Humanized Anti-CD3 binding molecules and uses thereof

Abstract

Described herein are humanized anti-CD3 antibodies, antigen binding fragments, and binding polypeptides including such binding domains, as well as nucleic acids encoding the same. These binding constructs bind to CD3 epsilon. The disclosure also features methods of making and using such humanized antibodies, antigen binding fragments, and binding polypeptides to treat various diseases such as cancer.

Description

[0001] HUMANIZED ANTI-CD3 BINDING MOLECULES AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of priority of U.S. Provisional Appl. No. 63 / 538,939 filed September 18, 2023, the contents of which are incorporated by reference herein in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 16, 2024, is named 45817-0160W01_SL.xml and is 106,006 bytes in size.

[0006] BACKGROUND

[0007] Cluster of differentiation 3 (CD3) is a multimeric protein complex, also known as the T3 complex, and comprises four distinct polypeptide chains, epsilon (s), gamma (y), delta (8) and zeta (Q, which assemble and function as three pairs of dimers (sy, s8, Q. The CD3 complex serves as a T cell co-receptor that associates non- covalently with the T cell receptor (TCR). The CD3-TCR complex plays an important role in, for example, the T cell-mediated immune response as it is involved in the recognition of antigens and subsequent signal transduction and activation of immunocompetent T lymphocytes. CD3 is thus an important therapeutic target for activation of T cells and targeting tumor cells. Accordingly, there remains a need for antibodies, antigen binding fragments, and binding proteins that bind to CD3 (e.g., with reduced immunogenicity compared to a reference or no immunogenicity) and nucleic acids encoding the same. SUMMARY

[0008] The present disclosure provides, inter alia, a binding molecule that comprises an antibody, antigen-binding fragment, and binding polypeptide(s) that binds CD3 and nucleic acids encoding the same. Such binding molecules, antibodies, antigen binding fragments, and nucleic acids (e.g., mRNA) encoding such binding proteins are useful in, for example, activating T cells and methods of treating diseases and / or disorders.

[0009] In one aspect, the disclosure features a binding molecule comprising a polypeptide or polypeptides that bind CD3 epsilon. The polypeptide or polypeptides comprise a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL), wherein the VH comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences set forth in SEQ ID NOs:2-4 and the VL comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences set forth in SEQ ID NOs:6-15; wherein the VH comprises the VH CDR 1, 2, and 3 of SEQ ID NO:2 and the VL comprises the VL CDR 1, 2, and 3 of SEQ ID NO:6. The VH does not comprise the amino acid sequence set forth in SEQ ID NO: 1 and the VL does not comprise the amino acid sequence set forth in SEQ ID NO: 5 as these are not humanized VH and VL. Also, the VH and VL are also not identical to any anti- CD3 binding VH and / or VL sequence listed in WO 2007 / 0422261, US 2017 / 0355767, US 10407501, US 9587021, or US 8236308.

[0010] In another aspect, the disclosure features a binding molecule comprising a polypeptide or polypeptides that bind CD3 epsilon. The polypeptide or polypeptides comprise a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL), wherein the VH comprises an amino acid sequence that is identical to any one of the amino acid sequences set forth in SEQ ID NOs:2-4 except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions therein and the VL comprises an amino acid sequence that is identical to any one of the amino acid sequences set forth in SEQ ID NOs:6-15 except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions therein, The VH comprises the VH CDR 1, 2, and 3 of SEQ ID NO:2 and the VL comprises the VL CDR 1, 2, and 3 of SEQ ID NO:6. The VH does not comprise the amino acid sequence set forth in SEQ ID NO: 1 and the VL does not comprise the amino acid sequence set forth in SEQ ID NO: 5 as these are not humanized VH and VL. Also, the VH and VL are also not identical to any anti-CD3 binding VH and / or VL sequence listed in WO 2007 / 0422261, US 2017 / 0355767, US 10407501, US 9587021, or US 8236308.

[0011] In some instances, the polypeptide or polypeptides bind human CD3 epsilon. In some instances, the polypeptide or polypeptides bind both human and cynomolgus CD3 epsilon.

[0012] In some instances, the humanized VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NOs: 2, 3 or 4, and the humanized VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 6 to 15. In one case, the humanized VH comprises an amino acid sequence that is identical to the sequence set forth in SEQ ID NO: 2 except for 1, 2, 3, or 4 amino acid substitutions in the framework regions, and the humanized VL comprises an amino acid sequence that is identical to the sequence set forth in SEQ ID NO: 6 except for 1, 2, 3, or 4 amino acid substitutions in the framework regions. In another case, the humanized VH comprises an amino acid sequence that is at identical to the sequence set forth in SEQ ID NO: 4 except for 1, 2, 3, or 4 amino acid substitutions in the framework regions, and the humanized VL comprises an amino acid sequence that is identical to the sequence set forth in SEQ ID NO: 6 except for 1, 2, 3, or 4 amino acid substitutions in the framework regions.

[0013] In some instances, the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO:6; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 7; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 8; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NOV; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 10; the humanized VH comprises an amino acid sequence of SEQ ID NO: 2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 11 ; the humanized VH comprises an amino acid sequence of SEQ ID NO: 2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 12; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 13; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 14; the humanized VH comprises an amino acid sequence of SEQ ID NO:2 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 15; the humanized VH comprises an amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises an amino acid sequence of SEQ ID NO:6; the humanized VH comprises an amino acid sequence of SEQ ID NO:3 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 7; the humanized VH comprises an amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 8; the humanized VH comprises an amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises an amino acid sequence of SEQ ID NOV; the humanized VH comprises an amino acid sequence of SEQ ID NOV and the humanized VL comprises an amino acid sequence of SEQ ID NOVO; the humanized VH comprises an amino acid sequence of SEQ ID NOV and the humanized VL comprises an amino acid sequence of SEQ ID NO: 11; the humanized VH comprises an amino acid sequence of SEQ ID NOV and the humanized VL comprises an amino acid sequence of SEQ ID NO: 12; the humanized VH comprises an amino acid sequence of SEQ ID NOV and the humanized VL comprises an amino acid sequence of SEQ ID NO: 13; the humanized VH comprises an amino acid sequence of SEQ ID NOV and the humanized VL comprises an amino acid sequence of SEQ ID NO: 14; the humanized VH comprises an amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 15; the humanized VH comprises an amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises an amino acid sequence of SEQ ID NO:6; the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NO:7; the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 8; the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NOV; the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 10; the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 11; the humanized VH comprises an amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 12; the humanized VH comprises an amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 13; the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 14; or the humanized VH comprises an amino acid sequence of SEQ ID NO:4 and the humanized VL comprises an amino acid sequence of SEQ ID NO: 15.

[0014] In certain instances, the humanized VH further comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 55. In some cases, this amino acid sequence is linked at the C-terminus of the humanized VH. In some cases, the humanized VH further comprises an amino acid sequence that is identical to the sequence set forth in SEQ ID NO:55 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some cases, this amino acid sequence is linked at the C-terminus of the humanized VH. In some instances, the humanized VH further comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 56. In some cases, this amino acid sequence is at the C-terminus of the CHI domain of the humanized VH. In some instances, the humanized VH further comprises an amino acid sequence that is identical to the sequence set forth in SEQ ID NO: 56 except for 1, 2, 3, or 4 amino acid substitutions. In some cases, this amino acid sequence is at the C-terminus of the CHI domain of the humanized VH.

[0015] In some instances, the humanized VL further comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 57. In some cases, this sequence is attached or linked at the C-terminus of the humanized VL. In some cases, the humanized VL further comprises an amino acid sequence that is identical to the sequence set forth in SEQ ID NO:57 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some cases, this sequence is attached or linked at the C-terminus of the humanized VL.

[0016] In certain instances, the binding molecule comprises: the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:21; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:22; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:23; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:24; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:25; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:26; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:27; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:28; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:29; the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 30; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:21; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:22; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:23; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:24; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:25; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:26; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:27; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:28; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:29; the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO: 30; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:21; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:22; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:23; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:24; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:25; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:26; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:27; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:28; the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:29; or the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:30.

[0017] In some instances, the polypeptide or polypeptides is a bivalent whole antibody, a multispecific antibody, a bispecific antibody, an antigen-binding fragment, a single-chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, a tandem scFv (taFv), or a fusion protein. In one case, the polypeptide or polypeptides is a Fab fragment. In another case, the polypeptide or polypeptides is a scFv.

[0018] In certain instances, the polypeptide or polypeptides that bind to CD3 epsilon is part of a bispecific antibody comprising a second binding domain that specifically binds a second antigen. In some cases, the polypeptide or polypeptides that bind to CD3 epsilon is a Fab.

[0019] In some instances, the binding molecule further comprises an agent or moiety selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a half-life extension moiety, or a moiety that facilitates the binding molecule crossing the blood brain barrier.

[0020] In some instances, the polypeptide or polypeptides bind CD3 epsilon with a KD of 100 nM or less. In some instances, the polypeptide or polypeptides bind CD3 epsilon with a KD of 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, or 0.01 nM or less. In some instances, the polypeptide or polypeptides bind CD3 epsilon with a konof from 103M^s'1to 105M^s'1. In certain instances, the polypeptide or polypeptides bind CD3 epsilon with a koffof from 10'3M^s'1to 10'5M^s'1.

[0021] In certain instances, the polypeptide or polypeptides are conjugated to a biologic. In some cases, the polypeptide or polypeptides are conjugated to the biologic via a peptide linker or chemical linker. In some cases, the biologic is an antibody, a cytokine, a growth factor, an enzyme, a polypeptide, a protein, a carbohydrate, or a nucleic acid.

[0022] In another aspect, the disclosure provides a binding molecule comprising a humanized Fab that specifically binds CD3E (e.g., human CD3E or human and cyno CD3E) comprising a first amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or 4; and a second amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, wherein the humanized Fab comprises the six CDRs based on any CDR definition set forth in Table 2, and wherein the humanized Fab does not comprise any VH or VL sequence listed in WO 2007 / 0422261, US 2017 / 0355767, US 10407501, US 95870 1, or US 8236308. In one case, the binding molecule comprises a humanized Fab comprising a first amino acid sequence that is 100% identical to SEQ ID NO: 2 and a second amino acid sequence that is 100% identical to SEQ ID NO: 6. In another case, the binding molecule comprises a humanized Fab comprising a first amino acid sequence that is 100% identical to SEQ ID NO: 4 and a second amino acid sequence that is 100% identical to SEQ ID NO: 6. In certain instances, the VH of the Fab is linked at its C- terminal to a human Ig CHI. In certain instances, the VH of the Fab is linked at its C- terminal to a human Ig CHI (e.g., human IgGl, IgG2, IgG3, or IgG4 CHI) and to the upper hinge region of a human Ig (e.g., human IgGl, IgG2, IgG3, or IgG4 upper hinge region) at the C-terminus of the CHI. In one case, the binding molecule comprises a first amino acid sequence that is 100% identical to SEQ ID NO: 17 and a second amino acid sequence that is 100% identical to SEQ ID NO:21. In another case, the binding molecule comprises a first amino acid sequence that is 100% identical to SEQ ID NO: 19 and a second amino acid sequence that is 100% identical to SEQ ID NO:21. In some cases, the binding molecule can include another antigen-binding polypeptide that binds to a different antigen (i.e., an antigen other than CD3). In some cases, the different antigen is a tumor antigen. In some cases, the another antigen-binding polypeptide is an Fab. In other cases, the another antigen-binding polypeptide is a scFv. In still other cases, the another antigen-binding polypeptide is a whole antibody.

[0023] In another aspect, the disclosure provides a binding molecule comprising a humanized scFv that specifically binds CD3E (e.g., human CD3E, or human and cyno CD3E) comprising a first amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or 4; and a second amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, wherein the humanized scFv comprises the six CDRs based on any CDR definition set forth in Table 2, and wherein the humanized scFv does not comprise any VH or VL sequence listed in WO 2007 / 0422261, US 2017 / 0355767, US 10407501, US 9587021, or US 8236308, and wherein the first amino acid sequence and the second amino acid sequence of the humanized scFv are linked by a linker (peptide or chemical). In some instances the linker is a glycine-serine linker such as (648)3 (SEQ ID NO:67). In one case, the binding molecule comprises a humanized scFv comprising a first amino acid sequence that is 100% identical to SEQ ID NO: 2 and a second amino acid sequence that is 100% identical to SEQ ID NO: 6. In another case, the binding molecule comprises a humanized scFv comprising a first amino acid sequence that is 100% identical to SEQ ID NO: 4 and a second amino acid sequence that is 100% identical to SEQ ID NO: 6. In some cases, the binding molecule can include another antigen-binding polypeptide that binds to a different antigen (i.e., an antigen other than CD3). In some cases, the different antigen is a tumor antigen. In some cases, the another antigen-binding polypeptide is an Fab. In other cases, the another antigen-binding polypeptide is a scFv. In still other cases, the another antigen-binding polypeptide is a whole antibody.

[0024] In another aspect, the disclosure provides a binding molecule comprising a humanized whole antibody (i.e., comprising two heavy chains and two light chains) that specifically binds CD3E (human CD3E or human and cyno CD3E) comprising a first amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or 4; and a second amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, wherein the humanized whole antibody comprises the six CDRs based on any CDR definition set forth in Table 2, and wherein the humanized whole antibody does not comprise any VH or VL sequence listed in WO 2007 / 0422261, US 2017 / 0355767, US 10407501, US 9587021, or US 8236308. In one case, the binding molecule comprises a humanized whole antibody comprising a first amino acid sequence that is 100% identical to SEQ ID NO: 2 and a second amino acid sequence that is 100% identical to SEQ ID NO:6. In another case, the binding molecule comprises a humanized whole antibody comprising a first amino acid sequence that is 100% identical to SEQ ID NO: 4 and a second amino acid sequence that is 100% identical to SEQ ID NO: 6. In some cases, the binding molecule can include another antigen-binding polypeptide that binds to a different antigen (i.e., an antigen other than CD3). In some cases, the different antigen is a tumor antigen. In some cases, the another antigen-binding polypeptide is an Fab. In other cases, the another antigen-binding polypeptide is a scFv. In still other cases, the another antigen-binding polypeptide is a whole antibody.

[0025] In another aspect, the disclosure features a multispecific molecule comprising a means for binding human CD3 epsilon, and a binding molecule that specifically binds to another antigen. In some cases, the another antigen is a tumor associated antigen. In some cases, the another antigen-binding polypeptide is an Fab. In other cases, the another antigen-binding polypeptide is a scFv. In still other cases, the another antigen-binding polypeptide is a whole antibody. In some cases, the multispecific molecule is a bispecific binding molecule.

[0026] In another aspect, the disclosure provides a pharmaceutical composition comprising a binding molecule described herein, and a pharmaceutically acceptable carrier.

[0027] In another aspect, the disclosure features a pharmaceutical composition comprising a means for binding human CD3 epsilon, and a pharmaceutically acceptable carrier.

[0028] In another aspect, provided herein is a nucleic acid or nucleic acids encoding a binding molecule described herein. Also provided is a vector or vectors comprising the nucleic acid or nucleic acids described herein. Also featured herein is a host cell comprising the nucleic acid or nucleic, or vector or vectors described herein.

[0029] In another aspect, this disclosure provides a method of making a binding molecule described herein. The method comprises culturing a host cell comprising the nucleic acid or nucleic, or vector or vectors described herein under conditions that facilitate expression of the binding molecule, and isolating the binding molecule. The method can further involve formulating the binding molecule as a sterile pharmaceutical composition.

[0030] Also featured is a method of treating a cancer or CD3 -related disease or disorder in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of the binding molecule or the pharmaceutical composition of claim described herein. In some instances, the cancer is T-ALL In some instances, the cancer is ovarian cancer. In some cases, the CD3 -related disease is Type I diabetes. In certain instances, the CD3 -related disease is an autoimmune disease. In some cases, the CD3 related disease is multiple myeloma (e.g., relapsing MM or refractory MM). In certain cases, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, Crohn’s disease, systemic sclerosis, or acute respiratory distress syndrome.

[0031] In another aspect the disclosure features an mRNA or mRNAs that encode a binding molecule described above.

[0032] In a further aspect, the disclosure features a polynucleotide or polynucleotides comprising an mRNA or mRNAs comprising a 5' UTR; an open reading frame (ORF) or ORFs encoding a binding molecule described herein; a stop codon; and a 3' UTR.

[0033] In one aspect, the disclosure features a 5 ’UTR, a polynucleotide or polynucleotides comprising means for encoding a human CD3 epsilon binding molecule, a stop codon, and a 3 ’UTR.

[0034] In some instances, the mRNA or mRNAs comprises a microRNA (miR) binding site. In some cases, the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and / or TLR8 and secretes pro-inflammatory cytokines and / or chemokines. In certain cases, the microRNA binding site is for a microRNA selected from miR- 122, miR- 126, miR- 142, miR- 144, miR- 146, miR- 150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof. In some cases, the microRNA binding site is for a microRNA selected from miR122-5p, miR126-3p, miR-142-3p, miR-142-5p, miR-155, or any combination thereof. In certain cases, the microRNA binding site is located in the 3' UTR of the mRNA or mRNAs.

[0035] In some instances, the 5' UTR comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 73. In certain instances, the 3' UTR comprises a nucleic acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 92.

[0036] In some instances, the mRNA or mRNAs comprises a 5' terminal cap. In certain cases, the 5' terminal cap comprises a CapO, Capl, ARC A, inosine, N1 -methylguanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof. In one instance, the 5 ’terminal cap comprises m7GpppGm.

[0037] In certain instances, the mRNA or mRNAs comprise a 3’ trailing sequence of linked nucleosides. In some cases, the 3’ trailing sequence of linked nucleosides is a poly-adenylate (poly- A) tail or a poly A-G quartet. In some cases, the 3’ trailing sequence of linked nucleosides is a poly-A tail and the poly-A tail is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 nucleotides in length, or at least about 100 nucleotides in length. In certain cases, the 3’ trailing sequence of linked nucleosides is a poly-A tail and the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length. In one instance, the poly A tail is about 100 nucleotides in length.

[0038] In some instances, the mRNA or mRNAs comprise at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. In some cases, the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (y), N1 -methylpseudouracil (ml\| / ), 1 -ethylpseudouracil, 2-thiouracil (s2U), 4’ -thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof. In some cases, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1 -methylpseudouracils. In one case, 100% of the uracils are N1 -methylpseudouracils. In some cases, the open reading frame consists of nucleosides selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine. In certain cases, the modified uridine is 1 -methylpseudouridine. In some cases, the mRNA or mRNAs comprise a 5 ’terminal cap comprising m7GpppGm and a poly-A region 100 nucleotides in length. In certain cases, all uracils of the polynucleotide are Nl- methylpseudouracils.

