Compositions Comprising Alpha Factor Prepro Sequences and Uses Thereof

Abstract

Disclosed herein are modified polypeptides comprising (i) a payload polypeptide and (ii) a wild-type or modified α-factor prepro sequence, wherein the wild-type or modified α-factor prepro sequence is operably linked to the payload polypeptide. Also provided herein are polynucleotides encoding the modified polypeptides of the disclosure, and compositions comprising such polynucleotides or modified polypeptides of the disclosure. Methods of making and using the disclosed polypeptides, polynucleotides, and compositions are also provided.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 296,824, filed January 5, 2022, the entire contents of which are incorporated herein by reference.

[0002] Protein secretion from live cells is a step in many medical and scientific processes. For example, biomanufacturing often relies on the secretion of large amounts of biological therapeutics from live cells for production. Additionally, mRNA vaccines rely on protein secretion from the cells of the vaccinated individual. Additionally, many scientific fields utilize live cell protein secretion for important experiments, including the study of protein function and structure. Thus, enhanced protein secretion may enable the production of more biologics at lower costs, smaller vaccine doses, and simpler experimental procedures for protein research. Summary of the Invention

[0003] The present disclosure provides techniques for increasing the secretion of polypeptides from mammalian cells, where the polypeptides are encoded by exogenous nucleic acids, e.g., DNA or RNA therapeutics. The present disclosure further recognizes that increasing the levels of polypeptide secretion from mammalian cells can be beneficial for several processes, including the manufacture of polypeptides (e.g., therapeutic polypeptides) and the expression of polypeptides in mammalian cells, tissues, or subjects following delivery of polynucleotides encoding such polypeptides.

[0004] One important approach to delivering therapeutic agents is to deliver a polynucleotide to a subject and then use the subject's cellular machinery to express a therapeutic polypeptide from the delivered polynucleotide. A recent example of this approach is the introduction of an mRNA-based vaccine for SARS-CoV-2, which delivers an mRNA encoding an antigenic polypeptide for expression in the subject. While these approaches have been successful, a challenge with such approaches is that the particular polypeptide delivered in this manner is required to be secreted from the cell following expression.

[0005] The present disclosure provides the insight that the α-factor prepro sequence can be used to increase polypeptide secretion from cells, particularly mammalian cells. Thus, the use of the α-factor prepro sequence can improve the secretion level of a payload polypeptide (e.g., a therapeutic polypeptide). Improved secretion levels of a payload polypeptide expressed from an administered polynucleotide can reduce the amount of polynucleotide that needs to be delivered (e.g., reducing associated costs and / or increasing patient comfort during administration).

[0006] Among the techniques described herein for increasing expression of a payload polypeptide are modified polypeptides comprising a payload polypeptide and a wild-type or modified α-factor prepro sequence, the wild-type or modified α-factor prepro sequence being operably linked to the payload polypeptide. Polynucleotides encoding such polypeptides are also provided. Additionally, cells and methods incorporating the described techniques are provided.

[0007] The present disclosure provides insight that the Arabidopsis root growth factor (GLV) secretory peptide sequences disclosed herein, alone or in combination with the α-factor prepro sequences disclosed herein, can be used, for example, to increase polypeptide secretion from plant cells, e.g., Arabidopsis cells. [Brief description of the drawings]

[0008] [Figure 1] Graph showing the measured secretion levels and ACE2 binding affinity of SARS-CoV-2 receptor binding domain (RBD; black circle), RBD variant_1 (white circle), RBD variant_1 with prepro sequence (white square), RBD variant_2 (grey circle), and RBD variant_2 with prepro sequence (grey square).

[0009] [Diagram 2] FIG. 1 is a bar graph showing the secretion levels of certain polypeptides with the native signal peptide (white bars) and the wild-type prepro secretory sequence (grey bars).

[0010] [Diagram 3] FIG. 1 is a bar graph showing the secretion levels of certain polypeptides with the native signal peptide (white bars) and the wild-type prepro secretory sequence (grey bars).

