D in FIG.36) to confirm antibody titers and correct assembly.

[0378] Briefly, a membrane was incubated with two HRP-conjugated detection antibodies: goat anti-human kappa LC at a 1:200 dilution (Thermo Fisher Scientific) and goat anti-human IgG, Fey-Fragment specific (Thermo Fisher Scientific) at a 1:500 dilution in 3% BSA Fraction V (Eurobio Scientific, Les Ulis, France). The membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 0.5 seconds and the data analyzed with Image Lab Software (Bio-Rad).Target Antigen ELISA for Mispairing Determination

[0379] An ELISA utilizing the target antigen was employed with a control antibody Fab to detect immunoglobulin chain mispairing. Antibody agents produced for each of Groups A, B, C, and D in FIG.36 were subjected to target antigen ELISA to quantify target antigen-specific binding titers.

[0380] For randomly biotinylated antigen ELISAs, target antigens were randomly biotinylated using the EZ-Link NHS-PEG4-Biotin kit (Thermo Fisher Scientific) according to the manufacturer's guidelines. The Pierce Biotin Quantitation kit (Thermo Fisher Scientific) was used to quantify the number of biotin molecules per target antigen, resulting in an average of 1-10 biotins attached to each target antigen.

[0381] Randomly biotinylated target antigen was added to ELISA plates (96 well Streptavidin plate, F-bottom) at 100 ng / 100 pL, diluted in lx Coating Buffer, and incubated overnight at 4 °C. Unbound trimers were removed by washing with PBS-T (PBS with 0.01 % Tween20), and plates were blocked with lxBIocking Buffer (Blocker™ Kasein in PBS from Thermo Scientific™ diluted to 0.18% Casein with lxPBS before usage) for one hour at 37°C, and then buffer was removed. Recombinant antibody agents, or cell culture supernatants were serially diluted lxBIocking Buffer, and then incubated for one hour at 37°C. Recombinant antibody agents or cell culture supernatants were removed and plates were washed three times with PBS-T. Horseradish peroxidase (HRP) labeled secondary against the human IgG (Jackson Immuno Research, 109-035-098) was added at 1:5,000 dilution in PBS-T and incubated at 37°C for 45 min. Plates were then washed three times with PBS-T. Ultra TMB-ELISA Substrate Solution (ThermoFisher Scientific) was added at 100 pL / well for colorimetric detection and quenched with lOOpL / well 25% sulphuric acid (Merck, 1.007.161.000). Absorption was measured at 450 nm. Two independent biological replicates (n = 2) were performed for all assays.Results:- 70 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)Gyros ELISA

[0382] As shown in FIGs. 37A-D, antibody agents formed for all samples (FIG. 37A shows results from Group A, FIG. 37B shows results from Group B, FIG. 37C shows results from Group C, and FIG. 37D shows results from Group D), except those samples that included only a Ab2-LS heavy chain in Groups A and C. The Abl CrossMabCHl-CLcv light chain a charge variant was not expected to pair as with the CHI domain of the Ab2 LS heavy chain Group C or the Ab2 CrossMabCHl-CLx heavy chain L / S utilized with Group D. However, intact antibodies were still detected when no Ab2 light chain was present in Group D, suggesting a potential mismatch between the Abl CrossMabCHl-CLcv light chain and the Ab2 CrossMabCHl-CLx heavy chain. The detectable concentration in Groups B and D in the HC only group is only due to a formation of a HC-HC dimer as shown in FIG. 38 and FIG. 39.Western Blot

[0383] Western blots performed on each of the Groups (A, B, C, and D) demonstrated each of the Groups resulted in antibody titers and with the correct assembly as shown in FIGs. 38 and 39.

[0384] FIG. 38 and FIG. 39 show exemplary Western Blot analysis of antibody agent mispairing under nonreducing conditions and reducing conditions, respectively, and stained for anti-human Fcg IgG + anti-human kappa light chain. Specifically, FIG. 38 and FIG. 39 show no visible antibody agent aggregate formation, and successful Ab2 monomer formation, and no detectible mispairing between Ab2HC and Abl LC where the CrossMabCH1'CLxformat is used (samples 14 and 28), and where the sample contains no Ab2 LC.Target Antigen ELISA