[0039] In another aspect, the disclosure provides a pharmaceutical composition comprising a polynucleotide or polynucleotides described herein, and a pharmaceutically acceptable carrier.

[0040] In yet another aspect, the disclosure features a lipid nanoparticle comprising an mRNA or mRNAs or polynucleotide or polynucleotides described herein. The LNP encapsulates the mRNA or mRNAs or polynucleotide or polynucleotides. In certain cases, the lipid nanoparticle has a mean particle size of from 80 nm to 160 nm. In some cases, wherein the lipid nanoparticle has a polydispersity index (PDI) of from 0.02 to 0.2 and / or a lipid: nucleic acid ratio of from 10 to 20. In certain cases, the lipid nanoparticle comprises a phospholipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and a sterol. In some cases, the phospholipid is l,2-distearoyl-sn-glycero-3- phosphocholine. In certain cases, the ionizable amino lipid is a compound of Formula (I). In some cases, the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99, 102, 105, 108, 111 , 114, 117, 120, 123, 126, 129, 132- tetratetracontaoxatetratriacontahectyl stearate.

[0041] . In certain cases, the sterol is cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7- dehydrocholesterol, 5 -dehydroepisterol, 7-dehydrositosterol, 20a,22R- dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7a-hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol, oxysterol, parkeol, saringosterol, spinasterol, sterol ester, trametenolic acid, zhankuic acid, or zymosterol. In a particular case, the sterol is cholesterol.

[0042] In one particular case, the LNP comprises a phospholipid that is 1 ,2-distearoyl-sn- gly cero-3 -phosphocholine, an ionizable amino lipid that is: 9 and a sterol that is cholesterol.

[0043] In another aspect, the disclosure features a vector or vectors comprising a polynucleotide or polynucleotides described herein.

[0044] In yet another aspect, the disclosure features a host cell comprising a polynucleotide or polynucleotides described herein, or the vector or vectors described herein. In some cases, the host cell is a eukaryotic cell. In certain cases, the eukaryotic cell is a mammalian cell. In some cases, the mammalian cell is a CHO cell or a HEK cell.

[0045] In a further aspect, the disclosure features a method of treating a cancer or CD3- related disease or disorder in a human subject in need thereof. The method involves administering to the human subject a therapeutically effective amount of the pharmaceutical composition (e.g., a polypeptide, or polynucleotide or polynucleotides (e.g., an mRNA or mRNAs), or an LNP comprising a nucleic acid payload) described herein. In some instances, the cancer is T-ALL. In some instances, the cancer is ovarian cancer. In some instances, the cancer is a solid tumor. In some cases, the CD3 -related disease is Type I diabetes. In certain instances, the CD3-related disease is an autoimmune disease. In some cases, the CD3 -related disease is multiple myeloma (e.g., relapsing MM or refractory MM). In certain cases, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, Crohn’s disease, systemic sclerosis, or acute respiratory distress syndrome.

[0046] Also provided is a kit comprising (i) a binding molecule, a mRNA or mRNAs, a polynucleotide or polynucleotides, a pharmaceutical composition (e.g., a polypeptide or a delivery vehicle, such as an a LNP, comprising a nucleic acid payload described herein), and (ii) a package insert instructing a user of the kit to administer the binding molecule; the pharmaceutical composition; to a human subject in need thereof. In certain instances, the human subject has or is at risk of developing cancer (e.g., a solid tumor such as ovarian cancer). In other instances, the human subject has an autoimmune disease. In certain cases, the autoimmune disease is Type I diabetes, rheumatoid arthritis, ulcerative colitis, Crohn’s disease, systemic sclerosis, or acute respiratory distress syndrome. In some cases, the human subject has multiple myeloma (e.g., relapsing MM or refractory MM).

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 shows western blot analysis to confirm expression of 44 Fab variants (including wild type VH and VL) after they were transiently transfected into an EXPL CHO cell line.

[0049] FIG. 2 shows results of SDS-PAGE and size exclusion chromatography (SEC) profile of five Fab constructs. FIG. 3 shows results of FACS binding titration to CD3+Jurkat cells.

[0050] FIG.4 is an alignment of the amino acid sequences of humanized SP34 VH and VL constructs with the amino acid sequences of murine VH and VL of parental SP34. The VH sequences from top to bottom are SEQ ID NOs.: 1-4 and the VL sequences from top to bottom are SEQ ID NOs. : 5-15. SEQ ID NO: 1 is the amino acid sequence of the VH of parental SP34 and SEQ ID NO:5 is the amino acid sequence of the VL of parental SP34. The amino acid sequences that are different between the sequences are underlined.

[0051] DETAILED DESCRIPTION

[0052] The compositions and methods of this disclosure feature humanized antibodies, antigen binding fragments, and binding polypeptides that bind to CD3 (e.g., with reduced immunogenicity or no immunogenicity compared to the reference parental SP34 antibody) as well as binding molecules comprising any of these humanized antibodies, antigen binding fragments, and binding polypeptides. The disclosure also provides polynucleotides (such as mRNA) encoding the disclosed binding constructs, and methods of using such humanized antibodies, antigen binding fragments, binding polypeptides, and polynucleotides. In some embodiments, the antibodies, antigen binding fragments, or binding polypeptides that bind to CD3 is part of a multispecific or bispecific antibody comprising a second binding domain that specifically binds a second antigen(s) (e.g., a tumor associated antigen(s)). In some embodiments, the antibodies are full-length antibodies that include a pair of heavy chains and a pair of light chains, each containing a variable domain and a constant region. In some embodiments, the antibodies are single chain Fv (scFv) molecules. In some embodiments, CD3-binding fragments are binding molecules comprising Fab. The above-described binding constructs can bind human CD3E or human and cynomolgus CD3 epsilon.

[0053] The compositions and methods of the disclosure exhibit a series of beneficial biochemical properties. For example, antibodies, antigen binding fragments, and / or binding polypeptides are capable of binding CD3 (e.g., CD3 epsilon) with high affinity and / or can carry a lower risk for inducing immune responses in humans (e.g., due to humanization).

[0054] Definitions

[0055] As used herein, the term “about” refers to a stated numerical term and a value that is no more than 10% above or below the value being described. For example, the term “about 5 nM” indicates disclosure of both the stated value of 5 nM and a range of from 4.5 nM to 5.5 nM.

[0056] “% identity” is the percentage of identical residues between two or more sequences and is determined over the full length of the sequences being compared. It is a function of the number of amino acids or nucleotide residues that are identical in the sequences being compared. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment of the sequences. The percentage of sequence identity is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for calculation of the percentage of sequence identity. Alignment for purposes of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs. For example, the BLAST program (Tatiana t al., FEMS Microbiol. Lett., 174:247-250 (1999)) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, can be used to obtain an optimal alignment of amino acid or nucleic acid sequences to calculate the percentage of sequence identity. CD3

[0057] Cluster of Differentiation 3 (CD3) is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+naive T cells) and T helper cells (CD4+naive T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD38 chain, and two CD3s chains. These chains associate with the T-cell receptor (TCR) and the CD3-zeta (^-chain) to generate an activation signal in T lymphocytes. The TCR, CD3-zeta, and the other CD3 molecules together constitute the TCR complex.

[0058] As part of the TCR-CD3 complex, CD3 epsilon is present on the surface of T- lymphocyte and plays an essential role in the adaptive immune response. When antigen presenting cells (APCs) activate T-cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains - i.e., CD3 delta (CD3D), CD3 epsilon (CD3E), CD3 gamma (CD3G) and CD3 zeta (CD3Z). All the CD3 chains contain immunoreceptor tyrosine-based activation motifs (IT AMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways. In addition to this role in signal transduction in T-cell activation, CD3E plays an essential role in T-cell development. It initiates the TCR-CD3 complex assembly by forming the two heterodimers CD3D / CD3E and CD3G / CD3E. It also participates in internalization and cell surface down-regulation of TCR-CD3 complexes via endocytosis sequences present in CD3E cytosolic region.

[0059] The amino acid sequence of mature human CD3E is provided below: QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIG SDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMS VATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPV PNPDYEP1RKGQRDLYSGLNQRRI (SEQ ID NO:31) The amino acid sequence of mature cynomolgus CD3E is provided below: QDGNEEMGSITQTPYQVSISGTT TLTCSQHI.GSEAQWQHNGKNKEDSGDRLFLP EFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVTVDI CITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEP1R KGQQDLYSGLNQRRI (SEQ ID NO:32)

[0060] Defining the Antigen Binding Regions of CD3 Antibodies

[0061] This disclosure features antibodies, antigen-binding fragments thereof, and binding polypeptides that bind CD3. In some instances, the antibodies, antigen-binding fragments thereof, and binding polypeptides bind CD3 epsilon. In some instances, the antibodies, antigen-binding fragments thereof, and binding polypeptides bind human CD3 epsilon. In some instances, the antibodies, antigen-binding fragments thereof, and binding polypeptides bind both human and cynomolgus CD3 epsilon.

[0062] The antibodies, antigen-binding fragments thereof, and binding polypeptides of this disclosure are related to the anti-CD3E antibody, SP34. See, e.g., US20170355767A1, which is incorporated by reference herein. More specifically, this disclosure relates to humanized versions of the parental murine SP34 antibody.

[0063] The amino acid sequence of the heavy chain variable domain (VH) of the anti- CD3E antibody, SP34, is provided below (VH CDRsl-3 are in bold and based on IMGT definition):

[0064] EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRS KYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFG NSYVSWFAYWGQGTLVTVSA (SEQ ID NO: 1)

[0065] The amino acid sequence of the light chain variable domain (VL) of the anti- CD3E antibody, SP34, is provided below (VL CDRsl-3 are based on IMGT definition): QAWTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTV L (SEQ ID NO: 5) Table 1 provides the six CDR sequences of SP34 based on common CDR definitions known in the art.

[0066] Table 1: CDR Sequences of SP34

[0067] This disclosure features binding molecules that comprise polypeptide or polypeptides that bind CD3, and more specifically

[0068] 5 CD3E (human CD3E or human and cyno CD3E). Provided herein are antibodies, antigen-binding fragments, and binding polypeptides

[0069] that bind CD3 and comprise the six CDRs, or the VH and VL of humanized SP34. Table 2 provides the six CDR sequences of the humanized SP34 binding molecules based on common CDR definitions known in the art.

[0070] Table 2: CDR Sequences of Humanized SP34

[0071] 5

[0072] This disclosure relates to a binding molecule that comprises antibodies, antigenbinding fragments, and binding polypeptide(s) that bind CD3 (e.g., human and / or cyno CD3) comprising the six CDRs of a humanized SP34 described herein. In some cases, these antibodies, antigen-binding fragments, and binding polypeptides bind CD3E (e.g., human and / or cyno CD3E). In some instances, the antibodies, antigen-binding fragments, and binding polypeptides comprise the six CDRs of a humanized SP34 based on the Kabat definition. In some instances, the antibodies, antigen-binding fragments, and binding polypeptides comprise the six CDRs of a humanized SP34 based on the Chothia definition. In some instances, the antibodies, antigen-binding fragments, and binding polypeptides comprise the six CDRs of a humanized SP34 based on the enhanced Chothia definition. In some instances, the antibodies, antigen-binding fragments, and binding polypeptides comprise the six CDRs of a humanized SP34 based on the contact definition. In some instances, the antibodies, antigen-binding fragments, and binding polypeptides comprise the six CDRs of a humanized SP34 based on the IMGT definition.

[0073] In some cases, the VH and VL CDRs in Table 2 can include any of the amino acid substitutions of any one or more of SEQ ID NOs.: 54 or 72 to 83 listed in Table 1 of US 2017 / 0355767, which is incorporated by reference herein.

[0074] This disclosure also relates to antibodies, antigen-binding fragments, and binding polypeptides that bind CD3 (e.g., human and / or cyno CD3) comprising the VH and VL of a humanized SP34 described herein. In some cases, these antibodies, antigen-binding fragments, and binding polypeptides bind CD3E (e.g., human and / or cyno CD3E). Table 3 provides the amino acid sequences of exemplary humanized VH and VL of humanized SP34.

[0075] Table 3: Humanized VH and VL sequences

[0076]

[0077] In some cases, the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in any one of SEQ ID NOs.:2-4. In some cases, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs.:2-4, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in any one of SEQ ID NOs.:6-15. In some cases, the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs.:6-15, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some instances, the variability in the VH and VL when present is in the framework regions and not in the CDRs. The VH and VL are not any one of the anti-CD3E binding VH or VL described in WO 2007 / 042261, US 2017 / 0355767, US 10407501 , US 9587021 , or US 8236308. For example, this disclosure does not encompass binding molecules comprising the VHs set forth in SEQ ID NOs: 6-9 or the VLs set forth in SEQ ID NOs: 10-12 of US 2017 / 0355767. The VH and VL are also not any of the anti-CD3E binding humanized VH and VL known in the art.

[0078] In some instances, the C-terminal of the VH is linked directly or via a linker (e.g., peptide, chemical) to a CHI of a human immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4). In some cases, the CHI comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:55).

[0079] In some cases, the CHI comprises an amino acid sequence that differs from SEQ ID NO:55 at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions a result of amino acid substitutions and / or deletions.

[0080] In some cases, the C-terminal of the CHI is linked to an upper hinge region of a human immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4). In some cases, the upper hinge region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence: EPKSCDKTHT (SEQ ID NO: 56). In some cases, the upper hinge comprises an amino acid sequence that differs from SEQ ID NO: 56 at 0, 1, 2, 3, or 4 positions a result of amino acid substitutions and / or deletions.

[0081] In some instances, the VL is linked directly or via a linker (e.g., peptide, chemical) to a CL of a human immunoglobulin (human lambda or human kappa). In some cases, the CL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence:

[0082] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC (SEQ ID NO: 57).

[0083] In some cases, the CL comprises an amino acid sequence that differs from SEQ ID NO:57 at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions a result of amino acid substitutions and / or deletions. In some cases, the CL comprises an S or SG at the C-terminus of SEQ ID NO: 57 Non-limiting examples of such CD3E binding polypeptides are provided in Table

[0084] 4 below.

[0085] Table 4: Humanized VH:CHl:Upper Hinge and VL:CL sequences

[0086]

[0087]

[0088]

[0089] In some cases, the VL constructs in Table 4 may each include a C-terminal glycine (G) following the last C-terminal serine.

[0090] In some cases, the VH:CH1 :upper hinge construct comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in any one of SEQ ID NOs.: 17-19. In some cases, the VH: CHI: upper hinge construct comprises an amino acid sequence set forth in any one of SEQ ID NOs. : 17- 19, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ammo acid substitutions. In some cases, the VL:CL construct comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequence set forth in any one of SEQ ID NOs.:21-30. In some cases, the VL:CL construct comprises an amino acid sequence set forth in any one of SEQ ID NOs.:21-30, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ammo acid substitutions. The variability in the VH:CH1 : upper hinge and VL when present is generally in the framework regions, constant region and / or hinge region and not in the CDRs. The VH and VL of these constructs are not any one of the anti-CD3E binding VH or VL described in WO 2007 / 042261 or US 2017 / 0355767. The VH and VL of these constructs are also not any of the anti-CD3E binding humanized VH and VL known in the art.

[0091] CD3E Binding Molecules

[0092] This disclosure also features binding molecules that comprise any of the polypeptide or polypeptides described above.

[0093] In some instances, the CD3E (human CD3E, or human and cyno CD3E) binding polypeptide or polypeptides described above are a humanized whole antibody (i.e., comprising two arms each having a heavy chain and a light chain), an antigen binding fragment thereof, or a binding polypeptide(s). In some cases, the polypeptide or polypeptides are selected from the group consisting of: a single-chain Ev molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a Fv fragment, a Fab domain or region, a F(ab’)2 molecule, a tandem scFv (taFv), or a fusion protein.

[0094] In certain cases, the disclosure provides a whole antibody that binds human CD3E or human and cyno CD3E. In certain cases the whole antibody is monospecific (i.e., it binds to human CD3E or human and cyno CD3E, but not another antigen). In some cases, the antibody comprises the hinge and Fc region of a human immunoglobulin such as human IgGl, human IgG2, human IgG3, or human IgG4. The constant regions can be engineered to reduce or improve effector function as needed. The hinge region can also be modified to reduce proteolytic cleavage, prevent or reduce arm shuffling, or otherwise improve the properties of the antibody. In some cases, the hinge and Fc region of IgG4PAA is employed.

[0095] In some cases, the disclosure features a scFv comprising a VH and a VL that binds human CD3E or human and cyno CD3E. The VH and VL can be linked together as VH-linker-VL or VL-linker-VH. The linker can be a peptide linker or a chemical linker. In some cases, the linker is a glycine serine linker such as (GGGGS)n wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:63). Non-limiting examples of other linkers suitable for linking the polypeptides of this disclosure include, but are not limited to, (GS)n (SEQ ID NO: 105), (GGS)n (SEQ ID NO: 106), (GGGS)n (SEQ ID NO:64), (GGSG)n (SEQ ID NO:65), or (GGSGG)n (SEQ ID NO:66), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some instances the length of the linker can be about 15 amino acids. In one instance the linker is (G4S)3(SEQ ID NO:67).

[0096] In some case, the polypeptide or polypeptides is a Fab region or domain that specifically binds CD3E. In some cases, the Fab may include the upper hinge region of a human immunoglobulin (e.g., human IgGl, human IgG2, human IgG3, human IgG4), or portions thereof. The upper hinge region can be modified to reduce proteolytic cleavage or otherwise improve the properties of the antibody.