[0011] [Figure 4]1 is a bar graph showing the measured secretion levels of RBD variant_1 polypeptides bound to specific signal peptides. "Native SP" refers to an RBD polypeptide bound to a wild-type RBD signal peptide. "Prepro" refers to an RBD polypeptide bound to a wild-type α-factor prepro sequence. "S1P_delSTE13" refers to an RBD polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with an S1P cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "PCSK9_delSTE13" refers to an RBD polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with a PCSK9 cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "Furin" refers to an RBD polypeptide bound to a modified α-factor prepro sequence in which the Kex2 cleavage site of the wild-type prepro sequence has been replaced with a Furin cleavage site. "delSTE13" refers to an RBD polypeptide bound to a modified α-factor preprosequence in which the Ste13 cleavage site of the wild-type preprosequence has been deleted. "PCSK4" refers to an RBD polypeptide bound to a modified α-factor preprosequence in which the Kex2 cleavage site has been replaced with a PCSK4 cleavage site. "PCSK4_delSTE13" refers to an RBD polypeptide bound to a modified α-factor preprosequence in which (i) the Kex2 cleavage site has been replaced with a PCSK4 cleavage site and (ii) the Ste13 cleavage site has been deleted. "Surf4_motif" refers to an RBD polypeptide bound to a modified α-factor preprosequence in which the amino-terminal residue of the wild-type preprosequence has been mutated to a tripeptide motif (herein referred to as "Surf4 motif") having high Surf4 binding affinity. "Furin_delSTE13" refers to an RBD polypeptide bound to a modified α-factor prepro sequence in which (i) its Kex2 cleavage site has been replaced with a Furin cleavage site, and (ii) its Ste13 cleavage site has been deleted.

[0012] [Diagram 5]1 is a bar graph showing the measured secretion levels of RBD variant_1 polypeptides bound to specific signal peptides. "Native SP" refers to an RBD polypeptide bound to a wild-type RBD signal peptide. "Prepro" refers to an RBD polypeptide bound to a wild-type α-factor prepro sequence. "S1P_PCSK9_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence is replaced with an S1P cleavage site and a PCSK9 cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "3x_S1P_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence is replaced with three (3) S1P cleavage sites, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_motif_SG_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by serine-glycine linkers, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "3x_Surf4_motif_SG_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by three (3) Surf4 motifs joined by serine-glycine linkers, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "3x_PCSK9_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence is replaced by three (3) PCSK9 cleavage sites, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted."Surf4_motif_furin_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by a Surf4 motif, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a Furin cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "SP1_furin_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site and a Furin cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "3x_PCSK4_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with three (3) PCSK4 cleavage sites, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "S1P_PCSK4_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with an S1P cleavage site and a PCSK4 cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "Surf4_motif_S1P_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence has been replaced with a Surf4 motif, (ii) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with an S1P cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "3x_Surf4_motif_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence has been replaced with three (3) Surf4 motifs, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted."Surf4_motif_PCSK4_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced with a Surf4 motif, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced with a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "3x_Surf4_motif_AA_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced with three (3) Surf4 motifs joined by alanine-alanine linkers, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "Furin_PCSK4_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with a Furin cleavage site and a PCSK4 cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "PCSK9_PCSK4_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with a PCSK9 cleavage site and a PCSK4 cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "2x_Surf4_motif_AA_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by an alanine-alanine linker, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "Surf4_motif_PCSK9_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by a Surf4 motif, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a PCSK9 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted."PCSK9_furin_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence is replaced with a PCSK9 cleavage site and a Furin cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_motif_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced with two (2) Surf4 motifs, and (ii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "S1P_PCSK9_furin_PCSK4_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with an S1P cleavage site, a PCSK9 cleavage site, a furin cleavage site, and a PCSK4 cleavage site, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted. "3x_furin_delSTE13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the Kex2 cleavage site of the wild-type prepro sequence has been replaced with three (3) furin cleavage sites, and (ii) the Ste13 cleavage site of the wild-type prepro sequence has been deleted.

[0013] [Figure 6]1 is a bar graph showing the measured secretion levels of RBD variant_1 polypeptides bound to specific signal peptides. "Native SP" refers to an RBD polypeptide bound to a wild-type RBD signal peptide. "Prepro" refers to an RBD polypeptide bound to a wild-type α-factor prepro sequence. "2x_Surf4_SG_3x_S1P_delSte13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced with two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced with three (3) S1P cleavage sites, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_3x_S1P_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by three (3) S1P cleavage sites, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_S1P_PCSK9_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site and a PCSK9 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted."2x_Surf4_SG_S1P_PCSK9_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site, a PCSK9 cleavage site, and a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_S1P_delSte13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_PCSK4_delSte13" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_S1P_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site and a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted."2x_Surf4_SG_3x_PCSK9_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by three (3) PCSK9 cleavage sites, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_Furin_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a Furin cleavage site and a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_3x_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by three (3) PCSK4 cleavage sites, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_PCSK9_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a PCSK9 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted."2x_Surf4_SG_PCSK9_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a PCSK9 cleavage site and a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_PCSK9_Furin_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a PCSK9 cleavage site and a Furin cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_S1P_Furin_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site and a Furin cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_S1P_PCSK9_Furin_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site, a PCSK9 cleavage site, and a Furin cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted."2x_Surf4_SG_Furin_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a Furin cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_S1P_Furin_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by an S1P cleavage site, a Furin cleavage site, and a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "2x_Surf4_SG_PCSK9_Furin_PCSK4_delSte13" refers to an RBD variant_1 polypeptide linked to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced by two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced by a PCSK9 cleavage site, a Furin cleavage site, and a PCSK4 cleavage site, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted.