[0385] FIG. 40 shows an exemplary binding curve obtained from the target antigen ELISA of positive control samples including a Abl-LS antibody or control LS antibody (fully assembled) and negative control samples including only a Ab2-LSx (in domain swap or "CrossMabCH1'CLx" format) heavy chain, a Ab2-LS heavy chain, or negative control antibody (antibody that does not target an epitope on the target antigen). FIG. 40 shows successful binding of the Abl-LS and positive control antibodies to target antigen, and no binding of the negative controls Ab2 HC without the Ab2 LC and negative control antibody that does not bind to the target antigen). FIG. 41 shows exemplary binding curves obtained from the target antigen ELISA for each of the Groups A, B, C, and D (FIG. 41A, FIG. 41B, FIG. 41C, and FIG. 41D, respectively). Overall, FIG. 41 shows no shift in the binding curves in any of the groups, indicating no detectable mispairing in all four groups.EQUIVALENTS

[0386] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of technologies described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims.- 71 - 13276319vl

Claims

Attorney Docket No.: 2013237-1583 (BNT REF: P2042W01)CLAIMS1. An antibody variant comprising:(i) a first immunoglobulin chain comprising a first variable domain and a first constant domain; and (ii) a second immunoglobulin chain comprising a second variable domain and a second constant domain;wherein the first constant domain is a heavy chain constant domain that is unable to homodimerize; and wherein the first variable domain and the second variable domain associate to form an antigen-binding domain that specifically binds to a target antigen.

2. The antibody variant of claim 1, wherein the first variable domain comprises a heavy chain variable domain.

3. The antibody variant of claim 1 or 2, wherein the first constant domain comprises a heavy chain constant domain.

4. The antibody variant of any one of claims 1-3, wherein the first constant domain comprises a CHI domain operably linked to a CH2 domain and a CH3 domain.

5. The antibody variant of claim 4, wherein the first constant domain is unable to homodimerize because of one or more mutations.

6. The antibody variant of any one of claims 1-5, wherein the second variable domain comprises a light chain variable domain.

7. The antibody variant of any one of claims 1-6, wherein the second constant domain comprises a light chain constant domain.

8. The antibody variant of any one of claims 1-7, wherein the antibody variant further comprises a third immunoglobulin chain comprising a third constant domain.

9. The antibody variant of claim 8, wherein the third constant domain comprises a heavy chain constant domain.

10. The antibody variant of claim 8 or 9, wherein the third constant domain comprises a CH2 domain and a CH3 domain.

11. The antibody variant of any one of claims 8-10, wherein the third constant domain is unable to homodimerize.- 72 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)12. The antibody variant of claim 11, wherein the third constant domain is unable to homodimerize because of one or more mutations.

13. The antibody variant of any one of claims 8-12, wherein the third immunoglobulin chain consists of the constant domain.

14. The antibody variant of any one of claims 8-12, wherein the third immunoglobulin chain does not comprise a functional variable domain.

15. The antibody variant of any one of claims 8-12, wherein the third immunoglobulin chain comprises a third variable domain that does not associate with the first or second variable domains.

16. The antibody variant of claim 15, wherein the third immunoglobulin chain comprises an unstructured amino acid sequence (e.g., GS linker).

17. The antibody variant of claim 15, wherein the third immunoglobulin chain comprises a single domain Fab fragment.

18. The antibody variant of claim 15, wherein the third immunoglobulin chain comprises a VHH or VNAR domain.

19. The antibody variant of claim 15, wherein the third immunoglobulin chain comprises a single-chain variable fragment (scFv).

20. The antibody variant of any one of claims 8-19, wherein the third immunoglobulin chain comprises fluorescent protein and / or a Tag.

21. The antibody variant of claim 20, wherein the third immunoglobulin chain comprises a green fluorescent protein (GFP).

22. The antibody variant of any one of claims 8-21, wherein the first immunoglobulin chain and the third immunoglobulin chain heterodimerize to form an Fc domain.

23. The antibody variant of claim 22, wherein the first immunoglobulin chain and the third immunoglobulin chain heterodimerize as a result of a knob-in-hole (KIH) mutation.

24. The antibody variant of claim 23, wherein the KIH mutation comprises Y349T and T394F, according to the EU numbering scheme.- 73 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)25. The antibody variant of claim 24, wherein the first constant domain comprises the Y349T mutation and the third constant domain comprises the T394F mutation.

26. The antibody variant of claim 24, wherein the first constant domain comprises the T394F mutation and the third constant domain comprises the Y349T mutation.