[0097] The above constructs each comprises the six CDRs according to any one CDR definition shown in Table 2. In some cases, the VH and VL CDRs in Table 2 can include any of the amino acid substitutions of any one or more of SEQ ID NOs.: 54 or 72 to 83 listed in Table 1 of US 2017 / 0355767, which is incorporated by reference herein. In addition, these constructs comprise a VH comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any sequence set forth in SEQ ID NOs:2-4 and a VL comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any n sequence set forth in SEQ ID NOs:6-l 5. None of these VH and VL comprise any of the anti-CD3 binding VH or VL domains set forth in SEQ ID NO: 1 and 5 of this disclosure, or any described in WO 2007 / 042261 or US 2017 / 0355767. The VH and VL of these constructs are also not any of the anti-CD3E binding humanized VH and VL already known in the art. In some instances, the Fab that binds human CD3E or human and cyno CD3E comprises a VH-comprising region and a VL-comprising region each of which is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any sequence set forth in Table 4. In certain instances, the disclosure features a binding molecule comprising a Fab that binds both human and cyno CD3E and comprises the amino acid sequences set forth in: SEQ ID NO:17 and SEQ ID NO:21; SEQ ID NO: 17 and SEQ ID NO:22; SEQ ID NO: 17 and SEQ ID NO:23; SEQ ID NO: 17 and SEQ ID NO:24; SEQ ID NO: 17 and SEQ ID NO:25; SEQ ID NO: 17 and SEQ ID NO:26; SEQ ID NO: 17 and SEQ ID NO:27; SEQ ID NO:17 and SEQ ID NO:28; SEQ ID NO: 17 and SEQ ID NO:29; SEQ ID NO: 17 and SEQ ID NO:30; SEQ ID NO: 18 and SEQ ID NO:21; SEQ ID NO: 18 and SEQ ID NO:22; SEQ ID NO: 18 and SEQ ID NO:23; SEQ ID NO: 18 and SEQ ID NO:24; SEQ ID NO: 18 and SEQ ID NO:25; SEQ ID NO: 18 and SEQ ID NO:26; SEQ ID NO: 18 and SEQ ID NO:27; SEQ ID NO: 18 and SEQ ID NO:28; SEQ ID NO: 18 and SEQ ID NO:29; SEQ ID NO: 18 and SEQ ID NO:30; SEQ ID NO: 19 and SEQ ID NO:21; SEQ ID NO:19 and SEQ ID NO:22; SEQ ID NO: 19 and SEQ ID NO:23; SEQ ID NO: 19 and SEQ ID NO:24; SEQ ID NO: 19 and SEQ ID NO:25; SEQ ID NO: 19 and SEQ ID NO: 26; SEQ ID NO: 19 and SEQ ID NO: 27; SEQ ID NO: 19 and SEQ ID NO:28; SEQ ID NO: 19 and SEQ ID NO:29; or SEQ ID NO: 19 and SEQ ID NO: 30. Multispecific Binding Molecules

[0098] Other binding molecules are also encompassed by this disclosure. In one aspect, this disclosure provides multispecific antibodies - that is antibodies that bind to at least two different epitopes on the same antigen or that bind two different antigens. In some cases, the binding molecule is a bispecific antibody (BsAb), which may have binding specificities that are directed towards CD3E, and to a different epitope on CD3E or to any other antigen, e.g., a tumor associated antigen. Multispecific antibodies typically comprise at least two different antigen-binding domains, wherein each antigen-binding domain is capable of specifically binding to a separate epitope or antigen (i.e., CD3, and a different epitope on CD3E or to any other antigen). Each antigen-binding domain of a bispecific antibody can comprise a heavy chain variable domain (VH) and a light chain variable domain (VL). In the context of a bispecific antigen-binding fragment comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), each antigen binding domain comprises at least three VH CDRs and three VL CDRs that specifically binds to a particular antigen (i.e., CD3E, and a different epitope on CD3E or to any other antigen). The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding fragment (e.g., bispecific Fab, bispecific scFv). In some cases, these may further be linked to an Fc domain. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate Fc domain. Bispecific antigen-binding fragments of the present disclosure may comprise two Fc domains that are each individually part of a separate antibody heavy chain. The first and second Fc domains may be of the same sequence, or the Fc domains may have one or more mutations in the CH3 domain intended for, e.g., the facilitation of heterodimerization and / or ease of purification of heterodimeric (i.e., bispecific) molecules.

[0099] In some cases, the multispecific antibody comprises a single polypeptide chain and comprise first and second scFv wherein the first scFv is joined by a linker to a second scFv. In some cases, the linker is a glycine serine linker such as (GGGGS)n wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:63). Non-limiting examples of other linkers suitable for linking the polypeptides of this disclosure include, but are not limited to, (GS)n (SEQ ID NO: 105), (GGS)n (SEQ ID NO: 106), (GGGS)n (SEQ ID NO: 64), (GGSG)n (SEQ ID NO:65), or (GGSGG)n (SEQ ID NO:66), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some instances the length of the linker can be about 15 amino acids. In one instance the linker is (G4S)s (SEQ ID NO:67). The scFvs may bind different epitopes of the same antigen or bind to different antigens.

[0100] In some cases, the multispecific antibody comprises multiple chains, e.g., comprising a first Fab and second Fab, wherein the first Fab and the second Fab bind different epitopes of the same antigen or bind to different antigens. In one instance, the first Fab specifically binds to CD3E. In some cases, the first Fab binds to both human and cyno CD3E.

[0101] In some instances, a bispecific antibody may be a tetrameric bispecific antibody. For example, it comprises a first heavy chain and first light chain that binds to a first epitope or first antigen and a second heavy chain and a second light chain that binds to a second epitope or second antigen.

[0102] In certain instances, the Fc region of the multispecific antibody is engineered to promote heterodimerization. This can be done by any method known in the art such as the use of knob-into-hole, electrostatic steering, or any other design strategies for heterodimerization of Fc. See, Ha et al., Front Immunol. 2016; 7: 394; Moore et al., Methods, 154:38-50 (2019); WO 2017 / 106462; and Yu et al., Nat Methods, 16, 1095- 1100 (2019) (all incorporated by reference herein). In certain instances, the Fc region is also engineered to improve its therapeutic activity. See e.g., Mimoto et al., Curr Pharm Biotechnol., 2016;17(15):1298-1314; Chen et al., Theranostics, 2021; 11(4): 1901-1917; Delidakis et al., Ann Rev. Biomed Engg., 24:249-274 (2022) (all incorporated by reference herein). In some embodiments, multispecific antibodies of the present disclosure are secreted (e.g., released from a cell, for example, into the extracellular milieu).

[0103] Multispecific antibodies of the present disclosure can include any of the anti- CD3E binding domains described above. Multispecific antibodies of the present disclosure can comprise binding specificities that are directed towards CD3E and any other antigen. The other antigen may be, for example, a tumor associated antigen, a cancer cell antigen / marker, an immune cell antigen (e.g., a T cell activation marker), a pathogenic antigen, or any other non-CD3 antigen.

[0104] The multispecific antibodies may be produced by any means known in the art for producing multispecific antibodies, so long as the resulting multispecific antibody retains the functional characteristic of being able to specifically bind CD3 and at least one other antigen. In some embodiments, the BsAbs may be created using the methods described in Labrijin et al., Proc. Natl. Acad. Sci. USA, 110(13): 5145-50 (2013). Briefly, the two parental Abs, each containing single matched point mutations in the CH3 domains, are separately expressed and then mixed under reducing conditions in vitro. This separates the Abs into half-molecules, followed by reassembly, to form bispecific antibodies, and is compatible with large-scale manufacturing of bispecific antibodies. However, this is simply one exemplary method for making a multispecific antibody. Those of skill in the art will be aware that other methods of producing multispecific antibodies are available, and the present disclosure is not intended to be limited solely to the methods of making and type of multispecific antibodies disclosed herein.

[0105] Other multispecific antibody formats or technologies may be used to make the multispecific antigen-binding molecules of the present disclosure. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen binding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl / IgG2, dual acting Fab (DAF)- IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-1 1 , and references cited therein, for a review of the foregoing formats).

[0106] The disclosed multispecific antibodies can be made from or incorporate the CDRs or variable regions from polyclonal, monoclonal, chimeric, human, partially or fully humanized, and / or recombinant antibodies. Thus, the “parent” antibodies for the disclosed multispecific antibodies are not particularly limited; however, they are preferably fully human or humanized. In some embodiments, the parent antibody can be a polyclonal antibody. In some embodiments, the parent antibody can be a monoclonal. In some embodiments, the parent antibody can be a human antibody. In some embodiments, the parent antibody can be a murine antibody.

[0107] Conjugates

[0108] The binding molecules of this disclosure can be fused, linked, or conjugated to other moieties. These moieties may be for detection or purification, or to add another therapeutic agent, radionuclide, or toxin to the binding molecules. In certain instances, the moiety is linked or conjugated to the C-terminal of the binding molecule. In some instances, the linker can be any of the Gly Ser linkers discussed above (e.g., (G4S)s (SEQ ID NO:67)) or the natural hinge of human IgA. In some instances a radionuclide may be directly conjugated to the binding molecule, or conjugated via a small chelator.

[0109] In some instances, a binding molecule is linked to a detection or purification tag. In some instances, the tag moiety is His6 (SEQ ID NO: 58), c-myc, hemagglutinin (HA), Flag, headlock tag, C-tag, ALFA tag, Avi tag, alkaline phosphatase or horseradish peroxidase. In some instances, a binding molecule is linked to a fluorophore (GFP, RFP. In some instances, a binding molecule is linked to a drug or biologic. In some instances a tumor targeted drug / therapy is conjugated to the binding molecule of this disclosure to create an ADC. ADCs exploit the targeted efficiency of antibodies combined with the action of the cytotoxic payload conjugated to it. This results in specific targeting of cancer cells and thus lessens off-target side effects. The payload can be any drug that kills or inhibits the growth of the cancer that is being targeted. In some instances the payload is multikinase inhibitor 178 or Mertansine (DM1). In some instances, a binding molecule is linked to a photosensitizer. In some instances, a binding molecule is linked to a nanoparticle (e.g., liposomes, micelles, albumin- based NPs, polymer based polymerosomes). In some instances, a binding molecule is linked to a toxic agent. In some instances, a binding molecule is linked to a radionuclide (e.g.,89Zr,68 / 67Ga,18F,99mTc). In some instances, a binding molecule is linked to a VHH, a Fab, a scFv, or a whole antibody. In some instances, binding molecule is linked to a multimerization module (e.g. dimerization, trimerization, tetramerization, or pentamerization module). See e.g., Sadeghnezhad, G. et al., Int J Mol Sci., 20, 4818, 2019; Zhang et al., J. Mol Biol., 335, 49-56, 2004 (both incorporated by reference herein). In some cases, the dimerization module is a human Ig hinge-Fc domain (e.g., from IgGl, IgG4, IgA). In some cases, the dimerization module is IgG4PAA. In some cases, the trimerization module, is e.g. a collagen trimerization moiety, particularly a human collagen moiety, and a lung surfactant protein D moiety. In some instances, a binding molecule is linked to a moiety that facilitates the polypeptide crossing the blood brain barrier (e.g., antitransferrin antibody, FC5, FC44, a VHH that binds IGF-1R, VHHs E9, ni3a, and pa2H). In some instances, a binding molecule is linked to a half-life extension moiety (e.g., PEG (linear and branched), human serum albumin, a VHH that binds human serum albumin, PASylation (PA600), human Ig Fc).

[0110] Properties of the Binding Molecules

[0111] Humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure may have an affinity for CD3E of, for example, from 0.01 nM to 100 nM (e.g., from 0.01 nM to 90 nM, from 0.1 nM to 80 nM, from 0.5 nM to 70 nM, from 1 nM to 60 nM, or about 50 nM). In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of from about 0.01 nM to about 90 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of from about 0.01 nM to about 80 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity CD3E of from about 0.01 nM to about 70 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of from about 0.01 nM to about 60 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of from about 0.01 nM to about 40 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of from about 0.01 nM to about 20 nM.

[0112] In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 100 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 95 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 90 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 85 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 80 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 75 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 70 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 65 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 60 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 55 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 50 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 45 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 40 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 35 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 30 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 25 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 20 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 15 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 10 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 5 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 1 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 0.5 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 0.1 nM. In some embodiments, humanized antibodies, antigen binding fragments, or binding polypeptides of the disclosure have an affinity for CD3E of about 0.01 nM.

[0113] The specific binding of a humanized antibody, antigen binding fragments, or binding polypeptides described herein to CD3E can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody, antigen binding fragment, or binding protein needed to achieve the equilibrium constant (KD) of the antibody- , antigen binding fragment-, or binding polypeptides - antigen complex dissociation. The equilibrium constant, KD, which describes the interaction of CD3E with an antibody, antigen binding fragment, or binding polypeptides described herein is the chemical equilibrium constant for the dissociation reaction of an antigen-antibody, -antigenbinding fragment, or - binding protein complex into solvent-separated antigen and antibody, antigen binding fragment, or binding polypeptides that do not interact with one another.

[0114] Humanized antibodies, antigen binding fragments, or binding polypeptides described herein include those that specifically bind to CD3E with a KD value of less than 100 nM (e.g., less than 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, or 0.01 nM). In some embodiments, the antibodies, antigen binding fragments, or binding polypeptides described herein specifically bind to CD3E with a KD value of less than 10 nM (e.g., less than 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, or 0.01 nM).

[0115] Humanized antibodies, antigen binding fragments, or binding polypeptides described herein can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the KD or EC50 of a humanized anti-CD3E antibody, antigen binding fragment, or binding protein include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, ELISA- based assays, gene expression assays, and protein expression assays, among others. ELISA represents a particularly useful method for analyzing humanized antibody, antigen binding fragment, or binding protein activity, as such assays typically require minimal concentrations of binding domains (e.g., humanized antibodies, antigen binding fragments, binding polypeptides). A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a humanized anti-CD3E antibody, antigen binding fragment described herein). Humanized antibodies, antigen binding fragments, or binding polypeptides described herein may bind CD3E and fragments thereof. Humanized antibodies, antigen binding fragments, or binding polypeptides described herein may additionally bind isolated peptides derived from CD3E that structurally pre-organize various residues in a manner that simulates the conformation of the above fragments in the native protein. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigen-antibody, antigen-antigen-binding fragment, or antigen-binding protein complex bound to a HRP-conjugated secondary antibody.

[0116] In addition to the thermodynamic parameters of a humanized CD3E-antibody, - antigen binding fragment, or -binding polypeptide interaction, it is also possible to quantitatively characterize the kinetic association and dissociation of a humanized antibody, antigen binding fragment, or binding proteins described herein with CD3E. This can be done, e.g., by monitoring the rate of humanized antibody-, antigen-binding fragment-, or binding polypeptide-antigen complex formation according to established procedures. For example, one can use surface plasmon resonance (SPR) to determine the rate constants for the formation (kon) and dissociation (koff) of a humanized antibody-, antigen-binding fragment-, or binding polypeptide-CD3 complex. These data also enable calculation of the equilibrium constant of (KD) of humanized antibody-, antigen-binding fragment-, or binding protein-CD3 complex dissociation, since the equilibrium constant of this unimolecular dissociation can be expressed as the ratio of the koff to konvalues. SPR is a technique that is particularly advantageous for determining kinetic and thermodynamic parameters of antigen-antibody, -antigen-binding fragment, or -binding protein interactions since the experiment does not require that one component be modified by attachment of a chemical label. Rather, the antigen is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of humanized antibody, antigen-binding fragment, or binding proteins. Humanized antibody-, antigen-binding fragment-, or binding polypeptide-antigen binding induces distortion in the angle of reflection of incident light at the metallic surface, and this change in refractive index over time as humanized antibody, antigen-binding fragment, or binding protein is introduced to the system can be fit to established regression models in order to calculate the association and dissociation rate constants of an antibody- or antigen binding-fragment- or binding protein- antigen interaction.

[0117] Humanized antibodies, antigen binding fragments, or binding polypeptides described herein may exhibit high konand low koa values upon interaction with CD3. For example, humanized antibodies, antigen-binding fragments, or binding proteins described herein may exhibit konvalues in the presence of CD3 of greater than 103T's-1(e.g., 1.0 x io3MV1, 1.5 x io3M1, 2.0 x 103MV1, 2.5 x 103MV1, 3.0 x 103MV1, 3.5 X IO3MV1, 4.0 x 103MV1, 4.5 x 103MV1, 5.0 x 103MV1, 5.5 x 103MV1, 6.0 x 103MV \ 6.5 x 103MV1, 7.0 x 103MV1, 7.5 x 103MV1, 8.0 x 103MV1, 8.5 x 103MV1, 9.0 x 103MV1, 9.5 x 103MV1, 1.0 x 104MV1, 1.5 x 104MV1, 2.0 x 104MV1, 2.5 x 104MV1, 3.0 x 104MV1, 3.5 x 104MV1, 4.0 x 104MV1, 4.5 x 104MV1, 5.0 x 104MV \ 5.5 x 104MV1, 6.0 x 104MV1, 6.5 X IO4MV1, 7.0 x 104MV1, 7.5 x 104MV1, 8.0 x 104MV1, 8.5 x 104MV1, 9.0 x 104MV1, 9.5 x 104MV1, or 1.0 X IO5MV1). Humanized antibodies, antigen binding fragments, or binding polypeptides described herein may exhibit low koff values when bound to CD3. For instance, humanized antibodies, antigen binding fragments, or binding proteins described herein may exhibit koff values of less than 10’3s'1when complexed to CD3 (e.g., 1.0 x 10’3s’1, 9.5 x 10’4s’1, 9.0 x 10’4s’1, 8.5 x 10’4s’1, 8.0 x 10 ’4s’1, 7.5 x 10’4s’1, 7.0 x 10’4s’1, 6.5 x 10’4s’1, 6.0 x 10’4s’1, 5.5 x 10’4s’1, 5.0 x 10’4s’1, 4.5 x 10’4s’1, 4.0 x 10’4s’1, 3.5 x 10’4s’1, 3.0 x 10’4s’1, 2.5 x 10’4s’1, 2.0 x 10’4s’1, 1.5 x 10’4s’1, 1.0 x 10’4s’1, 9.5 x IO’5s’1, 9.0 x IO’5s’1, 8.5 x IO’5s’1, 8.0 x IO’5s’1, 7.5 x IO’5s’1, 7.0 x IO’5s’1, 6.5 x IO’5s’1, 6.0 x IO’5s’1, 5.5 x IO’5s’1, 5.0 x IO’5s’1, 4.5 x IO’5s’1, 4.0 x IO’5s’1, 3.5 x IO’5s’1, 3.0 x IO’5s’1, 2.5 x IO’5s’1, 2.0 x IO’5s’1, 1.5 x IO’5s’1, or 1.0 x IO’5s’1).