[0014] [Figure 7]1 is a bar graph showing the measured secretion levels of RBD variant_1 polypeptides bound to specific signal peptides. "Prepro_2" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which (i) the amino terminal residue of the wild-type prepro sequence is replaced with two (2) Surf4 motifs joined by a serine-glycine linker, (ii) the Kex2 cleavage site of the wild-type prepro sequence is replaced with three (3) S1P cleavage sites, and (iii) the Ste13 cleavage site of the wild-type prepro sequence is deleted. "CATHC_3" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which a cathepsin C peptide is inserted at the C-terminus of prepro_2. "CATHC_2" refers to an RBD variant_1 polypeptide bound to a modified α-factor prepro sequence in which a cathepsin C peptide is inserted in the middle of prepro_2. "CFVII_2" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a clotting factor VII peptide is inserted in the middle of prepro_2. "CATHC_1" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a cathepsin C peptide is inserted at the N-terminus of prepro_2. "SORT1_3" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a sortilin 1 peptide is inserted at the C-terminus of prepro_2. "BDNF_2" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a brain-derived neurotrophic factor peptide is inserted in the middle of prepro_2. "CFVII_1" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a clotting factor VII peptide is inserted at the N-terminus of prepro_2. "BDNF_3" refers to an RBD variant_1 polypeptide linked to a modified alpha factor prepro sequence in which a brain-derived neurotrophic factor peptide has been inserted at the C-terminus of prepro_2. "CFVII_3" refers to an RBD variant_1 polypeptide linked to a modified alpha factor prepro sequence in which a coagulation factor VII peptide has been inserted at the C-terminus of prepro_2."BDNF_1" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a brain-derived neurotrophic factor peptide is inserted at the N-terminus of prepro_2. "SORT1_2" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a sortilin 1 peptide is inserted in the middle of prepro_2. "SORT1_1" refers to an RBD variant_1 polypeptide bound to a modified alpha-factor prepro sequence in which a sortilin 1 peptide is inserted at the N-terminus of prepro_2.

[0015] [Figure 8] (A-B) are bar graphs showing secretion levels of SARS-CoV-2 spike receptor binding domain (RBD) variant polypeptides containing specific signal peptides. (A) shows secretion of variant_1 and variant_2 in CHO-K1 Chinese hamster ovary cells with native spike signal peptide (white bars) and wild-type prepro secretory sequence (gray bars). Cells were treated with 50 ng of plasmid DNA. (B) shows secretion of 100 ng of variant_1 in Sf9 Spodoptera frugiperda cells with adipokinetic hormone signal peptide (white bars) and wild-type prepro secretory sequence (gray bars). Cells were treated with 100 ng of plasmid DNA.

[0016] [Figure 9] Bar graphs showing mucosal IgA and serum IgG antibody titers to SARS-CoV-2 spike in vaccinated mice. "SP(S)-RBD" refers to wild-type SARS-CoV-2 spike receptor binding domain (RBD) bound to the native signal peptide. "PreproRBD" refers to wild-type SARS-CoV-2 spike receptor binding domain (RBD) bound to the wild-type α-factor prepro sequence. "Prepro_2-RBD" refers to wild-type SARS-CoV-2 spike receptor binding domain (RBD) bound to the modified α-factor prepro sequence prepro_2.

[0017] [Figure 10] 1 is a bar graph showing mucosal IgA antibody titers to SARS-CoV-2 spike in vaccinated mice. "Prepro-RBD-Sbi(III-IV)" refers to wild-type SARS-CoV-2 spike receptor binding domain (RBD) fused to Staphylococcus aureus binder of IgG protein subunits III and IV bound to wild-type α-factor prepro sequence. "Prepro_2-RBD-Sbi(III-IV)" refers to wild-type SARS-CoV-2 spike receptor binding domain (RBD) fused to Staphylococcus aureus binder of IgG protein subunits III and IV bound to modified α-factor prepro sequence prepro_2.

[0018] [Figure 11] 1 is a bar graph showing the relative amount of RBD variant_1 polypeptide secreted from cells with intact uncleaved propeptide. "WT" refers to RBD variant_1 polypeptide bound to prepro_2 modified α-factor prepro sequence. "S1P_mut" refers to RBD variant_1 polypeptide bound to prepro_2 modified α-factor prepro sequence in which the S1P protease cleavage site has been removed.