27. The antibody variant of claim 23, wherein the KIH mutation comprises T366W, S354C, T366S, L368A, Y407V, and Y349C, according to the EU numbering scheme.

28. The antibody variant of claim 27 , wherein the first constant domain comprises the T366W and S354C mutations and the third constant domain comprises the T366S, L368A, Y407V, and Y349C mutations, according to the EU numbering scheme.

29. The antibody variant of claim 27 , wherein the first constant domain comprises the T366S, L368A, Y407V, and Y349C mutations and the third constant domain comprises the T366W and S354C mutations, according to the EU numbering scheme.

30. The antibody variant of any one of claims 8-29, wherein the first and / or third constant domains comprise an IgGl isotype.

31. The antibody variant of claim 30, wherein the first and / or third constant domains comprise a human IgGl isotype.

32. The antibody variant of any one of claims 8-31, wherein the first and / or third constant domains comprise one or more mutated amino acid residues that increase half-life of the antibody variant.

33. The antibody variant of claim 32, wherein the first and / or third constant domains comprise a combination of the following mutated amino acid residues: M252Y, S254T, and T256E, according to the EU numbering scheme.

34. The antibody variant of claim 32, wherein the first and / or third constant domains comprise a combination of the following mutated amino acid residues: M428L and N434S, according to the EU numbering scheme.

35. A combination comprising:the antibody variant of any one of claims 1-34, anda polypeptide comprising a fourth variable domain, wherein the first variable domain and the fourth variable domain are capable of associating to form a mispaired domain.

36. The combination of claim 35, wherein the fourth variable domain comprises an immunoglobulin light chain variable domain.- 74 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)37. The combination of claim 35 or 36, wherein the first immunoglobulin chain and / or the second immunoglobulin chain comprise one or more mutations to:(i) increase association between the first immunoglobulin chain and the second immunoglobulin chain; (ii) decrease association between the first immunoglobulin chain and the third immunoglobulin chain; and / or(iii) decrease association between the first immunoglobulin chain and the fourth variable domain.

38. The combination of any one of claims 35-37, wherein the third immunoglobulin chain and / or the fourth variable domain comprises one or more mutations to:(i) increase association between the first immunoglobulin chain and the second immunoglobulin chain; (ii) decrease association between the first immunoglobulin chain and the third immunoglobulin chain; and / or(iii) decrease association between the first immunoglobulin chain and the fourth variable domain.

39. The combination of claim 37, wherein the second constant domain comprises one or more charge variant mutations.

40. The combination of claim 39, wherein the one or more charge variant mutations comprises an amino acid at position 123 (according to the EU numbering scheme) that is substituted by the amino acid K, R or H.

41. The combination of claim 39 or 40, wherein the one or more charge variant mutations comprises an amino acid at position 124 (according to the EU numbering scheme) that is substituted by the amino acid K, R or H.

42. The combination of any one of claims 39-41, wherein the one or more charge variant mutations comprises any one of the following combinations of mutations according to the EU numbering scheme:(i) E123K and Q124R(ii) E123K and Q124K(iii) E123R and Q124R; and(iv) E123R and Q124K.

43. The combination of any one of claims 39-42, wherein the CHI domain of the first constant domain comprises one or more charge variant mutations.

44. The combination of claim 43, wherein one or more charge variant mutations in the CHI domain of the first constant domain comprises an amino acid at position 147 (EU numbering) that is substituted by the amino acid E or D, according to the EU numbering scheme.

45. The combination of claim 43 or 44, wherein the one or more charge variant mutations comprises a K213D mutation.- 75 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)46. The combination of any one of claims 43-45, wherein the one or more charge variant mutations in the CHI domain of the first constant domain comprises one of the following combinations of mutations: (i) K147E and K213D; or (ii) K147D and K213D.

47. The combination of claim 35 or 36, wherein the polypeptide comprises an immunoglobulin light chain variable domain operably linked to an immunoglobulin heavy chain CHI domain.

48. The combination of claim 47, wherein the CHI domain of the polypeptide comprises a Q124E mutation, according to the EU numbering scheme.

49. The combination of claim 37, wherein the first variable domain comprises a heavy chain variable domain and the first constant domain comprises a light chain constant domain.

50. The combination of claim 49, wherein the second immunoglobulin chain comprises a light chain variable domain and the second constant domain comprises a CHI domain.

51. The combination of claim 50, wherein the first constant domain comprises a Q124E mutation, according to the EU numbering scheme.