[0118] Methods of Humanization

[0119] Humanized antibodies, antigen binding fragments, or binding polypeptides described herein can include fully humanized antibodies, antigen binding fragments, or binding polypeptides that contain the six CDRs based on any particular CDR definition or a VH and a VL as described herein. As an example, one strategy that can be used to design humanized antibodies, antigen binding fragments, or binding polypeptides described herein is to align the sequences of the VH and / or VL of an antibody, antigen binding fragment, or binding protein (e.g., of the present disclosure) with the VH and / or VL of a consensus human antibody. Consensus human antibody heavy chain and light chain sequences are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 -3242 (1991); Tomlinson et al., J. Mol. Biol. 227:776-98 (1992); and Cox et al., Eur. J. Immunol. 24:827-836 (1994); the disclosure of which is incorporated herein by reference). In this way, the variable domain framework residues and CDRs (e.g. IMGT, Kabat) can be identified by sequence alignment. One can then substitute, for example, the CDRs of the consensus human antibody with the corresponding CDRs of an antibody or antigen binding fragment or binding polypeptide of the disclosure, thereby producing a humanized antibody, antigen binding fragment, or binding polypeptide.

[0120] In some embodiments, it may be desirable to import particular framework residues in addition to CDR sequences from an anti-CD3E antibody, antigen binding fragment, or binding protein into the VH and / or VL of a human antibody. For instance, US Patent No. 6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody, antigen binding fragment, or binding polypeptides. In some embodiments, framework residues may engage in non-covalent interactions with the antigen and thus contribute to the affinity of the antibody, antigen binding fragment, or binding proteins for the target antigen. In some embodiments, individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody, antigen binding domain, or binding proteins with the antigen. Certain framework residues may form the interface between VH and VL domains, and may therefore contribute to the global antibody, antigen binding domain, or binding protein structure. In some cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser / Thr) which may dictate antibody, antigen binding domain, or binding protein structure and antigen affinity upon attachment to carbohydrate moieties. In cases such as those described above, it may be beneficial to retain certain framework residues of an anti-CD3 antibody, antigen binding fragment, or binding polypeptide in, e.g., a humanized antibody or antigen binding fragment or binding polypeptide thereof, as various framework residues may promote high epitope affinity and improved biochemical activity of the antibody or antigen binding fragment or binding polypeptide thereof.

[0121] Humanized antibodies described herein also include antibody fragments, Fab domains, F(ab’) molecules, F(ab’)2 molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi-specific antibodies, bispecific antibodies, and heterospecific antibodies that contain one or more of the VH and VL sequences described herein, or a VH or VL having at least 85% sequence identity thereto e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto). These molecules can be expressed recombinantly, e.g., by incorporating polynucleotides encoding these proteins into expression vectors for transfection in a eukaryotic or prokaryotic cell using techniques described herein or known in the art, or synthesized chemically, e.g., by solid phase peptide synthesis methods described herein or known in the art.

[0122] Nucleic Acids. Host Cells, and Methods of Making

[0123] The disclosure provides a nucleic acid or nucleic acids that encode the binding molecules of this disclosure. The nucleic acid may be DNA or mRNA. The nucleic acid may be modified. The coding sequence may be modified to take into account the preferred codon usage of the expression host.

[0124] Also provided are vectors that comprise the above nucleic acids. In some instances the vector is an expression vector. In other instances, the vector is a viral vector.

[0125] Also provided are host cells that comprise the nucleic acid or vectors above. In some instances, the host cells are bacterial cells, yeast cells, insect cells, or mammalian cells. In some instances, the host cell is E. coli. In some instances, the host cell is Pichia pastoris or Saccharomyces cerevisiae. In some instances, the host cell is a CHO cell, a COS cell, a HEK293T cell, or a NIH3T3 cell.

[0126] The disclosure also features methods of making the binding molecules described herein. The method involves culturing the host cell under conditions that promote the expression of the binding molecule. In some instances, the binding molecule is purified. In certain instances, the binding molecule is formulated as a sterile pharmaceutical composition.

[0127] Also provided are pharmaceutical compositions comprising the binding molecules described herein, and a pharmaceutically acceptable carrier. In some instances, the pharmaceutically acceptable carrier is phosphate buffered saline. In some instances, the pharmaceutically acceptable carrier is sterile distilled water. mRNA Constructs In some aspects, the polynucleotides disclosed herein are or function as a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0128] The disclosure provides a polynucleotide or polynucleotides comprising an mRNA or mRNAs comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) or ORFs encoding a polypeptide or polypeptides or a multispecific antibody described herein; (iii) a stop codon; and (iv) a 3' UTR. In certain instances, the mRNA can include a 5’-cap. In certain instances, the mRNA can also include a 3’ trailing sequence of linked nucleosides such as a poly-A region or a poly A-G quartet.

[0129] The disclosure features compositions including an mRNA or mRNAs encoding a polypeptide or polypeptides described herein (e.g., a binding molecule comprising an anti-CD3E antibody, an anti-CD3E Fab, an anti-CD3E scFv, a a bispecific antibody comprising an anti-CD3E binding region, or a multispecific antibody comprising an anti- CD3E binding region ). In one instance, the mRNAs include (i) a 5 ’-cap structure; (ii) a 5 ’-UTR; (iii) an open reading frame encoding the polypeptide; (iv) a 3 ’ -untranslated region (3 ’-UTR); and (v) a poly-A region. mRNA: 5 ’-cap

[0130] The 5'-cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing. mRNA molecules may be 5'-end capped generating a 5'-ppp-5'-triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O- methylated. 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0131] Alterations to the nucleic acids of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, alternative nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional alternative guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0132] Additional alterations include, but are not limited to, 2'-O-methylation of the ribose sugars of 5'-terminal and / or 5 '-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.

[0133] 5 ’-cap structures include those described in International Patent Publication Nos. WO2008 / 127688, W02008 / 016473, and WO2011 / 015347, each of which is incorporated herein by reference in its entirety.

[0134] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / linked to a nucleic acid molecule.

[0135] For example, the Anti -Reverse Cap Analog (ARC A) cap contains two guanosines linked by a 5 '-5 '-triphosphate group, wherein one guanosine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'- guanosine (m7G-3 'mppp-G; which may equivalently be designated 3' O-Me- m7G(5')ppp(5')G)). The 3'-0 atom of the other, unaltered, guanosine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA or mmRNA). The N7- and 3'-O-methlyated guanosine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0136] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O- methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'- guanosine, m7Gm-ppp-G).

[0137] In some instances, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.

[0138] In some instances, the cap analog is a N7-(4-chlorophenoxyethyl) substituted dicnucleotide form of a cap analog known in the art and / or described herein. Nonlimiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5’)ppp(5’)G and a N7-(4- chlorophenoxyethyl)-m3'°G(5’)ppp(5’)G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 21:4570-4574 (2013); the contents of which are herein incorporated by reference in its entirety). In some instances, a cap analog of the present disclosure is a 4-chloro / bromophenoxy ethyl analog.

[0139] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5'-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability. Nucleic acids of the disclosure (e.g., mRNAs of the disclosure) may also be capped post-transcriptionally, using enzymes. 5’ cap structures produced by enzymatic capping may enhance binding of cap binding proteins, increase half-life, reduce susceptibility to 5' endonucleases and / or reduce 5' decapping, as compared to synthetic 5'-cap structures known in the art (or to a wild-type, natural or physiological 5'-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanosine cap nucleotide wherein the cap guanosine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5'cap analog structures known in the art. Cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0140] 5' terminal caps may include endogenous caps or cap analogs. A 5' terminal cap may include a guanosine analog. Useful guanosine analogs include inosine, N1 -methylguanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.

[0141] In some instances, the nucleic acids described herein may contain a modified 5’- cap. A modification on the 5 ’-cap may increase the stability of mRNA, increase the half- life of the mRNA, and could increase the mRNA translational efficiency. The modified 5 ’-cap may include, but is not limited to, one or more of the following modifications: modification at the 2’ and / or 3’ position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety. Untranslated Regions (UTRs)

[0142] Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide before a start codon (5' UTR) and after a stop codon (3' UTR) that are not translated. In some instances, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure comprising an open reading frame (ORF) encoding an antibody further comprises UTR (e.g., a 5'UTR or functional fragment thereof, a 3'UTR or functional fragment thereof, or a combination thereof).

[0143] Translation of a polynucleotide comprising an open reading frame encoding a polypeptide can be controlled and regulated by a variety of mechanisms that are provided by various cis-acting nucleic acid structures. For example, naturally-occurring, cis-acting RNA elements that form hairpins or other higher-order (e.g., pseudoknot) intramolecular mRNA secondary structures can provide a translational regulatory activity to a polynucleotide, wherein the RNA element influences or modulates the initiation of polynucleotide translation, particularly when the RNA element is positioned in the 5' UTR close to the 5’-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):515-526; Kozak (1986) Proc Natl Acad Sci 83:2850-2854).

[0144] Cis-acting RNA elements can also affect translation elongation, being involved in numerous frameshifting events (Namy et al., (2004) Mol Cell 13(2): 157-168). Internal ribosome entry sequences (IRES) represent another type of cis-acting RNA element that are typically located in 5' UTRs, but have also been reported to be found within the coding region of naturally-occurring mRNAs (Holcik et al. (2000) Trends Genet 16(10):469-473). In cellular mRNAs, IRES often coexist with the 5'-cap structure and provide mRNAs with the functional capacity to be translated under conditions in which cap-dependent translation is compromised (Gebauer et al., (2012) Cold Spring Harb Perspect Biol 4(7):a012245). Another type of naturally-occurring cis-acting RNA element comprises upstream open reading frames (uORFs). Naturally-occurring uORFs occur singularly or multiply within the 5' UTRs of numerous mRNAs and influence the translation of the downstream major ORF, usually negatively (with the notable exception of GCN4 mRNA in yeast and ATF4 mRNA in mammals, where uORFs serve to promote the translation of the downstream major ORF under conditions of increased eIF2 phosphorylation (Hinnebusch (2005) Annu Rev Microbiol 59:407-450)). Additional exemplary translational regulatory activities provided by components, structures, elements, motifs, and / or specific sequences comprising polynucleotides (e.g., mRNA) include, but are not limited to, mRNA stabilization or destabilization (Baker & Parker (2004) Curr Opin Cell Biol 16(3):293-299), translational activation (Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), and translational repression (Blumer et al., (2002) Meeh Dev 110(1 -2) : 97- 112). Studies have shown that naturally-occurring, cisacting RNA elements can confer their respective functions when used to modify, by incorporation into, heterologous polynucleotides (Goldberg-Cohen et al., (2002) J Biol Chem 277( 16) : 13635- 13640).

[0145] The present disclosure provides synthetic polynucleotides comprising a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity. In some instances, the disclosure provides a polynucleotide comprising a 5’ untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3’ UTR, and at least one modification, wherein the at least one modification provides a desired translational regulatory activity, for example, a modification that promotes and / or enhances the translational fidelity of mRNA translation. In some instances, the desired translational regulatory activity is a cis-acting regulatory activity. In some instances, the desired translational regulatory activity is an increase in the residence time of the 43 S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon. In some instances, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some instances, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some instances, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some instances, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome. In some instances, the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some instances, the desired translational regulatory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some instances, the desired translational regulatory activity is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some instances, the desired translational regulatory activity is inhibition or reduction in the production of aberrant translation products. In some instances, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.

[0146] Accordingly, the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and / or an RNA secondary structure(s) that provides a desired translational regulatory activity as described herein. In some aspects, the mRNA comprises an RNA element that comprises a sequence and / or an RNA secondary structure(s) that promotes and / or enhances the translational fidelity of mRNA translation. In some aspects, the mRNA comprises an RNA element that comprises a sequence and / or an RNA secondary structure(s) that provides a desired translational regulatory activity, such as inhibiting and / or reducing leaky scanning. In some aspects, the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and / or an RNA secondary structure(s) that inhibits and / or reduces leaky scanning thereby promoting the translational fidelity of the mRNA.

[0147] In some instances, the RNA element comprises natural and / or modified nucleotides. In some instances, the RNA element comprises of a sequence of linked nucleotides, or derivatives or analogs thereof that provides a desired translational regulatory activity as described herein. In some instances, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a desired translational regulatory activity as described herein. RNA elements can be identified and / or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g. stem-loop), by the location of the element within the RNA molecule (e.g., located within the 5’ UTR of an mRNA), by the biological function and / or activity of the element (e.g., “translational enhancer element”), and any combination thereof.

[0148] In some aspects, the disclosure provides an mRNA having one or more structural modifications that inhibits leaky scanning and / or promotes the translational fidelity of mRNA translation, wherein at least one of the structural modifications is a GC-rich RNA element. In some aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA. In one embodiment, the GC- rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5 ’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

[0149] In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60- 70% cytosine, 50%-60% cytosine, 40-50% cytosine, 30-40% cytosine bases. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

[0150] In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40- 50% cytosine, or 30-40% cytosine. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

[0151] In some instances, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is >50% cytosine. In some instances, the sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.

[0152] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of about 3-30, 5-25, 10-20, 15-20 or about 20, about 15, about 12, about 10, about 6 or about 3 nucleotides, or derivatives or analogues thereof, wherein the sequence comprises a repeating GC-motif, wherein the repeating GC-motif is [CCG]n, wherein n = 1 to 10 (SEQ ID NO: 107), n= 2 to 8, n= 3 to 6, or n= 4 to 5. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, 3, 4 or 5. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, or 3. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 2. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 3. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 4. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 5.

[0153] In another aspect, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element comprises any one of the sequences set forth in Table 5. In one embodiment, the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5 ’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

[0154] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence VI [CCCCGGCGCC (SEQ ID NO: 68)] as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence VI as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5 ’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence VI as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other instances, the GC-rich element comprises the sequence VI as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

[0155] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V2 [CCCCGGC] as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence V2 as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence V2 as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other instances, the GC-rich element comprises the sequence V2 as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12- 15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

[0156] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence EK [GCCGCC] as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence EK as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence EK as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other instances, the GC-rich element comprises the sequence EK as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12- 15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

[0157] In yet other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence VI [CCCCGGCGCC (SEQ ID NO: 68)] as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 5: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:71). The skilled artisan will of course recognize that all Us in the RNA sequences described herein will be Ts in a corresponding template DNA sequence, for example, in DNA templates or constructs from which mRNAs of the disclosure are transcribed, e.g., via IVT.

[0158] In some instances, the GC-rich element comprises the sequence VI as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR sequence shown in Table 5. In some instances, the GC-rich element comprises the sequence VI as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 5: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:71).

[0159] In other instances, the GC-rich element comprises the sequence VI as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5 ’ UTR of the mRNA, wherein the 5 ’ UTR comprises the following sequence shown in Table 5: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:71). In some instances, the 5’ UTR comprises the following sequence set forth in

[0160] Table 5:

[0161] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGC

[0162] CGCCACC (SEQ ID NO: 72).

[0163] Table 5

[0164]

[0165] In another aspect, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a stable RNA secondary structure comprising a sequence of nucleotides, or derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop. In one embodiment, the stable RNA secondary structure is upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located 12-15 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure has a deltaG of about -30 kcal / mol, about -20 to -30 kcal / mol, about -20 kcal / mol, about -10 to -20 kcal / mol, about -10 kcal / mol, about -5 to -10 kcal / mol.

[0166] In another embodiment, the modification is operably linked to an open reading frame encoding a polypeptide and wherein the modification and the open reading frame are heterologous.

[0167] In another embodiment, the sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.

[0168] RNA elements that provide a desired translational regulatory activity as described herein can be identified and characterized using known techniques, such as ribosome profiling. Ribosome profiling is a technique that allows the determination of the positions of PICs and / or ribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science 324(5924):218-23, incorporated herein by reference). The technique is based on protecting a region or segment of mRNA, by the PIC and / or ribosome, from nuclease digestion. Protection results in the generation of a 30-bp fragment of RNA termed a ‘footprint’. The sequence and frequency of RNA footprints can be analyzed by methods known in the art (e.g., RNA-seq). The footprint is roughly centered on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at positions where the PIC and / or ribosome exhibits decreased processivity and fewer footprints where the PIC and / or ribosome exhibits increased processivity (Gardin et al., (2014) eLife 3:e03735). In some instances, residence time or the time of occupancy of the PIC or ribosome at a discrete position or location along an polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.

[0169] A UTR can be homologous or heterologous to the coding region in a polynucleotide. In some instances, the UTR is homologous to the ORF encoding the antibody. In some instances, the UTR is heterologous to the ORF encoding the antibody. In some instances, the polynucleotide comprises two or more 5'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences. In some instances, the polynucleotide comprises two or more 3'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.

[0170] In some instances, the 5 'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized.

[0171] In some instances, the 5 'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., N1 methylpseudouracil or 5-methoxyuracil.

[0172] UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and / or translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods. In some instances, a functional fragment of a 5'UTR or 3'UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.

[0173] Natural 5'UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G. 5'UTRs also have been known to form secondary structures that are involved in elongation factor binding.

[0174] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a polynucleotide. For example, introduction of 5'UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or antibody, can enhance expression of polynucleotides in hepatic cell lines or liver. Likewise, use of 5'UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CDl lb, MSR, Fr-1, i-NOS), for leukocytes (e g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A / B / C / D).

[0175] In some instances, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. In some instances, the 5’UTR and the 3’UTR can be heterologous. In some instances, the 5'UTR can be derived from a different species than the 3'UTR. In some instances, the 3'UTR can be derived from a different species than the 5'UTR.

[0176] Co-owned International Patent Application No. PCT / US2014 / 021522 (Publ. No. WO / 2014 / 164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide of the present disclosure as flanking regions to an ORF.

[0177] Exemplary UTRs of the application include, but are not limited to, one or more 5'UTR and / or 3'UTR derived from the nucleic acid sequence of: a globin, such as an CLOT 0-globin (e.g., aXenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin?); a HSD17B4 (hydroxysteroid (17-0) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a sindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein (e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucose transporter (e.g., hGLUTl (human glucose transporter 1)); an actin (e.g., human a or 0 actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomerase (e.g., a 5'UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9); an ATP synthase (e.g., ATP5A1 or the 0 subunit of mitochondrial H+-ATP synthase); a growth hormone e (e.g., bovine (bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1 al (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A); a 0-F1- ATPase, a creatine kinase, a myoglobin, a granulocyte-colony stimulating factor (G- CSF); a collagen (e.g., collagen type I, alpha 2 (Coll A2), collagen type I, alpha 1 (Coll Al), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1 (C0I6AI)); a ribophorin (e.g., ribophorin I (RPNI)); a low density lipoprotein receptor-related protein (e.g., LRP1); a car diotrophin- like cytokine factor (e.g., Nntl); calreticulin (Calr); a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plodl); and a nucleobindin (e.g., Nucbl).