[0019] [Figure 12]Bar graph showing SARS-CoV-2 pseudotype virus neutralizing N50 antibody titers in sera from mice vaccinated with SARS-CoV-2 strain RBD linked to various signal peptides. "SP(S)-RBD-Sbi(III-IV)" refers to the SARS-CoV-2 spike receptor binding domain (RBD) fused to the Staphylococcus aureus binder of IgG protein subunits III and IV linked to the native spike signal peptide. "Prepro-RBD-Sbi(III-IV)" refers to the SARS-CoV-2 spike receptor binding domain (RBD) fused to the Staphylococcus aureus binder of IgG protein subunits III and IV linked to the wild-type α-factor prepro sequence. "Wuhan / D614G" refers to the SARS-CoV-2 wild-type RBD (white bars). "Omicron(BA.1)" refers to the SARS-CoV-2 Omicron BA.1 strain RBD (gray bars).

[0020] [Figure 13] This consists of bar graphs showing mucosal IgA and serum IgG antibody titers to SARS-CoV-2 wild-type spike in serum from vaccinated mice. "SP(S)-RBD-Sbi(III-IV)" refers to the wild-type SARS-CoV-2 spike receptor-binding domain (RBD) fused to the Staphylococcus aureus binder of IgG protein subunits III and IV bound to the native spike signal peptide. "Prepro-RBD-Sbi(III-IV)" refers to the wild-type SARS-CoV-2 spike receptor-binding domain (RBD) fused to the Staphylococcus aureus binder of IgG protein subunits III and IV bound to the wild-type α-factor prepro sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Specific Definitions About or Approximately: As used herein, the terms "about" and "approximately" when used herein with respect to a value refer to a similar value in the context of a reference value. In general, those skilled in the art who are familiar with the context will understand the associated degree of dispersion encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" or "approximately" may encompass a range of values ​​that is 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0022] Administering: As used herein, the term "administering" or "administration" generally refers to administering a composition to a subject to achieve delivery of an agent that is or is contained in the composition. Those skilled in the art will recognize the various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (e.g., may be and include one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a particular organ (e.g., in the liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may include only a single dose. In some embodiments, administration may include the application of a fixed number of doses. In some embodiments, administration may include intermittent administration (e.g., multiple doses separated in time) and / or periodic administration (e.g., individual doses separated by a common time interval). In some embodiments, administration may include continuous administration (e.g., perfusion) over at least a selected period of time.

[0023] α-factor prepro sequence: As used herein, the phrase "alpha-factor prepro sequence" or "α-factor prepro sequence" refers to a signal peptide or a portion thereof that is part of the yeast mating pheromone α-factor precursor protein (also referred to as "prepro-α-factor"). The α-factor prepro sequence facilitates the translocation of a polypeptide comprising the α-factor prepro sequence or a portion thereof into the secretory pathway that allows for the secretion of the polypeptide. For example, in some embodiments, a polypeptide can comprise an α-factor prepro sequence and a payload polypeptide, where the α-factor prepro sequence facilitates the translocation of the polypeptide into the secretory pathway that allows for the secretion of the payload polypeptide. The α-factor prepro sequence comprises a C-terminal portion of about 19 amino acids, also referred to as the α-factor pre sequence, and an N-terminal portion of about 72 amino acids, also referred to as the α-factor pro sequence. In some embodiments, the α-factor prepro sequence comprises the sequence of SEQ ID NO: 2, or a fragment or variant thereof. In some embodiments, the variant of SEQ ID NO: 2 comprises SEQ ID NO: 70. In some embodiments, the α-factor prepro sequence comprises the α-factor pre sequence, or a fragment or variant thereof. In some embodiments, the α-factor pre sequence is provided as SEQ ID NO: 68. In some embodiments, the α-factor preprosequence comprises the α-factor prosequence or a fragment or variant thereof. In some embodiments, the α-factor prosequence is provided as SEQ ID NO:69.

[0024] Amino acid: in its broadest sense as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a natural amino acid. In some embodiments, an amino acid is a non-natural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in natural peptides. "Non-standard amino acid" refers to an amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or amino-terminal amino acid in a polypeptide, may include structural modifications compared to the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid, compared to one containing the otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid, compared to one containing the otherwise identical unmodified amino acid. As is clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

[0025] Antigen: As used herein, the term "antigen" refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., including T cells whose receptors specifically interact with the antigen). In some embodiments, an antigen comprises at least one epitope of a target protein. In some embodiments, an epitope may be a linear epitope. In some embodiments, an epitope may be a conformational epitope. In some embodiments, an antigen binds to an antibody and may or may not elicit a specific physiological response in an organism. In general, an antigen may be or include any chemical entity, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments, other than a biological polymer [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or includes a polypeptide. In some embodiments, the antigen is or comprises a glycan. In general, those skilled in the art will understand that the antigen may be provided in isolated or pure form, or alternatively, in crude form (e.g., together with other substances, such as in an extract (e.g., a cell extract) or other relatively crude preparation of an antigen-containing source). In some embodiments, the antigen utilized in accordance with the present invention is provided in crude form. In some embodiments, the antigen is a recombinant antigen.