52. A composition for use in a mispairing assay comprising the combination of any one of claims 35-51.

53. A composition comprising one or more nucleic acids (e.g., polyribonucleotides) encoding the combination of any one of claims 35-51.

54. A kit comprising the antibody variant of any one of claims 1-34, the combination of any one of claims 40-56, or the composition of claim 52 or 53.

55. A method comprising:(i) measuring the binding affinity of one or more antibody agents to a target antigen,wherein the one or more antibody agents are in a composition comprising:(A) a first immunoglobulin chain comprising a first variable domain and a first constant domain; (B) a second immunoglobulin chain comprising a second variable domain and a second constant domain;(C) a third immunoglobulin chain comprising a third constant domain; and(D) a polypeptide comprising a third variable domain;wherein the first constant domain is a heavy chain constant domain that is unable to homodimerize; andwherein the first variable domain and the second variable domain are capable of associating to form an antigen-binding domain that specifically binds to the target antigen and wherein the first variable- 76 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)domain and the third variable domain are capable of associating to form a mispaired antigen-binding domain;(ii) comparing the binding affinity of the one or more antibody agents to the binding affinity measured in a control composition, wherein the control composition does not comprise the polypeptide;wherein the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of mispairing between the first and third variable domains, andwherein a decrease in binding affinity measured in the composition compared to the control composition indicates the detection of mispairing between the first and third variable domains.

56. The method of claim 55, wherein the first, second, and third immunoglobulin chains and the polypeptide are present in the composition as first, second, third, and fourth nucleic acid sequences encoding the first, second, and third immunoglobulin chains and polypeptide, respectively.

57. The method of claim 56, wherein the method further comprises the step of:expressing the first, second, and third immunoglobulin chains and polypeptide from the first, second, third, and fourth nucleic acid sequences, respectively, thereby producing the composition.

58. The method of claim 57, wherein expressing the first, second, and third, immunoglobulin chains and the polypeptide from the first, second, third, and fourth nucleic acid sequences, respectively, comprises transfecting a host cell with the first, second, third and fourth nucleic acid sequences and culturing the host cell under conditions such that the first, second, and third immunoglobulin chains and polypeptide are expressed.

59. The method of any one of claims 55-58, wherein the third constant domain comprises a heavy chain constant domain.

60. The method of claim 59, wherein the third constant domain comprises a CH2 domain and a CH3 domain.

61. The method of claim 59 or 60, wherein the third constant domain is unable to homodimerize.

62. The antibody variant of claim 61, wherein the third constant domain is unable to homodimerize because of one or more mutations.

63. The method of any one of claims 59-62, wherein the third immunoglobulin chain consists of the heavy chain constant domain.

64. The method of any one of claims 59-63, wherein the third immunoglobulin chain does not comprise a functional variable domain.

65. The method of any one of claims 59-62, wherein the third immunoglobulin chain comprises a third variable domain that does not associate with the first or second variable domains.- 77 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)66. The method of claim 65, wherein the third immunoglobulin chain comprises a VHH domain.

67. The method of claim 65, wherein the third immunoglobulin chain comprises a single-chain variable fragment (scFv).

68. The method of any one of claims 64-67, wherein the third immunoglobulin chain comprises fluorescent protein and / or a Tag.

69. The method of claim 68, wherein the fluorescent protein comprises a green fluorescent protein (GFP).

70. The method of any one of claims 55-69, wherein the first immunoglobulin chain and the third immunoglobulin chain heterodimerize to form an Fc domain.

71. The method of claim 70, wherein the first immunoglobulin chain and the third immunoglobulin chain heterodimerize as a result of a knob-in-hole (KIH) mutation.

72. A method comprising:(i) measuring the binding affinity of one or more antibody agents to a target antigen,wherein the one or more antibody agents are in a composition comprising:(A) a first immunoglobulin chain comprising a first variable domain and a first constant domain;(B) a second immunoglobulin chain comprising a second variable domain and a second constant domain; and(C) a third immunoglobulin chain comprising a third variable domain and a third constant domain; andwherein the first variable domain and the second variable domain associate to form an antigenbinding domain that specifically binds to the target antigen;wherein the first variable domain and the third variable domain associate to a mispaired antigenbinding domain;(ii) comparing the binding affinity of the one or more antibody agents to the binding affinity measured in a control composition, wherein the control composition does not comprise the third immunoglobulin chain, wherein the same or similar binding affinity measured in the composition compared to the control composition indicates no detection of mispairing between the first and third variable domains, andwherein a decrease in binding affinity measured in the composition compared to the control composition indicates detection of mispairing between the first and third variable domains.