[0178] In some instances, the 5'UTR is selected from the group consisting of a 0-globin 5’UTR; a 5'UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 a polypeptide (CYBA) 5'UTR; a hydroxysteroid (17-0) dehydrogenase (HSD17B4) 5'UTR; a Tobacco etch virus (TEV) 5'UTR; a Venezuelen equine encephalitis virus (TEEV) 5'UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5'UTR; a heat shock protein 70 (Hsp70) 5'UTR; a eIF4G 5'UTR; a GLUT1 5'UTR; functional fragments thereof and any combination thereof.

[0179] In some instances, the 3'UTR is selected from the group consisting of a 0-globin 3’UTR; a CYBA 3'UTR; an albumin 3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; a hepatitis B virus (HBV) 3'UTR; a-globin 3'UTR; a DEN 3'UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; an elongation factor 1 al (EEF1A1) 3'UTR; a manganese superoxide dismutase (MnSOD) 3'UTR; a 0 subunit of mitochondrial H(+)- ATP synthase (0-mRNA) 3'UTR; a GLUT1 3'UTR; a MEF2A 3'UTR; a 0-Fl-ATPase 3'UTR; functional fragments thereof and combinations thereof.

[0180] Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the present disclosure. In some instances, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. In some instances, variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR. Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.

[0181] UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5' and / or 3' UTR can be inverted, shortened, lengthened, or combined with one or more other 5' UTRs or 3' UTRs.

[0182] In some instances, the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5’UTR or 3’UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For example, a double betaglobin 3'UTR can be used (see US2010 / 0129877, the contents of which are incorporated herein by reference in its entirety).

[0183] In certain instances, the polynucleotides of the disclosure comprise a 5' UTR and / or a 3' UTR selected from any of the UTRs disclosed herein. In some instances, the 5' UTR comprises:

[0184] 5' UTR-001 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:73);

[0185] 5' UTR-002 (Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:77);

[0186] 5' UTR-004 (Upstream UTR) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC) (SEQ ID NO:78);

[0187] 5' UTR-005 (Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:79); 5' UTR-007 (Upstream UTR) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC) (SEQ ID NO: 80);

[0188] 5' UTR-008 (Upstream UTR)

[0189] (GGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:81);

[0190] 5' UTR-009 (Upstream UTR)

[0191] (GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO: 82);

[0192] 5' UTR-010, Upstream (GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO: 83);

[0193] 5' UTR-011 (Upstream UTR)

[0194] (GGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO: 84);

[0195] 5' UTR-012 (Upstream UTR)

[0196] (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAUAAGAGCCACC) (SEQ ID NO: 85);

[0197] 5' UTR-013 (Upstream UTR) (GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO: 86);

[0198] 5' UTR-014 (Upstream UTR) (GGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAAAAGAGCCACC) (SEQ ID NO: 87); 5' UTR-015 (Upstream UTR)

[0199] (GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO: 88);

[0200] 5' UTR-016 (Upstream UTR)

[0201] (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUAAGAGCCACC) (SEQ ID NO: 89);

[0202] 5' UTR-017 (Upstream UTR);

[0203] (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUUAAGAGCCACC) (SEQ ID NO:90); or

[0204] 5' UTR-018 (Upstream UTR) 5' UTR

[0205] (UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGG AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:91).

[0206] In some instances, the 3' UTR comprises:

[0207] 3 UTR:

[0208] UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:92)

[0209] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0210] (UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGG CCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG CACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC) (SEQ ID NO:93);

[0211] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0212] (UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUAC ACAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG CACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC) (SEQ ID NO:94);

[0213] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0214] (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUA AAGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG CACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC) (SEQ ID NO:95);

[0215] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0216] (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCC UCCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUG CACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC) (SEQ ID NO:96);

[0217] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0218] (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCC UCCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUG CACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC) (SEQ ID NO:97);

[0219] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0220] (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCC UCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGU AGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC) (SEQ ID NO:98);

[0221] 142-3p 3' UTR (UTR including miR142-3p binding site)

[0222] (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCC UCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUG AAUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC) (SEQ ID NO:99);

[0223] 3’UTR-018

[0224] GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAG GGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (SEQ ID NO: 100);

[0225] 3' UTR (miR142 and miR126 binding sites variant 1)

[0226] (UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGG CCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG

[0227] CACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC) (SEQ ID NO: 101);

[0228] 3' UTR (miR142 and miR126 binding sites variant 2)

[0229] (UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGG CCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUG

[0230] CACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC) (SEQ ID NO: 102);

[0231] 3 ’UTR (miR142-3p binding site variant 3)

[0232] UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUA GGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 103; or

[0233] 3’UTR (miR142-3p binding site variant 3, DNA sequence) TGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCC CCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTCCATAAAGTAGGA AACACTACAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO: 104).

[0234] In certain instances, the 5'UTR and / or 3'UTR sequence of the present disclosure comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5'UTR sequences comprising any of SEQ ID NOs: 73, 74 and 77- 91 and / or 3'UTR sequences comprises any of SEQ ID NOs: 92-104, and any combination thereof.

[0235] The polynucleotides of the present disclosure can comprise combinations of features. For example, the ORF can be flanked by a 5'UTR that comprises a strong Kozak translational initiation signal and / or a 3'UTR comprising an oligo(dT) sequence for templated addition of a poly- A tail. A 5'UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different UTRs (see, e.g., US2010 / 0293625, herein incorporated by reference in its entirety).

[0236] Other non-UTR sequences can be used as regions or subregions within the polynucleotides of the present disclosure. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the present disclosure. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some instances, the polynucleotide of the present disclosure comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some instances, the polynucleotide comprises an IRES instead of a 5’UTR sequence. In some instances, the polynucleotide comprises an ORF and a viral capsid sequence. In some instances, the polynucleotide comprises a synthetic 5'UTR in combination with a nonsynthetic 3'UTR.

[0237] In some instances, the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, "TEE," which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some instances, the 5'UTR comprises a TEE.

[0238] In one aspect, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or capindependent translation.

[0239] In some instances, a 5'UTR and / or 3'UTR comprising at least one TEE described herein can be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector.

[0240] In some instances, a 5'UTR and / or 3'UTR of a polynucleotide of the present disclosure comprises a TEE or portion thereof described herein. In some instances, the TEEs in the 3'UTR can be the same and / or different from the TEE located in the 5'UTR.

[0241] In some instances, a 5'UTR and / or 3'UTR of a polynucleotide of the present disclosure can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In one embodiment, the 5'UTR of a polynucleotide of the present disclosure can include 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1- 30, 1-25, 1-20, 1-15, 1-10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequences. The TEE sequences in the 5'UTR of the polynucleotide of the present disclosure can be the same or different TEE sequences. A combination of different TEE sequences in the 5'UTR of the polynucleotide of the present disclosure can include combinations in which more than one copy of any of the different TEE sequences are incorporated.

[0242] In some instances, the 5'UTR and / or 3'UTR comprises a spacer to separate two TEE sequences. As a non-limiting example, the spacer can be a 15 nucleotide spacer and / or other spacers known in the art. As another non-limiting example, the 5'UTR and / or 3'UTR comprises a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more than 10 times in the 5'UTR and / or 3'UTR, respectively. In some instances, the 5'UTR and / or 3'UTR comprises a TEE sequence-spacer module repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0243] In some instances, the spacer separating two TEE sequences can include other sequences known in the art that can regulate the translation of the polynucleotide of the present disclosure, e.g., miR binding site sequences described herein (e.g., miR binding sites and miR seeds). As a non-limiting example, each spacer used to separate two TEE sequences can include a different miR binding site sequence or component of a miR sequence (e.g., miR seed sequence).

[0244] In some instances, a polynucleotide of the present disclosure comprises a miR and / or TEE sequence. In some instances, the incorporation of a miR sequence and / or a TEE sequence into a polynucleotide of the present disclosure can change the shape of the stem loop region, which can increase and / or decrease translation. See e.g., Kedde et al., Nature Cell Biology 2010 12(10):1014-20, herein incorporated by reference in its entirety). mRNA: Coding Region

[0245] Provided herein are mRNA(s) that encode the coding region(s) of the binding molecules of the disclosure. In some instances, the disclosure provides mRNA(s) that encode coding regions of a polypeptide(s) which is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any of the CD3E binding molecules described herein, provided that the binding molecule does not comprise a VH set forth in SEQ ID NO: 1 and a VL set forth in SEQ ID NO: 5 or any of the anti-CD3 binding VH and VL listed in WO 2007 / 042261 or US 2017 / 0355767, and provided that the CD3E binding polypeptide(s) has the three VH CDRs and three VL CDRs based on any one CDR definitions set out in Table 2. In certain instances, the disclosure provides coding regions that encode a polypeptide(s) which includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations of any of the sequences provided or referenced herein. In some instances, the mRNA(s) comprise a codon optimized nucleic acid sequence, wherein the open reading frame (ORF) of the codon optimized nucleic acid sequence is derived from a binding molecule described herein. For example, the polynucleotides can comprise a codon optimized ORF(s) encoding a polypeptide(s) comprising a binding molecule described herein.

[0246] In certain instances, the mRNA has one ORF that encodes a CD3E binding polypeptide comprising the amino acid sequences of VH CDRs 1-3 and VL CDRs 1-3 or the VH and VL of an anti-CD3E binding molecule of this disclosure. In other instances, a first mRNA comprises a first ORF comprising the amino acid sequences of VH CDRs 1-3 or a VH or a VH-CH1 or a VH:CH1 :upper hinge of this disclosure and a second mRNA comprises a second ORF comprising the amino acid sequences of VL CDRs 1-3, or a VL or a VL-CL of this disclosure.

[0247] The nucleic acid molecules of the disclosure may include one or more alterations. Herein, in a nucleotide, nucleoside, or polynucleotide (such as the mRNA of the disclosure), the terms “alteration” or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides. The alterations may be various distinct alterations. In some instances, where the nucleic acid is an mRNA, the coding region, the flanking regions, and / or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations. In some instances, an alternative polynucleotide introduced to a cell may exhibit reduced degradation in the cell, as compared to an unaltered polynucleotide. The polynucleotides can include any useful alteration, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone). In certain instances, alterations (e.g., one or more alterations) are present in each of the sugar and the internucleoside linkage. Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs) (e.g., the substitution of the 2’ OH of the ribofuranosyl ring to 2’H), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional alterations are described herein.

[0248] Modified Nucleic Acids

[0249] According to Aduri et al., (Aduri, R. et al., Journal of Chemical Theory and Computation. 3(4): 1464-75(2006)), there are 107 naturally occurring nucleosides, including 1 -methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2- methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6- threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6- isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6- dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6- hydroxynorvalylcarbamoyladenosine, 1 ,2-O-dimethyladenosine, N6,2-O- dimethyladenosine, 2-O-methyladenosine, N6,N6,O-2-trimethyladenosine, 2-methylthio- N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-methyladenosine, 2- methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 2- thiocytidine, 3 -methylcytidine , N4-acetylcytidine, 5 -formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5- formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O- dimethylcytidine, N4,N4,2-O-trimethylcytidine, 1 -methylguanosine, N2,7- dimethylguanosine, N2-methylguanosine, 2-O-ribosylphosphate guanosine, 7- methylguanosine, under modified hydroxywybutosine, 7-aminomethyl-7- deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, 4- demethylwyosine, epoxyqueuosine, hydroxywybutosine, isowyosine, N2, 7,2-0- trimethylguanosine, N2,2-O-dimethylguanosine, 1,2-0-dimethylguanosine, 2-0- methylguanosine, N2,N2,2-O-trimethylguanosine, N2,N2,7-trimethylguanosine, peroxywybutosine, galactosyl-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3- carboxypropyl)uridine, 3 -methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5- methylaminomethyluridine, 5-carboxymethyluridine, 5- carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5- taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2- thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5- (isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5- carboxymethylaminomethyl-2-O-methyluridine, 5-carbamoylmethyl-2-O-methyluridine, 5-methoxycarbonylmethyl-2-O-methyluridine, 5-(isopentenylaminomethyl)-2-O- methyluridine, 5,2-O-dimethyluridine, 2-0-methyluridine, 2-thio-2-O-methyluridine, uridine 5-oxyacetic acid, 5 -methoxy carbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5- carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5- methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1- methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1 -methylpseudouridine, 3- methylpseudouridine, 2-0-methylpseudouridine, inosine, 1 -methylinosine, 1,2-0- dimethylinosine, and 2-0-methylinosine. Each of these may be components of nucleic acids (e.g., mRNA) of the present disclosure.

[0250] Nucleosides Containing Modified Sugars

[0251] The alternative nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be altered on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted Ci-6 alkyl; optionally substituted Ci-6 alkoxy; optionally substituted Ce-io aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted Ce-io aryloxy; optionally substituted Ce-io aryl-Ci-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA) in which the 2'-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4’ -carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein

[0252] Generally, RNA includes the sugar group ribose, which is a 5 -membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multi cyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R- GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl- (3'— >2')), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.

[0253] Alterations on the Nucleobase

[0254] The present disclosure provides for alternative nucleosides and nucleotides. As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

[0255] Exemplary non-limiting alterations include an amino group, a thiol group, an alkyl group, a halo group, or any described herein. The alternative nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more alternative or alternative nucleosides).

[0256] In some instances, a nucleic acid of the disclosure (e.g., an mRNA) includes one or more 2’-0Me nucleotides, 2’ -methoxy ethyl nucleotides (2’-M0E nucleotides), 2’-F nucleotide, 2’-NH2 nucleotide, 2 ’fluoroarabino nucleotides (FANA nucleotides), locked nucleic acid nucleotides (LNA nucleotides), or 4’-S nucleotides.

[0257] The alternative nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or alternative nucleotides including non-standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil. The alternative nucleosides and nucleotides can include an alternative nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties (e.g., resistance to nucleases and stability), and these properties may manifest through disruption of the binding of a major groove binding partner.

[0258] In some instances, the alternative nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (\| / ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio- uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5- bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5- carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl- uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl- 2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl- 2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5- carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine (rm5U), 1 -taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4-thio-pseudouridine, 5-methyl- uridine (m5U, i.e., having the nucleobase deoxythymine), 1 -methyl-pseudouridine 5-methyl-2-thio-uridine (m5s2U), 1 -methyl-4-thio-pseudouridine 4-thio-l- methyl-pseudouridine, 3 -methyl-pseudouridine (m3\| / ), 2 -thio- 1 -methyl-pseudouridine, 1- methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio- uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (\| / m), 2-thio-2'-O- methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O- methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5- (isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2 ’-F-ara- uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine.

[0259] In one instance, the nucleic acid is modified to contain 1 -methylpseudouridine (m1\| / ) in lieu of uridine at each instance.

[0260] In some instances, the alternative nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine include 5 -azacytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (CC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5- halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2- thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4- thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio- zebularine, 2-methoxy-cytidine, 2-methoxy- 5 -methyl- cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, lysidine (kzC), a-thio- cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O- methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl- cytidine (FCm), N4,N4,2'-O-trimethyl-cytidine (m Cm), 1 -thio-cytidine, 2’-F-ara- cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0261] In some instances, the alternative nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2, 6- diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza- 2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyl-adenosine (m1A), 2- methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6- threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl- adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl- adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl- adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m Am), l,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2’-F-ara- adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0262] In some instances, the alternative nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1 -methylinosine (m1!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (02yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7- cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,

[0263] 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine,

[0264] 7-methyl-inosine, 6-methoxy-guanosine, 1 -methyl-guanosine (m'G), N2-methyl- guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m2,2’7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio- guanosine, a-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl- guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), l-methyl-2'-O- methyl-guanosine (m'Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O- methyl-inosine (Im), l,2'-O-dimethyl-inosine (m' lm), 2'-O-ribosylguanosine (phosphate) (Gr(p)) , 1 -thio-guanosine, O6-methyl-guanosine, 2’-F-ara-guanosine, and 2’-F- guanosine.

[0265] The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine, or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In some instances, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5 -methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3 -deazaguanine, deazaadenine, 7- deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a]l,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1 ,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and / or derivatives thereof (e.g., A includes adenine or adenine analogs (e.g., 7-deaza adenine)).

[0266] In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5- trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5- hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5- bromo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5- phenyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5 -methoxy-uracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5- methoxy-uracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines.

[0267] In some instances, the polynucleotides of the disclosure contain 1-methyl- pseudouracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5 -trifluor omethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5- hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5- bromo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5 -iodo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl- pseudouracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-fluoro- cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, N4-acetyl- cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-formyl- cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-aminoallyl- cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-carboxy- cytosine, and cytosine as the only uracils and cytosines.

[0268] In some instances, the polynucleotides of the disclosure contain 5-methoxy- uridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5- hy dr oxymethyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5- bromo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy- uridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-fluoro- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5 -formyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5- methoxy-uridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy- uridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines.

[0269] In some instances, the polynucleotides of the disclosure contain 1-methyl- pseudouridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5 -bromo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-methoxy- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-ethyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-phenyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-ethnyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, N4-methyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-fluoro- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, N4-acetyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-formyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-aminoallyl- cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1 -methyl-pseudouridine, uridine, 5-carboxy- cytidine, and cytidine as the only uridines and cytidines.

[0270] In some instances, the polynucleotides of the disclosure contain the uracil of one of the nucleosides of Table 6 and uracil as the only uracils. In other instances, the polynucleotides of the disclosure contain a uridine of Table 6 and uridine as the only uridines.

[0271] Table 6 - Exemplary Uracil-containing Nucleosides

[0272]

[0273] In some instances, the polynucleotides of the disclosure contain the cytosine of one of the nucleosides of Table 7 and cytosine as the only cytosines. In other instances, the polynucleotides of the disclosure contain a cytidine of Table 7 and cytidine as the only cytidines. Table 7 - Exemplary Cytosine Containing Nucleosides

[0274]

[0275] Alterations on the Internucleoside Linkage

[0276] The alternative nucleotides, which may be incorporated into a polynucleotide molecule, can be altered on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.

[0277] The alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0278] The alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl and / or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., the alpha (a), beta (0) or gamma (y) position) can be replaced with a sulfur (thio) and a methoxy.

[0279] The replacement of one or more of the oxygen atoms at the a position of the phosphate moiety (e.g., a-thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. While not wishing to be bound by theory, phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0280] In specific instances, an alternative nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio- cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l- thiophosphate)-pseudouridine).

[0281] Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.