[0026] Biological sample: As used herein, the term "biological sample" generally refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or body fluid. In some embodiments, the biological sample may be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; suspended nucleic acid; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymphatic fluid; gynecological body fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; lavage or washings; e.g., ductal or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces; other body fluids; secretions; and / or excretions; and / or cells therefrom, and the like. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells from the individual from whom the sample is obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of a biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), and the like. In some embodiments, as the context will allow, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto). For example, filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to a procedure (e.g., amplification or reverse transcription of mRNA, separation and / or purification of specific components, etc.).

[0027] Characteristic portion: As used herein, the term "characteristic portion" in its broadest sense refers to a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion that is found in the substance and in related substances that share the particular characteristic, attribute, or activity, but not in related substances that do not share the particular characteristic, attribute, or activity. In certain embodiments, a characteristic portion shares at least one functional property with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion that contains a contiguous stretch of amino acids, or a collection of contiguous stretches of amino acids, that are characteristic of the protein or polypeptide. In some embodiments, each such contiguous stretch typically contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g., a protein, antibody, etc.) is a portion that shares at least one functional characteristic with the related intact substance in addition to the sequence and / or structural identity specified above. In some embodiments, a characteristic portion may be biologically active.

[0028] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to each other, but are sufficiently similar to allow a comparison between them. As a result, those skilled in the art will understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, a set of comparable conditions, situations, individuals, or populations is characterized by a number of substantially identical characteristics and one or a few different characteristics. Those skilled in the art will understand what degree of identity is required in any given situation for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable in context. For example, those skilled in the art will understand that a set of situations, individuals, or populations is comparable to each other when it is characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of situations, individuals, or populations are caused by or indicate variations in the varying characteristics.

[0029] Determining: Many methodologies described herein include a step of "determining." Those skilled in the art will understand upon reading this specification that such "determining" can be utilized or performed by using any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, the determining involves the manipulation of the physical sample. In some embodiments, the determining involves the consideration and / or manipulation of data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, the determining involves receiving relevant information and / or materials from a source. In some embodiments, the determining involves comparing one or more characteristics of the sample or entity to a comparable reference.

[0030] Delivery / Contacting: As used interchangeably herein, the terms "delivery," "delivering," or "contacting" refer to the introduction of a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell. The target cell can be cultured in vitro or ex vivo or present within a subject (in vivo). The method of introducing a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell can vary depending on the in vitro, ex vivo, or in vivo application. In some embodiments, the fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in cell culture by in vitro transfection. In some embodiments, the fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell via a delivery vehicle (e.g., a nanoparticle, a liposome, and / or a complex with a cell-penetrating agent). In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell of a subject by administering the fusion polynucleotide (e.g., as described herein) or the fusion polypeptide (e.g., as described herein) to the subject.

[0031] Functional: As used herein, the term "functional" is used to refer to forms or fragments of an entity that exhibit particular properties and / or activities.

[0032] Fragment: A "fragment" of a material or entity described herein comprises a discrete portion of the whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, a fragment is composed of such discrete portions. In some embodiments, a fragment is composed of or comprises characteristic structural elements or portions found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. In some embodiments, a polynucleotide or polypeptide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) found throughout the entire polynucleotide or polypeptide. In some embodiments, a polynucleotide or polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the entire polynucleotide or polypeptide. The entire polypeptide or polynucleotide may in some embodiments be referred to as the "parent" of the polynucleotide or polypeptide fragment.

[0033] Modified: In general, the term "modified" refers to aspects that have been manipulated by the hand of man. For example, in some embodiments, a small molecule may be considered modified if its structure and / or production is designed and / or implemented by the hand of man. Similarly, in some embodiments, a polynucleotide may be considered "modified" if two or more sequences that are not linked together in that order in nature are manipulated by the hand of man and directly linked to each other in a modified polynucleotide. For example, in some embodiments of the invention, a modified polynucleotide contains a regulatory sequence found in nature that is operably linked to a first coding sequence, but not to a second coding sequence, and that is operably linked to a second coding sequence by the hand of man. Equivalently, a cell or organism is considered "engineered" if it has been manipulated such that its genetic information has been altered (e.g., new genetic material not previously present has been introduced by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations, or mating protocols). As is common practice and understood by those of skill in the art, the expression products of modified polynucleotides, and / or the progeny of modified polynucleotides or cells, will usually still be referred to as "modified" even if actual manipulation was performed on an earlier entity.

[0034] Epitope: as used herein includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts another conformation (e.g., linearized).