73. The method of claim 72, wherein the composition further comprises:(D) a fourth immunoglobulin chain comprising a fourth variable domain and a fourth constant domain.- 78 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)74. The method of claim 73, wherein the third variable domain and the fourth variable domain associate to form an antigen-binding domain to a second target antigen.

75. The method of any one of claims 72-74, wherein the first, second, third, and fourth immunoglobulin chains are present in the composition as first, second, third, and fourth nucleic acid sequences encoding the first, second, third, and fourth immunoglobulin chains, respectively.

76. The method of claim 75, wherein the method further comprises the step of:expressing the first, second, third, and fourth immunoglobulin chains from the first, second, third, and fourth nucleic acid sequences, respectively, thereby producing the composition.

77. The method of claim 76, wherein expressing the first, second, third, and fourth immunoglobulin chains from the first, second, third, and fourth nucleic acid sequences, respectively, comprises transfecting a host cell with the first, second, third and fourth nucleic acid sequences and culturing the host cell under conditions such that the first, second, third, and fourth immunoglobulin chains are expressed.

78. The method of any one of claims 56-77, wherein the first, second, third and / or fourth nucleic acids are polyribonucleotides.

79. The method of claim 78, wherein the polyribonucleotides comprise a polyribonucleotide encoding each of the first, second, third, and fourth immunoglobulin chains.

80. The method of claim 78 or 79, wherein the polyribonucleotides are fully or partially encapsulated within lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

81. The method of any one of claims 56-80, wherein the first, second, third, and / or fourth nucleic acids comprise a DNA template.

82. The method of claim 81, wherein the DNA template is encoded in a vector (e.g., viral vectors, plasmid vectors, bacteriophage vectors, cosmids, phagemids, and / or artificial chromosomes).

83. The method of claim 82, wherein the vector comprises a viral vector.

84. The method of claim 83, wherein the viral vector comprises an adeno-associated viral (AAV) vector.

85. The method of any one of claims 56-77 and 81-84, wherein the first, second, third, and fourth nucleic acid sequences are comprised in a single multicistronic expression cassette.- 79 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)86. The method of any one of claims 55-85, wherein the method comprises a method of characterizing the risk of mispairing of immunoglobulin chains.

87. The method of any one of claims 55-86, wherein the composition comprises two or more antibody agents and the method characterizes the risk of mispairing between antibody variable domains of the antibody agents.

88. The method of any one of claims 55-85, wherein the method comprises a method of determining if immunoglobulin chains in a composition mispair.

89. The method of claim 88, wherein the composition comprises two or more antibody agents and the method determines if antibody variable domains of two different antibody agents in a composition mispair.

90. The method of any one of claims 55-85, wherein the method comprises a method of determining the suitability of one or more antibody agents for manufacture, administration, use as a therapeutic.

91. The method of claim 90, wherein the therapeutic comprises two or more antibody agents.

92. The method of claim 91, wherein the therapeutic comprises two or more antibody agents delivered as nucleic acids encoding the antibody agents.

93. The method of claim 90, wherein the therapeutic comprises a bispecific antibody.

94. The method of claim 93, wherein the bispecific antibody is delivered as a nucleic acid encoding the bispecific antibody.

95. The method of any one of claims 56-94, wherein the composition comprises a relative amount of first nucleic acid compared to the third nucleic acid is between about 1:0.1 to about 1:100.

96. The method of any one of claims 56-95, wherein measuring binding affinity comprises performing an enzyme-linked immunosorbent assay (ELISA).

97. The method of claim 96, wherein the ELISA comprises a plate pre-coated with target antigen.

98. The method of claim 96 or 97, wherein the ELISA further comprises a detection antibody.

99. The method of claim 98, wherein the detection antibody comprises an anti-human Fc antibody linked to an enzyme.

100. The method of claim 99, wherein the enzyme comprises horseradish peroxidase (HRP).- 80 - 13276319vlAttorney Docket No.: 2013237-1583 (BNT REF: P2042W01)101. The method of claim 100, wherein measuring binding affinity comprises measuring color change produced from a reaction between the enzyme and a substrate added to the sample.- 81 - 13276319vl

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