[0282] Combinations of Alternative Sugars, Nucleobases, and Intemucleoside Linkages

[0283] The polynucleotides of the disclosure can include a combination of alterations to the sugar, the nucleobase, and / or the internucleoside linkage. These combinations can include any one or more alterations described herein. mRNA: Poly-A tail

[0284] During RNA processing, a long chain of adenosine nucleotides (poly(A) tail) is normally added to mRNA molecules to increase the stability of the mRNA. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then poly(A) polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that is between 100 and 250 residues long (SEQ ID NO: 109).

[0285] Methods for the stabilization of RNA by incorporation of chain-terminating nucleosides at the 3 ’-terminus include those described in International Patent Publication No. WO2013 / 103659, incorporated herein in its entirety.

[0286] Poly(A) tail deadenylation by 3' exonucleases is a key step in cellular mRNA degradation in eukaryotes. By blocking 3' exonucleases, the functional half-life of mRNA can be increased, resulting in increased protein expression. Chemical and enzymatic ligation strategies to modify the 3' end of mRNA with reverse chirality adenosine (LA10) and / or inverted deoxythymidine (IdT) are known to those of skill in the art and have been demonstrated to extend mRNA half-life in cellular and in vivo studies. In some instances, the poly(A)tail of the mRNA includes a 3’ LA10 or IdT modification. For example, as described in International Patent Publication No. WO2017 / 049275, the tail modifications of which are incorporated by reference in their entirety.

[0287] Additional strategies have been explored to further stabilize mRNA, including: chemical modification of the 3’ nucleotide (e.g., conjugation of a morpholino to the 3’ end of the poly(A)tail); incorporation of stabilizing sequences after the poly(A) tail (e.g., a co-polymer, a stem-loop, or a triple helix); and / or annealing of structured oligos to the 3' end of an mRNA, as described, for example, in International Patent Publication No. WO2017 / 049286, the stabilized linkages of which are incorporated by reference in their entirety. Annealing an oligonucleotide (e.g., an oligonucleotide conjugate) with a complex secondary structure (e.g., a triple-helix structure or a stem-loop structure) at the 3 ’end may provide nuclease resistance and increase half-life of mRNA.

[0288] Unique poly(A) tail lengths may provide certain advantages to the RNAs of the present disclosure. Generally, the length of a poly(A) tail of the present disclosure is greater than 30 nucleotides in length. In some instances, the poly(A) tail is greater than 35 nucleotides in length. In some instances, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In some instances, the length is at least 50 nucleotides. In some instances, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 65 nucleotides. In another embodiment, the length is at least 70 nucleotides. In some instances, the length is at least 80 nucleotides. In some instances, the length is at least 90 nucleotides. In some instances, the length is at least 100 nucleotides. In some instances, the length is at least 120 nucleotides. In some instances, the length is at least 140 nucleotides. In some instances, the length is at least 160 nucleotides. In some instances, the length is at least 180 nucleotides. In some instances, the length is at least 200 nucleotides. In some instances, the length is at least 250 nucleotides. In some instances, the length is at least 300 nucleotides. In some instances, the length is at least 350 nucleotides. In some instances, the length is at least 400 nucleotides. In some instances, the length is at least 450 nucleotides. In some instances, the length is at least 500 nucleotides. In some instances, the length is at least 600 nucleotides. In some instances, the length is at least 700 nucleotides. In some instances, the length is at least 800 nucleotides. In some instances, the length is at least 900 nucleotides. In some instances, the length is at least 1000 nucleotides. In some instances, the length is at least 1100 nucleotides. In some instances, the length is at least 1200 nucleotides. In some instances, the length is at least 1300 nucleotides. In some instances, the length is at least 1400 nucleotides. In some instances, the length is at least 1500 nucleotides. In some instances, the length is at least 1600 nucleotides. In some instances, the length is at least 1700 nucleotides. In some instances, the length is at least 1800 nucleotides. In some instances, the length is at least 1900 nucleotides. In some instances, the length is at least 2000 nucleotides. In some instances, the length is at least 2500 nucleotides. In some instances, the length is at least 3000 nucleotides.

[0289] In some instances, the poly(A) tail may be 80 nucleotides, 120 nucleotides, or 160 nucleotides in length. In some instances, the poly(A) tail may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length.

[0290] In some instances, the poly(A) tail is designed relative to the length of the mRNA. This design may be based on the length of the coding region of the mRNA, the length of a particular feature or region of the mRNA, or based on the length of the ultimate product expressed from the RNA. When relative to any additional feature of the RNA (e.g., other than the mRNA portion which includes the poly(A) tail), poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature. The poly(A) tail may also be designed as a fraction of the mRNA to which it belongs. In this context, the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly (A) tail.

[0291] In some instances, engineered binding sites and / or the conjugation of nucleic acids or mRNA for poly(A) binding protein may be used to enhance expression. The engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the nucleic acids and / or mRNA. As a nonlimiting example, the nucleic acids and / or mRNA may include at least one engineered binding site to alter the binding affinity of poly(A) binding protein (PABP) and analogs thereof. The incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof.

[0292] Additionally, multiple distinct nucleic acids or mRNA may be linked together to the PABP (poly(A) binding protein) through the 3 '-end using nucleotides at the 3'- terminus of the poly(A) tail. Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr, and day 7 post-transfection. As a non-limiting example, the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site.

[0293] In some instances, a poly(A) tail may be used to modulate translation initiation. While not wishing to be bound by theory, the poly-A tail recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis.

[0294] In some instances, a poly(A) tail may also be used in the present disclosure to protect against 3 ’-5’ exonuclease digestion.

[0295] In some instances, the nucleic acids or mRNA of the present disclosure are designed to include a poly-A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant nucleic acid or mRNA may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the poly-A-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone (SEQ ID NO: 110).

[0296] In some instances, the nucleic acids or mRNA of the present disclosure may include a poly(A) tail and may be stabilized by the addition of a chain terminating nucleoside. The nucleic acids and / or mRNA with a poly(A) tail may further include a 5 ’cap structure.

[0297] In some instances, the nucleic acids or mRNA of the present disclosure may include a poly-A-G Quartet. The nucleic acids and / or mRNA with a poly-A-G Quartet may further include a 5 ’cap structure.

[0298] In some instances, the chain terminating nucleoside which may be used to stabilize the nucleic acid or mRNA including a poly(A) tail or poly-A-G Quartet may be, but is not limited to, those described in International Patent Publication No.

[0299] WO2013103659, incorporated herein by reference in its entirety. In some instances, the chain terminating nucleosides which may be used with the present disclosure includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxy cytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such as 2', 3'- dideoxyadenosine, 2', 3 '-di deoxyuridine, 2',3'-dideoxycytosine, 2', 3'- dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or a -O- methylnucleoside.

[0300] In some instances, the mRNA which includes a poly(A) tail or a poly-A-G Quartet may be stabilized by an alteration to the 3 ’region of the nucleic acid that can prevent and / or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013 / 103659, incorporated herein by reference in its entirety).

[0301] In yet another embodiment, the mRNA, which includes a poly(A) tail or a poly- A-G Quartet, may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’, 3 ’-dideoxynucleoside 3'-O-methylnucleosides, 3'-O- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein

[0302] Synthesis of Polynucleotides

[0303] The polynucleotide molecules (e.g., mRNA) for use in accordance with the disclosure may be prepared according to any useful technique, as described herein. The alternative nucleosides and nucleotides used in the synthesis of polynucleotide molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0304] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,JH or13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography) .

[0305] Preparation of polynucleotide molecules of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, (1991), which is incorporated herein by reference in its entirety.

[0306] The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out (i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

[0307] Resolution of racemic mixtures of alternative polynucleotides or nucleic acids (e.g., polynucleotides or mRNA molecules) can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art. Alternative nucleosides and nucleotides (e.g., building block molecules) can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res. 22(1): 72-78 (1994); Fukuhara et al., Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each of which are incorporated by reference in their entirety.

[0308] If the polynucleotide includes one or more alternative nucleosides or nucleotides, the polynucleotides of the disclosure may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly altered in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof. In some embodiments, all nucleotides X in a polynucleotide of the disclosure (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0309] Different sugar alterations, nucleotide alterations, and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. An alteration may also be a 5' or 3' terminal alteration. The polynucleotide may contain from 1% to 100% alternative nucleosides, nucleotides, or internucleoside linkages (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i. e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100. In some embodiments, the remaining percentage is accounted for by the presence of A, G, U, or C.

[0310] When referring to percentage incorporation by an alternative nucleoside, nucleotide, or internucleoside linkage, in some embodiments the remaining percentage necessary to total 100% is accounted for by the corresponding natural nucleoside, nucleotide, or internucleoside linkage. In other embodiments, the remaining percentage necessary to total 100% is accounted for by a second alternative nucleoside, nucleotide, or internucleoside linkage.

[0311] Nanoparticles

[0312] The present disclosure also provides LNP compositions. The lipid nanoparticle compositions described herein may be used for the delivery of therapeutic and / or prophylactic agents, e.g., mRNAs, to mammalian cells or organs. For example, the lipid nanoparticles described herein have little or no immunogenicity. For example, the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0313] In some instances, the present application provides pharmaceutical compositions comprising: (a) a delivery agent comprising a lipid nanoparticle; and (b) a polynucleotide encoding a binding molecule of this disclosure (e.g., a Fab that binds CD3E; a bispecific Fab that binds CD3E and a second antigen; a whole antibody that binds CD3E; multispecific antibody that binds CD3E and on or more antigens). Lipid Nanoparticles

[0314] In some instances, polynucleotides of the present disclosure (e.g., mRNA) are included in a lipid nanoparticle (LNP). Lipid nanoparticles according to the present disclosure may comprise: (i) an ionizable lipid (e.g., an ionizable amino lipid; (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG- modified lipid. In some instances, lipid nanoparticles according to the present disclosure further comprise one or more polynucleotides of the present disclosure (e.g., mRNA).

[0315] The lipid nanoparticles according to the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see, for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551;

[0316] PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129;

[0317] PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426;

[0318] PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117;

[0319] PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[0320] In some instances, the lipid nanoparticle comprises: (i) 20 to 60 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 25 to 55 mol.% sterol or other structural lipid, (iii) 5 to 25 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 0.5 to 15 mol.% PEG-modified lipid.

[0321] In some instances, the lipid nanoparticle comprises: (i) 40 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 30 to 45 mol.% sterol or other structural lipid, (iii) 5 to 15 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1 to 5 mol.% PEG-modified lipid.

[0322] In some instances, the lipid nanoparticle comprises: (i) 45 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 35 to 45 mol.% sterol or other structural lipid, (iii) 8 to 12 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1.5 to 3.5 mol.% PEG-modified lipid. In the following sections, “Compounds” numbered with an “I-” prefix (e.g., “Compound 1-1,” “Compound 1-2,” “Compound 1-3,” “Compound I- VI,” etc., indicate specific ionizable lipid compounds. Likewise, compounds numbered with a “P-” prefix (e.g., “Compound P-I,” etc.) indicate a specific PEG-modified lipid compound.

[0323] Ionizable Amino Lipids

[0324] In some instances, the lipid nanoparticle of the present disclosure comprises an ionizable cationic lipid (e.g., an ionizable amino lipid) that is a compound of Formula (I): r its N-oxide, or a salt or isomer thereof, wherein R’ais R’branched; wherein wherein ? denotes a point of attachment; wherein Ract, RaP, Ray, and Ra5are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;

[0325] R2and R3are each independently selected from the group consisting of C1-14 alkyl and

[0326] C2-14 alkenyl;

[0327] R4is selected from the group consisting of -(CH2)n0H, wherein n is selected from the group consistin wherein ? denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of Ci -6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0328] M and M’ are each independently selected from the group consisting of -C(O)O- and

[0329] -OC(O)-;

[0330] R’ is a C1-12 alkyl or C2-12 alkenyl;

[0331] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0332] In some instances, in Formula (I), R’ais R’branched; denotes a point of attachment; Ract, RaP, Ray, and Ra5are each H; R2and R3are each Ci-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each - C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0333] In some instances, in Formula (I), R’ais R’branched; ^’branched -sdenotes a point of attachment; Ract, RaP, Ray, and Ra5are each H; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 3; and m is 7. In some instances of the compounds of Formula (I), R’ais R’branched; point of attachment; Ractis C2-12 alkyl; RaP, Ray, and Ra5are each H; R2and R3are each C1-14 alkyl; R4is alkyl); n2 is 2; R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a Ci -12 alkyl; 1 is 5; and m is 7.

[0334] In some instances of the compounds of Formula (I), R’ais R’branched; denotes a point of attachment; Ract, RaP, and Ra5are each H; Rayis C2-12 alkyl; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0335] In some instances, the compound of Formula (I) is selected from:

[0336]

[0337] In some instances, the compound of Formula (I) is:

[0338] In some instances, the compound of Formula (I) is: (Compound 1-2).

[0339] In some instances, the compound of Formula (I) is: (Compound 1-3).

[0340] Phospholipids The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0341] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0342] Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid- containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[0343] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0344] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[0345] In some instances, a phospholipid of the present disclosure comprises 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1 ,2-diphytanoyl- sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0346] In certain instances, a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC.

[0347] Alternative Lipids

[0348] In certain instances, a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain instances, a phospholipid useful.

[0349] In certain instances, an alternative lipid is used in place of a phospholipid of the present disclosure.

[0350] In certain instances, an alternative lipid of the present disclosure is oleic acid. In certain instances, the alternative lipid is one of the following:

[0351]

[0352] Structural Lipids

[0353] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term "structural lipid" refers to sterols and also to lipids containing sterol moieties.

[0354] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some instances, the structural lipid is a sterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. In certain instances, the structural lipid is a steroid. In certain instances, the structural lipid is cholesterol. In certain instances, the structural lipid is an analog of cholesterol. In certain instances, the structural lipid is alpha-tocopherol.

[0355] In some instances, the structural lipids may be one or more of the structural lipids described in U.S. Application No. 62 / 520,530.

[0356] Polyethylene Glycol (PEG)-Lipids

[0357] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.

[0358] As used herein, the term “PEG-lipid” refers to polyethylene glycol (PEG)- modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3 -amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0359] In some instances, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3 -amine (PEG-c-DMA).

[0360] In some instances, the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, and mixtures thereof.

[0361] In some instances, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about Ci6. In some instances, a PEG moiety, for example an mPEG-N H2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some instances, the PEG-lipid is PEG2k-DMG

[0362] In some instances, the PEG-lipid has the formula:

[0363] (134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99, 102, 105, 108, 111 , 114, 117, 120, 123, 126, 129, 132- tetratetracontaoxatetratriacontahectyl stearate).

[0364] In some instances, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. PEG-lipids are known in the art, such as those described in U.S. Patent No. 8,158,601 and International Publ. No. WO 2015 / 130584 A2, which are incorporated herein by reference in their entirety.

[0365] In general, some of the other lipid components (e.g., PEG lipids) of various Formulae, described herein may be synthesized as described International Patent Application No. PCT7US2016 / 000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.

[0366] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0367] In some instances, PEG lipids useful in the present disclosure can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain instances, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG- OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain instances, the PEG- OH lipid includes one or more hydroxyl groups on the PEG chain. In certain instances, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present disclosure. In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0368] In some instances, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. 62 / 520,530.

[0369] In some instances, a PEG lipid of the present disclosure comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, and mixtures thereof. In some instances, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and / or PEG-DPG.

[0370] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.

[0371] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid.

[0372] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[0373] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[0374] and an alternative lipid comprising oleic acid.

[0375] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid.

[0376] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid.

[0377] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[0378] a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid.

[0379] In some instances, a LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1. In some instances, a LNP of the present disclosure comprises an N:P ratio of about 6: 1. In some instances, a LNP of the present disclosure comprises an N:P ratio of about 3:1. In some instances, a LNP of the present disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of from about 10: 1 to about 100: 1. In some instances, a LNP of the present disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 20: 1. In some instances, a LNP of the present disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10: 1. In some instances, a LNP of the present disclosure has a mean diameter from about 50nm to about 150nm. In some instances, a LNP of the present disclosure has a mean diameter from about 70nm to about 120nm.

[0380] Other Lipid Composition Components

[0381] The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. Lor example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. Lor example, a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof). A polymer can be included in and / or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and / or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[0382] The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt / wt).

[0383] In some instances, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1,21:1,22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt / wt). In some instances, the wt / wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20: 1 or about 15:1.

[0384] In some instances, the pharmaceutical composition disclosed herein can contain more than one polypeptides. For example, a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).

[0385] In some instances, the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid: polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20: 1, from about 5: 1 to about 25: 1, from about 5: 1 to about 30: 1, from about 5: 1 to about 35:1, from about 5: 1 to about 40: 1, from about 5: 1 to about 45: 1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35: 1, from about 10:1 to about 40: 1, from about 10: 1 to about 45: 1, from about 10: 1 to about 50: 1, from about 10: 1 to about 55:1, from about 10: 1 to about 60:1, from about 10: 1 to about 70:1, from about 15: 1 to about 20:1, from about 15:1 to about 25: 1, from about 15: 1 to about 30:1, from about 15: 1 to about 35: 1, from about 15:1 to about 40: 1 , from about 15:1 to about 45:1, from about 15: 1 to about 50:1, from about 15:1 to about 55: 1, from about 15:1 to about 60: 1 or from about 15:1 to about 70: 1.

[0386] In some instances, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg / ml to 2 mg / ml such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml or greater than 2.0 mg / ml.

[0387] Nanoparticle Compositions

[0388] In some instances, the pharmaceutical compositions disclosed herein are Formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as a compound as described herein, and (ii) a polynucleotide encoding a binding molecule described herein. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding a binding molecule of the disclosure.

[0389] Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0390] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some instances, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain instances, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and / or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

[0391] In some instances, a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some instances, the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid. In some instances, the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG-modified lipid.

[0392] In some instances, the lipid nanoparticle comprises 47-49 mol.% ionizable cationic lipid (e.g. ionizable amino lipid, e.g., Compound 1-1, Compound 1-2, or Compound 1-3), 10-12 mol.% non-cationic lipid (e.g., phospholipid, e.g., DSPC), 38-40 mol.% sterol (e.g., cholesterol) or other structural lipid, and 1-3 mol.% PEG-modified lipid (e.g., PEG-DMG or Compound P-I).

[0393] For instance, in some instances, the lipid nanoparticle (“LNP-1”) may comprise the following components at the following molar ratios:

[0394] (i) 45-50 mol.% Compound 1-1

[0395] (ii) 35-45 mol.% sterol (e.g., cholesterol);

[0396] (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and

[0397] (iv) 1.5-3.5 mol.% PEG-lipid.