[0035] Functional: As used herein, a "functional" biological molecule is a form of a biological molecule in which it exhibits a characteristic property and / or activity. A biological molecule can have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0036] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second sequences, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be performed using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated in the ALIGN program (version 2.0). In some exemplary embodiments, comparison of nucleic acid sequences with the ALIGN program uses a PAM120 weighted residual table, a gap length penalty of 12, and a gap penalty of 4.Alternatively, the NWSgapdna.CMP matrix can be used to determine the percent identity between two nucleotide sequences using the GAP program in the GCG software package.

[0037] Improve, increase, suppress, or reduce: As used herein, the terms "improve," "increase," "suppress," "reduce," or their grammatical equivalents, refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or a measurement in the presence of a suitable comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system known or expected to respond in a particular way in the presence of the relevant agent or treatment.

[0038] Nucleic Acid, Oligonucleotide, Polynucleotide: As used herein, the terms "nucleic acid" and "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymer of three or more nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single-stranded. In some embodiments, a nucleic acid is double-stranded. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., "peptide nucleic acids." In some embodiments, a nucleic acid includes one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid includes one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in the natural residue.In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., by in vivo or in vitro replication in a recombinant cell or system, or by chemical synthesis). In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4 In some embodiments, the nucleic acid sequence is greater than or equal to 1,000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length. When nucleotide numbers are used as an indication of size, for example, of a fusion polynucleotide, a particular nucleotide number refers to the number of nucleotides on one strand, for example, of the fusion polynucleotide.

[0039] Operably linked: As used herein, refers to a juxtaposition in a relationship permitting the described components to function in their intended manner. A control element that is "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control elements. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest; in some embodiments, the control element acts in trans or otherwise remote from the functional element of interest.

[0040] Polypeptide: The term "polypeptide", which is used interchangeably herein with the term "protein", generally has the art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art intend that the term "polypeptide" is sufficiently general to encompass not only polypeptides having the complete sequences described herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, a polypeptide may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, a polypeptide may be a protein.

[0041] Polyribonucleotide: As used herein, the term "polyribonucleotide" refers to a polymer of three or more ribonucleotides. In some embodiments, a polyribonucleotide is single-stranded. In some embodiments, a polyribonucleotide is double-stranded. In some embodiments, a polyribonucleotide includes both single-stranded and double-stranded portions. In some embodiments, a polyribonucleotide may include a backbone structure as described in the definition of "nucleic acid / oligonucleotide" above. A polyribonucleotide may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA) oligonucleotide. In some embodiments, a polyribonucleotide is an mRNA oligonucleotide, the polyribonucleotide typically includes a poly(A) region at its 3' end. In some embodiments, a polyribonucleotide is an mRNA oligonucleotide, the polyribonucleotide typically includes an art-recognized cap structure at its 5' end, for example, for recognition and binding to ribosomes of the mRNA to initiate translation. In some embodiments, a polyribonucleotide includes an RNA oligonucleotide. When ribonucleotide numbers are used as an indicator of size, for example for a polyribonucleotide, the particular nucleotide number refers to the number of ribonucleotides on a single strand.

[0042] Recombinant: as used herein, is intended to refer to a polypeptide that is designed, modified, prepared, expressed, created, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise engineered to express a gene(s) or genetic component(s) that encodes and / or directs the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or a polypeptide that is prepared, expressed, created, or isolated by any other means, including by splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements arise from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources, e.g., in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0043] Reference: as used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. Those skilled in the art will understand when there is sufficient similarity to demonstrate reliance and / or comparison to a particular possible reference or control.

[0044] Sample: As used herein, the term "sample" generally refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, semen, endolymphatic fluid, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum, semen, serum, smegma, phlegm, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and / or cellular fluid. In some embodiments, the biological fluid may be or include phytoexudates. In some embodiments, the biological tissue or sample may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage or washing (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or washing). In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto). For example, filtration using a semipermeable membrane.Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques (e.g., amplification or reverse transcription of nucleic acids, separation and / or purification of specific components, etc.).

[0045] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is suffering from a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is one who has one or more characteristics that characterize a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or has been administered a diagnosis and / or therapy.

[0046] Treatment: As used herein, "treatment" (also referred to as "treat" or "treating") refers to the administration of any therapy that partially or completely alleviates, improves, relieves, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of subjects who do not show signs of the associated disease, disorder, and / or condition and / or of subjects who show early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of subjects who show one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0047] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity, but that is structurally distinct from the reference entity in the presence or level of one or more chemical moieties as compared to the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on the degree of structural identity with the reference entity. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, a reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of a reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of a reference polypeptide. In some embodiments, a variant polypeptide exhibits a reduced level of one or more biological activities as compared to a reference polypeptide.