[0398] For instance, in some instances, the lipid nanoparticle (“LNP-1 A”) may comprise the following components at the following molar ratios:

[0399] (i) 45-50 mol.% Compound 1-1

[0400] (ii) 35-45 mol.% Cholesterol;

[0401] (iii) 8-12 mol.% DSPC; and

[0402] (iv) 1.5-3.5 mol.% PEG-lipid.

[0403] For instance, in some instances, the lipid nanoparticle (“LNP- IB”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound 1-1

[0404] (ii) 35-45 mol.% Cholesterol;

[0405] (iii) 8-12 mol.% DSPC; and

[0406] (iv) 1.5-3.5 mol.% PEG-lipid.

[0407] In some instances, the lipid nanoparticle (“LNP-2”) may comprise the following:

[0408] (i) 45-50 mol.% Compound 1-2;

[0409] (ii) 35-45 mol.% sterol (e.g., Cholesterol);

[0410] (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and

[0411] (iv) 1.5-3.5 mol.% PEG-lipid.

[0412] In some instances, the lipid nanoparticle (“LNP-2A”) may comprise the following:

[0413] (i) 45-50 mol.% Compound 1-2;

[0414] (ii) 35-45 mol.% Cholesterol;

[0415] (iii) 8-12 mol.% DSPC; and

[0416] (iv) 1.5-3.5 mol.% PEG-lipid.

[0417] For instance, in some instances, the lipid nanoparticle (“LNP-2B”) may comprise the following components at the following molar ratios:

[0418] (i) 45-50 mol.% Compound 1-2;

[0419] (ii) 35-45 mol.% Cholesterol;

[0420] (iii) 8-12 mol.% DSPC; and

[0421] (iv) 1.5-3.5 mol.% PEG-lipid.

[0422] In some instances, the lipid nanoparticle (“LNP-3”) may comprise the following:

[0423] (i) 45-50 mol.% Compound 1-3;

[0424] (ii) 35-45 mol.% sterol (e.g., Cholesterol);

[0425] (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and

[0426] (iv) 1.5-3.5 mol.% PEG-lipid.

[0427] In some instances, the lipid nanoparticle (“LNP-3 A”) may comprise the following: (i) 45-50 mol.% Compound 1-3;

[0428] (ii) 35-45 mol.% Cholesterol;

[0429] (iii) 8-12 mol.% DSPC; and

[0430] (iv) 1.5-3.5 mol.% PEG-lipid.

[0431] In some instances, the lipid nanoparticle (“LNP-3B”) may comprise the following:

[0432] (i) 45-50 mol.% Compound 1-3;

[0433] (ii) 35-45 mol.% Cholesterol;

[0434] (iii) 8-12 mol.% DSPC; and

[0435] (iv) 1.5-3.5 mol.% PEG-lipid.

[0436] In some instances, the LNP has a poly dispersity value of less than 0.4. In some instances, the LNP has a net neutral charge at a neutral pH. In some instances, the LNP has a mean diameter of 50-150 nm. In some instances, the LNP has a mean diameter of 80-100 nm.

[0437] As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.

[0438] In some instances, a lipid nanoparticle (LNP) may comprise an ionizable amino lipid. As used herein, the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some instances, an ionizable amino lipid may be positively charged or negatively charged. An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain instances, an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

[0439] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given their ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[0440] The ionizable amino lipid is sometimes referred to in the art as an “ionizable cationic lipid”. In some instances, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

[0441] In addition to these, an ionizable amino lipid may also be a lipid including a cyclic amine group.

[0442] In some instances, the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety. In yet another embodiment, the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXXII of US Patent No. 7,404,969; each of which is herein incorporated by reference in their entirety.

[0443] In some instances, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety. In some instances, the lipid may be synthesized by methods known in the art and / or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.

[0444] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0445] The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.

[0446] As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

[0447] In some instances, the polynucleotide encoding a polypeptide(s) described herein are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm.

[0448] In some instances, the nanoparticles have a diameter from about 10 to 500 nm. In some instances, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[0449] In some instances, the largest dimension of a nanoparticle composition is 1 pm or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

[0450] A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some instances, the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20. The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.

[0451] For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

[0452] The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N:P ratio can serve as a useful metric.

[0453] As the N:P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

[0454] In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2: 1 to about 30: 1, such as 2:1, 3: 1, 4:1, 5: 1, 6:1, 7: 1, 8:1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18:1, 20:1, 22: 1, 24:1, 26: 1, 28:1, or 30: 1. In certain instances, the N:P ratio can be from about 2: 1 to about 8:1. In other instances, the N:P ratio is from about 5: 1 to about 8: 1. In certain instances, the N:P ratio is between 5: 1 and 6: 1. In one specific aspect, the N:P ratio is about is about 5.67:1.

[0455] In some instances, the polynucleotides described herein can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some instances, the polynucleotides can be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround or encase. As it relates to the formulation of the compounds of the present disclosure, encapsulation can be substantial, complete or partial. The term "substantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent. "Partial encapsulation" or “partially encapsulate” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent.

[0456] In some instances, the therapeutic nanoparticle polynucleotide can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time can include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle of the polynucleotides described herein can be formulated as disclosed in Inti. Pub. No. W02010075072 and U.S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety.

[0457] In some instances, the therapeutic nanoparticle polynucleotide can be formulated to be target specific, such as those described in Inti. Pub. Nos. WO2008121949, W02010005726, W02010005725, WO2011084521 and WO2011084518; and U.S. Pub. Nos. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in its entirety.

[0458] The LNPs can be prepared using microfluidic mixers or micromixers. Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM) (see, Zhigaltsev et al., Langmuir. 28:3633-40 (2012); Belliveau et al., Molecular Therapy-Nucleic Acids. I:e37 (2012); Chen et al., J. Am. Chem. Soc. 134(16):6948-51 (2012); each of which is herein incorporated by reference in its entirety). Exemplary micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany. In some instances, methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Pub. Nos.

[0459] US20040262223 and US20120276209, each of which is incorporated herein by reference in their entirety.

[0460] In some instances, the polynucleotides described herein can be Formulated in lipid nanoparticles using microfluidic technology (see, Whitesides, George M., Nature 442: 368-373 (2006); and Abraham et al., Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety). In some instances, the polynucleotides can be Formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[0461] In some embodiment, the nanoparticles described herein are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Pub. No. US20130172406, herein incorporated by reference in its entirety. The stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, or combinations thereof.

[0462] Methods of Treatment

[0463] The binding molecules or polynucleotides described herein can be administered to a subject in need thereof. In some aspects, the subject in need thereof is a subject with a disease, disorder, and / or condition that may be treated with the binding molecules or polynucleotides described herein. Specifically, a disease or disorder which would benefit from targeting cells expressing anti-CD3 (e.g., T cells) for activation and / or targeting of any other antigen in combination with targeting cells expressing anti-CD3 (e.g., by using a multispecific antibody of the present disclosure).

[0464] In some aspects, the subject in need thereof is a subject with an infection, an inflammatory condition, an autoimmune condition, and / or cancer.

[0465] In some aspects, the subject in need thereof is a subject with cancer. Certain cancers that may be treated in accordance with the binding molecules of the present disclosure include, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, and myeloid leukemia), lymphoma (e.g., Burkitt lymphoma (non-Hodgkin lymphoma), cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, Sezary syndrome, AIDS-related lymphoma, follicular lymphoma, diffuse large B-cell lymphoma), melanoma, merkel cell carcinoma, mesothelioma, myeloma (e.g., multiple myeloma), myelodysplastic syndrome, papillomatosis, paraganglioma, pheochromocytoma, pleuropulmonary blastoma, retinoblastoma, sarcoma (e.g., Ewing sarcoma, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, vascular sarcoma), Wilms' tumor, and / or cancer of the adrenal cortex, anus, appendix, bile duct, bladder, bone, brain, breast, bronchus, central nervous system, cervix, colon, endometrium, esophagus, eye, fallopian tube, gall bladder, gastrointestinal tract, germ cell, head and neck, heart, intestine, kidney (e.g., Wilms' tumor), larynx, liver, lung (e.g., non-small cell lung cancer, small cell lung cancer), mouth, nasal cavity, oral cavity, ovary, pancreas, rectum, skin, stomach, testes, throat, thyroid, penis, pharynx, peritoneum, pituitary, prostate, rectum, salivary gland, ureter, urethra, uterus, vagina, or vulva. In some aspects, the subject in need thereof is a subject with multiple myeloma. In some aspects, the subject in need thereof is a subject with a solid tumor. In some aspects, the subject in need thereof is a subject with ovarian cancer.

[0466] In some aspects, the technologies of the present disclosure can be practiced in a subject in need thereof that is likely to benefit from administration of technologies of the present disclosure (e.g., a subject with an autoimmune disease, a subject with cancer).

[0467] In some instances, a subject in need thereof is a human. In some embodiments, the human is male. In some instances, the human is female. In some instances, the human is an adult (e.g., 18 or more years of age). In some instances, the adult is greater than 18 years old, greater than 25 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 55 years old, greater than 60 years old, greater than 65 years old, greater than 70 years old, greater than 75 years old, greater than 80 years old, greater than 85 years old, greater than 90 years old, greater than 95 years old, greater than 100 years old, or greater than 105 years old in age. In some instances, the human is a child. In some instances, the child is greater than 2 years old, greater than 3 years old, greater than 4 years old, greater than 5 years old, greater than 6 years old, greater than 7 years old, greater than 8 years old, greater than 9 years old, greater than 10 years old, greater than 11 years old, greater than 12 years old, greater than 13 years old, greater than 14 years old, greater than 15 years old, or greater than 16 years old in age.

[0468] In some aspects, a subject in need thereof is administered a binding molecule(s) or a polynucleotide(s) of the present disclosure. These therapeutic agents can be administered to a subject (e.g., a mammalian subject, such as a human) by a variety of routes. In some embodiments, the binding molecule, humanized antibody, antigen binding fragment, binding polypeptide or nucleic acid is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, intrathecally, intracerebroventricularly, transdermally, or orally.

[0469] The most suitable route for administration in any given case will depend on the particular therapeutic agent administered, the patient, pharmaceutical formulation methods, and various patient-specific parameters, such as the patient's age, body weight, sex, severity of the diseases being treated, the patient’s diet, and the patient’s excretion rate.

[0470] An appropriate dosage of humanized anti-CD3 antibodies, antigen binding fragments, or binding proteins, or nucleic acids encoding the same of the present disclosure will vary with the particular condition, disease and / or disease being treated, various subject-specific parameters (e.g., age, weight, physical condition of the subject), severity of the particular condition, disease, and / or disorder being treated, the nature of current or combination therapy (if any), the specific route of administration and other factors within the knowledge and expertise of a health practitioner. In some embodiments, a maximally tolerated dose of technologies described herein is to be used, e.g., the highest safe dose according to sound medical judgement. In some embodiments, technologies described herein are administered in an effective amount, e.g., a dose sufficient to provide one or more medically desirable results.

[0471] Kits Also included herein are kits that contain a binding molecule(s) and / or polynucleotides (e.g., mRNA) encoding the same. In some instances, the kits provided herein contain one or more cells engineered to express and secrete a polypeptide(s) comprising an anti-CD3E binding molecule of the disclosure, such as a cell containing a nucleic acid molecule(s) of the disclosure.

[0472] A kit described herein may include reagents that can be used to produce a pharmaceutical composition of the invention. Optionally, kits described herein may include reagents that can induce the expression of a polypeptide(s) comprising an anti- CD3E binding molecule of the disclosure within cells (e.g., bacterial, yeast, insect, or mammalian cells).

[0473] Other kits described herein may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E. Coli cell or an immune cell) so as to express and secrete a polypeptide(s) comprising an anti-CD3E binding molecule of the disclosure. For example, a kit may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art. The kit may also contain a nucleic acid encoding the desired binding molecule as well as reagents for expressing the binding molecule in the cell.

[0474] A kit described herein may also provide a polypeptide(s) comprising an anti- CD3E binding molecule of the disclosure, or a nucleic acid encoding the same (e.g., mRNA) in combination with a package insert describing how the binding molecule, or nucleic acid may be administered to a subject, for example, for the treatment of a disease, disorder and / or condition (e.g, cancer).

[0475] EXAMPLES

[0476] Example 1 : Materials and Methods

[0477] In silico design'. SP34 is an anti-CD3 antibody isolated from mouse. It binds both human and cyno monkey CD3. It binds to CD3e alone. The mouse SP34 antibody was humanized similar to the method of Hanf et al. (Methods 2014 Jan 1 ;65(1): 68-76). Briefly sequences of the complementarity-determining regions (CDRs) of SP34 were annotated using the IMGT numbering scheme. Each nucleotide sequence of SP34 was generated and used to identify the nearest human germline VH or VL sequences by searching for similar sequences with the NCBI IgBLAST program. Common J and D gene sequences were attached to the VEI / VL as the acceptor. Next the most similar human VEI / VL sequences are identified using BLASTp and used to choose the nearest framework sequences into which the CDR sequences are grafted replacing the human CDRs.

[0478] Rosetta or Alpha fold was used to create the structural 3D homology model of the appropriate CDRs that were grafted into the acceptor framework. The framework residues that were potentially structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues back to mouse, or by mutating some residues on the CDRs’ backside to human or to a de novo designed sequence. CDR stabilizing or overall fold stabilizing sequences were then back-mutated to the corresponding mouse sequence to maintain the biophysical properties and target binding affinity. A list of humanized variants of VH and VL were selected based on modeled structure. Fab fragments were produced by combining VH and VL variants for biophysical characterization and affinity determination. The Fab was constructed by coexpression of heavy and light chains. The heavy chain comprises the VH domain followed by human IgGl CHI domain which in turn is followed by the upper hinge region of human IgGl and the light chain comprises the VL domain followed by human constant lambda chain.

[0479] Small Scale Fab Expression'. The Fab-His molecules with Flag tag fused to the N- terminus of heavy chain and His tag fused to the C-terminus of the light chain were transiently expressed in ExpiCHO cells using the EpxiF ectamine CHO (Thermofisher) transfection reagents. The ExpiCHO-S cells were cultured in ExpiCHO expression medium (ThermoFisher Scientific) in a shaker incubator set at 125 rpm, 37° C and 8% C02. The day prior to transfection, ExpiCHO-S cells were seeded at 3 x 106cells per ml in 1 ml of ExpiCHO Expression medium. On the day of transfection cells were split using pre- warmed (37° C) ExpiCHO expression media to a density of 6 x 106cells per ml. Using the manufacturer’s recommended protocol, a total 1 pg of plasmids at 1 : 1 ratio between heavy and light chain and 3.2 pL of ExpiFectamine CHO reagent were mixed in 40 pl of cold Opti-PRO SFM (Thermo Fisher Scientific), after incubating the mixture for 2 minutes it was then slowly added to the cells. One day (~20h) post-transfection, 240 pl of ExpiCHO Feed and 6 pl of ExpiCHO Enhancer were added to the ExpiFectamine- transfected cultures. Culture supernatants were harvested when cell viability dropped below 60% (-day 8). The Fab expression is evaluated with western blot by anti-Flag and anti-His antibody. koff Ranking and Affinity Determination by SPR: SPR koff ranking were carried out on a Biacore T200 (Cytiva) at 25°C. 5000RU goat anti-human Fab polyclonal antibody from Abcam(ab98531) was covalently coated to CM5 sensor chip by amine-coupling using the coupling kit (Cytiva). Expressed Fab-His soups diluted 1: 100 in HBS-EP+ buffer were injected over flow cell at the flow rate of 30 pl / min. A buffer injection served as a negative control. Channel 1 was blocked by ethanolamide and served as a reference channel. CD3e / CD3d heterodimer were injected over each flow cell at the flow rate of 30 pl / min in HBS-EP+ buffer at 2 concentrations: 200 nM and 100 nM for 3min, followed by dissociation in the same buffer for 5 min. A buffer injection served as a negative control. Upon completion of each association and dissociation cycle, surfaces were regenerated with 10 nM glycine pH 2.5 solution. The association rates (ka), dissociation rate constants (ka), and affinity constants (KD) were calculated using Biacore T200 evaluation software. Each fit was evaluated by the agreement between experimental data and the calculated fits, where the Chi2 values were below 10% of Rmax. Fab expression and purification: The Fab-His molecules with His tag fused to the C- terminus of the light chain were transiently expressed in ExpiCHO cells using the ExpiFectamine CHO (Thermofisher) transfection reagents. The ExpiCHO-S cells were cultured in ExpiCHO expression medium (ThermoFisher Scientific) in a shaker incubator set at 125 rpm, 37° C and 8% CO2. The day prior to transfection, ExpiCHO-S cells were seeded at 3 x 106cells per ml in 30 ml of ExpiCHO Expression medium. On the day of transfection cells were split using pre- warmed (37° C) ExpiCHO expression media to a density of 6 x 106cells per ml. Using the manufacturer’s recommended protocol, a total 20 pg of plasmids at 1:1 ratio between heavy and light chain and 72 pL of ExpiFectamine CHO reagent were mixed in 2.4 ml of cold Opti-PRO SFM (Thermo Fisher Scientific), after incubating the mixture for 2 minutes it was then slowly added to the cells. One day (~20 h) post-transfection, 7.2 ml of ExpiCHO Feed and 180 pl of ExpiCHO Enhancer were added to the ExpiFectamine-transfected cultures. Culture supernatants were harvested when cell viability dropped below 60% (-day 8), clarified by centrifugation at 3,000 rpm for 30 min and filtered using a 0.2 pm filter (Thermo Fisher Scientific).

[0480] Expressed single domain antibodies with a 6xHis (SEQ ID NO: 58) C-terminal tag were purified from ExpiCHO media by capture on TALON (TAKRA) cobalt immobilized metal affinity chromatography (IMAC) resin. Resin was prewashed in 50 mM sodium phosphate buffer pH7.4 (wash buffer) with 15x the volume of resin, then centrifuged for 2 min @ 700 x g to pellet the resin. The supernatant discarded and the resin wash repeated. To the filtered media containing the expressed protein was added 3 mL of IMAC resin, then allowed to bind overnight with gentle shaking. The media resin mixture was gravity loaded into a 25 ml column. Columns containing IMAC resin were then washed with 10 column volumes (-30 ml) of wash buffer (PBS pH 7.4 (Life Tech cat# 10010-023), 2 mM imidazole) then 2x with 10ml of wash buffer. Protein was then eluted using a total of 7.5 mL 150mM imidazole / PBS pH 7 in three 2.5 mL aliquots. Proteins were then dialyzed exhaustively using Slide- A-Lyzer® 10 or 3K as appropriate, (Dialysis cassette, Pierce) versus 1 x PBS (100 mM NaP04 pH 6.8, 200 mM NaCl).

[0481] Western Blot: SDS PAGE was run on each sample using gradient gels NuPAGE Bis-Tris 4-12% gradient gels using a MES running buffer (Thermo Fisher Scientific). Samples were prepared with reducing sample buffer and briefly heated to 95° C. Molecular weight standards (Blue Plus protein, Thermo Fisher) were included on the SDS-PAGE. The gel was transferred to nitrocellulose membrane using iBLot 2 gel transfer device following supplier manual (Thermal Fisher). The membrane was stained with 1 : 1 mixture of HRP conjugated anti -Flag M2 antibody (Abeam) and HRP Penta.His antibody (Qiagen) using iBind following company manual (Thermo Fisher).

[0482] Size Exclusion Chromatography (SEC): Analysis of native molecule homogeneity and determination of molecular weight was done using Size Exclusion Chromatography with Light Scattering (SEC-LS) when indicated. Size exclusion chromatography (SEC) was carried out on a Zenix SEC 300 4.6 x 300 mm (Sepax Technologies) in 20 mM sodium phosphate pH 7.2, 150 mM NaCl (PBS), 0.05% NaAzide at a flow rate of 0.35ml / min using an Agilent 1260 UPLC. In addition to UV detection, the eluent was monitored with a refractive index detector (Waters, Milford, MA). Light scattering was monitored using a Wyatt Dawn 18 angle, coupled with an Optrex refractometer. Intact mass of molecules was determined mass spectrometry (Merrigen, Lowell MA).

[0483] FACS Binding Affinity: Jurkat cell line that stably expresses CD3 were resuspended in FACS buffer (PBS pH 7.4 + 2% FBS +1 mM EDTA) at 4,000,000 cells / ml, and 50 pl of the cell suspension was mixed in a 96-well V bottom polypropylene plate (Nunc) with 50 p 1 of Fab-His protein in the same buffer. After one-hour incubation at 4°C, pelleted cells were washed 2x by ice cold FACS buffer and then resuspended in lOOul anti-His-iFluor 647 from GenScript at 1 : 100 dilution in FACS buffer, incubated 15 min at 4°C, centrifuged at 1000 rpm for 3 min; cell pellets were washed, fixed with 200 pl / well 1% paraformaldehyde for lOmin at room temperature, centrifuged, the fix solution was decanted and cell pellets were resuspended in 200 pl FACS buffer for analysis. Cellular fluorescence was determined on Canto equipped with FlowJo software (Becton- Dickinson, Franklin Lakes, NJ). The receptor EC50 values were determined by fitting the data with a single site total binding model using GraphPad Prism 6.0 (La Jolla, CA).

[0484] Melting Temperature (Tm) Determination with Differential Scanning Fluorimetry (DSF): Tmwere determined with Protein Thermal Shift Dye Kit (Thermo 4461146). Fab proteins were diluted to 0.3 mg / ml with PBS. Protein Thermal Shift Dye (lOOOx) was diluted to 8x in a tube. A MicroAmp Fast Optical 96-Well Reaction Plate was put on ice followed by adding 5 pl buffer, 12.5 pl protein and then 2.5 pl 8x dye in each well. The plate was sealed and then centrifuged at 1000 rpm for 1 min. Fluorescence measurements were performed using a StepOnePlus™ Real-Time PCR System instrument. During the DSF experiment, the temperature was increased from 25 °C to 99 °C at continuous ramp mode. Tmwas defined as the temperature corresponding to the maximum value of the first derivative of fluorescence transition.

[0485] Example 2: SP34 Humanization

[0486] The mouse SP34 antibody (see e.g., WO 2007 / 042261, which is incorporated by reference herein) is a cross species anti-CD3 antibody. It can be used in different types of antibody designs (e.g., bispecific antibody design). Here, SP34 was humanized based on the method of Hanf et al. (2014, cited above). Following in silico analysis, three variants of VH (Flag-SP34-VHCHl-hl to Flag-SP34-VHCHl-h3) and 10 variants of VL (SP34- VLClam-hl-His to SP34-VLClam-hlO-His) were designed. Amino acid sequences for such variants of VH and VL are shown in Table 3. In addition, FIG. 4 provides an alignment of the amino acid sequences of these humanized VH and VL with the murine VH and VL of parental SP34. By combining heavy and light chains, 44 Fab variants (including wild type VH and VL) were transiently transfected into EXPI-CHO cell line and the expression was confirmed by western blot (FIG. 1). Two 26- lane gel were run as described in the methods with two panels in each gel. Each panel contains one marker lane and one positive expression control lane and 11 samples with the same heavy chain. The position of marker lane is different in the two gels to set the two gels apart. The constructs were designed in that the heavy chain was labeled with Flag tag and the light chain was labeled with His tag. The detection antibody is the mixture of anti-flag and anti-His antibodies so that heavy chain (28kDa band), light chain (24 kDa) and intact heterodimer (52 kDa band) that did not get completed reduced are all stained. Heavy chain band and light chain band were not completely resolved due to the resolution of the gel. All transfections showed equivalent expression level.

[0487] BiaCore binding assay was carried out to rank the humanized variants based on dissociation constant koff. CM5 chip were loaded with 5000RU goat anti-human Fab polyclonal antibody from Abcam(ab98531). The soup is captured and stabilized for 1 min. 200nM and lOOnM CD3e / d was flowed for 3 min and dissociated for 5 min. The experiment was carried out with un-purified samples with limited concentration series. Therefore, all numbers generated in this experiment were used to rank the molecules only. The absolute value of KD should not be taken as the final determination of affinity. The experiment used three controls: wild type SP34 (HC-hO / LC-hO), purified Control 1 humanized SP34 and UCHT1. The amino acid sequence of Control 1 humanized SP34 is provided below: VH Amino Acid Sequence: EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRS I<YNNYATYYADSVI<DRFTISRDDSI<SSLYLQMNNLI<TEDTAMYYCVRHGNFGN SYVSWFAYWGQGTLVTVSS (SEQ ID NO:59)

[0488] VL Amino Acid Sequence: QAWTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAFRGLIGGTN

[0489] I<RAPGVPARFSGSLIGDI<AALTITGAQADDESIYFCALWYSNLWVFGGGTI<LTV

[0490] L (SEQ ID NO:60) The amino acid sequence of UCHT1 is provided below:

[0491] VH Amino Acid Sequence:

[0492] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINP

[0493] YKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSD WYFDVWGQGTTLTVFS (SEQ ID NO:61)

[0494] VL Amino Acid Sequence:

[0495] MDIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRL

[0496] HSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK (SEQ ID NO:62)

[0497] SPR confirmed all humanized variants have similar or slower koff to CD3e / d heterodimer comparing to wild type SP34 (Table 8).

[0498] Table 8: BiaCore Koff Ranking

[0499] All constructs with HC-h3 exhibited a one magnitude slower dissociation than other heavy chains. There was no clear trend for light chain variants. Since SP34 mainly used heavy chain to interact with CD3e, it is expected that the light chain mainly stabilizes heavy chain folding instead of making direct contribution to the interaction.

[0500] FACS binding analysis with crude Fab expression soup to Jurkat cells showed similar results (Table 9). CHO cells that do not express CD3 were used as a negative control for binding and in all cases humanized variants showed negative FACS binding. Purified UCHT1 and Control 1 humanized SP34 were used as positive controls. The purified control antibodies do show a higher percentage of staining with higher MFI, most likely due to the higher concentration than 1: 10 diluted expression soup.

[0501] Table 9: FACS Binding

[0502] Based on small scale koff ranking and FACS binding assay, 5 variants were selected for purification and characterization. Since the light chain utilized did not make a big difference in the koff ranking assay, the most human sequence (LC-hl) was selected to pair with 3 different heavy chain variants, which are B2, C2 and D2 variants. Two more variants (Bl 1 and Cl 1) were also selected with the least human light chain. B2 and Cl 1 were repeated to ensure data reproducibility. Parental SP34 (Al) was also included as a positive control. SDS PAGE and SEC profile confirms that all 5 constructs perform similarly as parental Fab (Al) (FIG. 2), especially for B2 and D2 variants. Other three have small percentage of light chain overexpression.

[0503] FACS binding titration to CD3+ Jurkat cells (FIG. 3) indicated B2 and D2 having slightly better EC50 than parental SP34. C2 and Bl 1 have slightly worse EC50 while Cl 1 is ~3 fold worse. Bmax is correlated with EC50 values for each variant. BiaCore binding kinetics confirmed similar trend at binding to CD3e / CD3d heterodimer (Table 10). Expression, retention of thermostability and affinity demonstrate a successful humanization of the SP34 murine monoclonal antibody. B2 was selected for further engineering due to the similarity to parental SP34 and humanness and named as “ModSP34”. ModSP34 comprises a VH and a VL with SEQ ID NOs: 2 and 6, respectively (see, Table 3). The corresponding Fab comprises SEQ ID Nos.: 17 and 21 (see, Table 4).

[0504] Table 10: BiaCore Binding of Purified Fab

[0505]

Claims

WHAT IS CLAIMED IS:

1. A binding molecule comprising a polypeptide or polypeptides that bind CD3 epsilon, the polypeptide or polypeptides comprising a humanized heavy chain variable domain (VH) and a humanized light chain variable domain (VL), wherein the VH comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences set forth in SEQ ID NOs:2-4 and the VL comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 6- 15; wherein the VH comprises the VH CDR 1, 2, and 3 of SEQ ID NO:2 and the VL comprises the VL CDR 1, 2, and 3 of SEQ ID NO:6; wherein the VH does not comprise the amino acid sequence set forth in SEQ ID NO: 1 and the VL does not comprise the amino acid sequence set forth in SEQ ID NO:5; and wherein the VH and VL are not identical to any VH or VL sequence listed in WO 2007 / 0422261, US 2017 / 0355767, US10407501, US 9587021 , or US 8236308, optionally wherein the polypeptide or polypeptides bind both human and cynomolgus CD3 epsilon.

2. The binding molecule of claim 1, wherein the humanized VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NOs: 2, 3 or 4, and / or the humanized VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 6 to 15.

3. The binding molecule of claim 1 or 2, wherein:(a) the humanized VH comprises the amino acid sequence of SEQ ID NO:2 and the humanized VL comprises the amino acid sequence of SEQ ID NO:6;(b) the humanized VH comprises the amino acid sequence of SEQ ID NO: 2 and the humanized VL comprises the amino acid sequence of SEQ ID NO:7;(c) the humanized VH comprises the amino acid sequence of SEQ ID NO:2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 8;(d) the humanized VH comprises the amino acid sequence of SEQ ID NO: 2 and the humanized VL comprises the amino acid sequence of SEQ ID NOV;(e) the humanized VH comprises the amino acid sequence of SEQ ID NO:2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 10;(f) the humanized VH comprises the amino acid sequence of SEQ ID NO:2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 11 ;(g) the humanized VH comprises the amino acid sequence of SEQ ID NO: 2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 12;(h) the humanized VH comprises the amino acid sequence of SEQ ID NO: 2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 13;(i) the humanized VH comprises the amino acid sequence of SEQ ID NO:2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 14;(j) the humanized VH comprises the amino acid sequence of SEQ ID NO:2 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 15;(aa) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO:6;(bb) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO:7;(cc) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 8;(dd) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NOV;(ee) the humanized VH comprises the amino acid sequence of SEQ ID NOV and the humanized VL comprises the amino acid sequence of SEQ ID NOVO;(ff) the humanized VH comprises the amino acid sequence of SEQ ID NOV and the humanized VL comprises the amino acid sequence of SEQ ID NO: 11 ;(gg) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 12;(hh) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 13;(ii) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 14;(jj) the humanized VH comprises the amino acid sequence of SEQ ID NO: 3 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 15;(aaa) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO:6;(bbb) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 7;(ccc) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 8;(ddd) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NOV;(eee) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 10;(fff) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 11 ;(ggg) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 12;(hhh) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 13;(iii) the humanized VH comprises the amino acid sequence of SEQ ID NO: 4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 14; or (jjj) the humanized VH comprises the amino acid sequence of SEQ ID NO:4 and the humanized VL comprises the amino acid sequence of SEQ ID NO: 15.

4. The binding molecule of any one of claims 1 to 3, wherein the humanized VH further comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:55, that is linked at the C-terminus of the humanized VH.

5. The binding molecule of claim 4, wherein the humanized VH further comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 56, which is at the C- terminus of the CHI domain of the humanized VH.

6. The binding molecule of any one of claims 1 to 5, wherein the humanized VL further comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 57, which is at the C-terminus of the humanized VL.

7. The binding molecule of any one of claims 1 to 6, comprising:(a) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:21;(b) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 22;(c) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 23;(d) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 24;(e) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:25;(f) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:26;(g) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:27;(h) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:28;(i) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:29;(j) the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO:30;(aa) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:21;(bb) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:22;(cc) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:23;(dd) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:24;(ee) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO.25;(ff) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:26;(gg) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:27;(hh) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:28;(ii) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO:29;(jj) the amino acid sequence of SEQ ID NO: 18 and the amino acid sequence of SEQ ID NO: 30;(aaa) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:21;(bbb) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 22;(ccc) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 23;(ddd) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 24;(eee) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:25;(fff) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 26;(ggg) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 27;(hhh) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:28;(iii) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence ofSEQ ID NO: 29; or(jjj) the amino acid sequence of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO:30.

8. The binding molecule of any one of claims 1 to 7, wherein the polypeptide or polypeptides that bind to CD3 epsilon is a Fab.

9. The binding molecule of any one of claims 1 to 8, further comprising an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, ananoparticle, a toxic agent, a radionuclide, a VHH, an Fab, a scFv, a multimerization module, a hinge and Fc domain of an IgG antibody, a half-life extension moiety, or a moiety that facilitates the binding molecule crossing the blood brain barrier.

10. A pharmaceutical composition comprising the binding molecule of any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

11. A nucleic acid or nucleic acids encoding the binding molecule of any one of claims 1 to 9.

12. A vector or vectors comprising the nucleic acid or nucleic acids of claim 11.

13. A host cell comprising the nucleic acid or nucleic acids of claim 11 or the vector or vectors of claim 12.

14. A method of making a binding molecule, the method comprising culturing the host cell of claim 13 under conditions that facilitate expression of the binding molecule, and isolating the binding molecule, optionally further comprising formulating the binding molecule as a sterile pharmaceutical composition.

15. A method of treating a cancer or CD3-related disease or disorder in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the binding molecule of any one of claims 1 to 9, or the pharmaceutical composition of claim 10, optionally wherein the cancer is a solid tumor (e.g., ovarian cancer) or T cell acute lymphoblastic leukemia (T-ALL), and the CD3 -related disease is Type 1 diabetes, an autoimmune disease (e.g., multiple myeloma, relapsed or refractory MM), or transplant rejection.

16. A messenger RNA (mRNA) or mRNAs comprising an open reading frame (ORF) or ORFs encoding the binding molecule of any one of claims 1 to 9.

17. A polynucleotide or polynucleotides comprising an mRNA or mRNAs comprising:(i) a 5' UTR;(ii) an open reading frame (ORF) or ORFs encoding the binding molecule of any one of claims 1 to 9;(iii) a stop codon; and(iv) a 3' UTR.

18. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of claim 17, wherein the mRNA or mRNAs comprises a microRNA (miR) binding site, optionally wherein the microRNA binding site is located in the 3' UTR of the mRNA or mRNAs.

19. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of any one of claims 17 or 18 wherein the 5' UTR comprises a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 73.

20. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of any one of claims 17 to 19, wherein the 3' UTR comprises a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 92.

21. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of any one of claims 17 to 20, wherein the mRNA or mRNAs comprises a 5' terminal cap, optionally wherein the cap comprises m7GpppGm.

22. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of any one of claims 17 to 21, wherein the mRNA or mRNAs comprise a poly-A tail, optionally wherein the poly A tail is about 100 nucleotides in length.

23. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of any one of claims 17 to 22, wherein all of the uracils of the mRNA are N1 - methylpseudouracils.

24. The mRNA or mRNAs of claim 16 or the polynucleotide or polynucleotides of any one of claims 17 to 22, wherein all of the uracils in the mRNA are 5-methoxyuracils.

25. A pharmaceutical composition comprising the mRNA or mRNAs of claim 16, or the polynucleotide or polynucleotides of any one of claims 17 to 24, and a pharmaceutically acceptable carrier.

26. A lipid nanoparticle comprising the mRNA or mRNAs of claim 16, or the polynucleotide or polynucleotides of any one of claims 17 to 24.

27. The LNP of claim 26, wherein the lipid nanoparticle comprises a phospholipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and a sterol, optionally wherein the LNP comprises 48 mole ratio % of the ionizable amino lipid, 2 mole ratio % of the PEG-lipid, 11 mole ratio % of the phospholipid, and 39 mole ratio % of the sterol.

28. The LNP of claim 27, wherein the phospholipid is l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

29. The LNP of claim 27 or 28, wherein the ionizable amino lipid is a compound of Lormula (I), optionally Compound 1-1.

30. The LNP of any one of claims 27 to 29, wherein the PEG lipid is 134-hydroxy- 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99, 102, 105, 108, 111 , 114, 117, 120, 123, 126, 129, 132- tetratetracontaoxatetratriacontahectyl stearate.

31. The LNP of any one of claims 27 to 30, wherein the sterol is cholesterol.

32. The LNP of claim 27, wherein: the phospholipid is l,2-distearoyl-sn-glycero-3 -phosphocholine, the ionizable amino lipid is:the sterol is cholesterol.

33. A method of treating a cancer or CD3 -related disease or disorder in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the mRNA or mRNAs of claim 16, the polynucleotide or polynucleotides of any one of claims 17 to 24, the pharmaceutical composition of claim 25, or the LNP of any one of claims 26 to 32, optionally wherein the cancer is a solid tumor (e.g., ovarian cancer) or a T cell acute lymphoblastic leukemia (T-ALL), and the CD3-related disease is Type 1 diabetes, an autoimmune disease (e.g., multiple myeloma, relapsed or refractory MM), or transplant rejection.

34. A kit comprising (i) the binding molecule of any one of claims 1-9, the mRNA or mRNAs of claim 16, the polynucleotide or polynucleotides of any one of claims 17 to 24, the pharmaceutical composition of claim 25, or the LNP of any one of claims 26 to 32, and (ii) a package insert instructing a user of the kit to administer the binding molecule; the mRNA or mRNAs; the polynucleotide or polynucleotides; the pharmaceutical composition; or the LNP to a human subject in need thereof.

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