[0048] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis (e.g., mRNA synthesis), and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly practiced in the art or as described herein. The techniques and procedures described above may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and described throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose.

[0049] Detailed Description of Specific Embodiments Protein secretion from living cells is a critical step in many medical and scientific processes. Enhancement of protein secretion may enable more biologics production at lower cost, fewer vaccine doses, and easier experimental procedures for protein research. Strategies including codon optimization and expression boosters such as strong promoters and the addition of N-terminal signal peptide sequences that trigger translocation to the ER have been used to enhance protein secretion from living cells. However, there is still room to further increase protein secretion.

[0050] The present disclosure shows that association of the α-factor prepro sequence or fragments thereof (e.g., the α-factor pre sequence or the α-factor pro sequence) with a wide range of polypeptides promotes secretion of those proteins from mammalian cells (e.g., human cell lines). Polypeptides evaluated include synthetic SARS-CoV-2 antigens, influenza hemagglutinins, HER2-targeted single chain antibodies, and biotherapeutics. Furthermore, the present disclosure demonstrates that optimization of the α-factor prepro sequence or fragments thereof can even further enhance secretion of associated polypeptides from human cell lines.

[0051] The present disclosure also provides the insight that the Arabidopsis root growth factor (GLV) secretory peptide sequences disclosed herein, alone or in combination with the α-factor prepro sequence disclosed herein, when associated with a wide range of polypeptides, promotes secretion of those proteins from plant cells, e.g., Arabidopsis cells.

[0052] α-factor prepro sequence Soluble secretory proteins are transported from the cytoplasm to the ER, then to the Golgi apparatus, and finally to secretory vesicles, which fuse with the plasma membrane to secrete their cargo from the cell. The yeast mating pheromone α-factor is a secretory peptide that binds to a receptor in compatible yeast cell types enabling fusion (Naider et al., incorporated herein by reference in its entirety). α-factor is synthesized as a precursor protein called "prepro α-factor" that consists of a signal peptide, a propeptide region, and four repeats of the active peptide pheromone. The signal peptide promotes translocation to the ER, where it is cleaved. The propeptide region allows for export from the ER and transport to the Golgi apparatus (Otte et al., incorporated herein by reference in its entirety). Proteolytic processing in the Golgi apparatus generates the mature α-factor peptide. The α-factor sequence, including both the signal peptide and propeptide regions (prepro), has been shown to increase expression 20-fold when fused to human lysozyme and expressed in yeast cells (Oka et al., incorporated herein by reference in its entirety).

[0053] Polypeptides In particular, the present disclosure provides a polypeptide. The polypeptide disclosed herein is modified to include a payload polypeptide and a wild-type or modified α-factor prepro sequence or a fragment thereof (e.g., α-factor pre sequence or α-factor pro sequence), wherein the wild-type or modified α-factor prepro sequence or a fragment thereof is operably linked to the payload polypeptide. In some embodiments, the wild-type or modified α-factor prepro sequence or a fragment thereof (e.g., α-factor pre sequence or α-factor pro sequence) can mediate the transport of the payload polypeptide. In some embodiments, the wild-type or modified α-factor prepro sequence or a fragment thereof (e.g., α-factor pre sequence or α-factor pro sequence) can mediate the transport of the payload polypeptide to the cell membrane, for example, for secretion outside the cell.

[0054] The present disclosure provides insight, for example, that wild-type or modified α-factor prepro sequences or fragments thereof (e.g., α-factor pre sequence or α-factor pro sequence) can mediate transport of payload polypeptides to the cell membrane, e.g., for secretion out of a cell (e.g., a mammalian cell), at a higher level than other signal peptides. In some embodiments, the secretion level can be the secretion rate. In some embodiments, the secretion level can be the amount of secreted polypeptide.

[0055] The present disclosure provides the recognition that increased secretion levels can be useful in a number of ways in the development of therapeutics, including the manufacture of polypeptides (e.g., polypeptide therapeutics) and the use of therapeutic polypeptides expressed in vivo from delivered polynucleotides. Thus, in some embodiments, the payload polypeptide is a therapeutic polypeptide. Multiple therapeutic polypeptides are known in the art, and the techniques disclosed herein can be used for a wide variety of therapeutic polypeptides. The present disclosure recognizes that biological therapeutics are becoming increasingly important to address health concerns. The present disclosure recognizes that multiple biological polypeptides can be used as payload polypeptides in the modified polypeptides described herein.

[0056] For example, in some embodiments, the payload polypeptide may comprise one or more antigens. In some embodiments, the one or more antigens are one or more viral antigens. In some embodiments, the one or more viral antigens are one or more SARS-CoV-2 antigens. In some embodiments, the one or more viral antigens comprise a SARS-CoV-2 receptor binding domain or a fragment thereof. In some embodiments, the one or more viral antigens comprise a SARS-CoV-2 receptor binding domain variant or a fragment thereof. In some embodiments, the one or more viral antigens comprise a SARS-CoV-2 spike protein or a fragment thereof. In some embodiments, the one or more viral antigens comprise a SARS-CoV-2 spike protein variant or a fragment thereof. In some embodiments, the one or more viral antigens are one or more influenza antigens, e.g., influenza A antigens.

[0057] The use of this technology for expressing antigens is not limited to viral antigens, other antigens can be encoded by the polypeptides disclosed herein.For example, one or more antigens can be one or more bacterial antigens.In some embodiments, one or more antigens are one or more cancer antigens.In some embodiments, one or more antigens are one or more archaeal antigens.

[0058] In some embodiments, the payload polypeptide comprises an antibody or a fragment thereof. An antibody refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, naturally occurring intact antibodies are tetramers of about 150 kD, composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each), which bind to each other to form what is commonly referred to as a "Y-shaped" structure. An antibody tetramer is composed of two heavy-light chain dimers, in which the heavy and light chains are bound to each other by a single disulfide bond; two other disulfide bonds connect the hinge regions of the heavy chains to each other, such that the dimers bind to each other to form a tetramer. Although standard antibodies are tetramers, the antibodies provided herein can take a variety of formats. For example, the antibody may be in a format selected from, but is not limited to, an intact IgA, IgG, IgE, or IgM antibody; or a bispecific or multispecific antibody (such as, for example, Zybodies®).In some embodiments, the antibody fragment can be selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide Fc fusions; single domain antibodies (e.g., shark single domain antibodies or fragments thereof, such as IgNAR); cameroid antibodies, masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®; Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., attachment of glycans) that it has when produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0059] In some embodiments, the payload polypeptide may comprise a recombinant polypeptide (e.g., a fusion polypeptide) comprising an extracellular receptor. In some embodiments, the payload polypeptide may comprise a recombinant polypeptide (e.g., a fusion polypeptide) comprising an immunoglobulin constant region. In some embodiments, the payload polypeptide may comprise a recombinant polypeptide (e.g., a fusion polypeptide) comprising an extracellular receptor and an immunoglobulin constant region. For example, in some embodiments, the payload polypeptide may comprise aflibercept or a fragment thereof. In some embodiments, the aflibercept may have the sequence provided at https: / / go.drugbank.com / drugs / DB08885, which is incorporated herein by reference in its entirety.

[0060] In some embodiments, the payload polypeptide may comprise a recombinant polypeptide (e.g., a fusion polypeptide) comprising a receptor agonist or antagonist. In some embodiments, the payload polypeptide may comprise a recombinant polypeptide (e.g., a fusion polypeptide) comprising an immunoglobulin constant region. In some embodiments, the payload polypeptide may comprise a recombinant polypeptide (e.g., a fusion polypeptide) comprising a receptor agonist or antagonist and an immunoglobulin constant region. For example, in some embodiments, the payload polypeptide may comprise dulaglutide or a fragment thereof. In some embodiments, dulaglutide may have the sequence provided at https: / / go.drugbank.com / drugs / DB09045, which is incorporated herein by reference in its entirety.

[0061] In some embodiments, the payload polypeptide may comprise secreted embryonic alkaline phosphatase (SEAP) or a fragment thereof. In some embodiments, the payload polypeptide may comprise trastuzumab, trastuzumab scFv, or a fragment of trastuzumab. In some embodiments, the payload polypeptide may comprise pertuzumab, pertuzumab scFV, or a fragment of pertuzumab. In some embodiments, the payload polypeptide may comprise GB235, GB235 scFv, or a fragment of GB235. In some embodiments, the payload polypeptide may comprise adalimumab, adalimumab scFv, or a fragment of adalimumab. In some embodiments, the payload polypeptide may comprise pembrolizumab, pembrolizumab scFV, or a fragment of pembrolizumab. In some embodiments, the payload polypeptide may comprise aflibercept or a fragment thereof. In some embodiments, the payload polypeptide may comprise dupilumab, dupilumab scFv, or a fragment of dupilumab. In some embodiments, the payload polypeptide may comprise ustekinumab, ustekinumab scFv, or a fragment of ustekinumab. In some embodiments, the payload polypeptide may comprise nivolumab, nivolumab scFv, or a fragment of nivolumab. In some embodiments, the payload polypeptide may comprise bevacizumab, bevacizumab scFv, or a fragment of bevacizumab. In some embodiments, the payload polypeptide may comprise etanercept or a fragment thereof. In some embodiments, the payload polypeptide may comprise tisagenlecleucel or a fragment thereof. In some embodiments, the payload polypeptide may comprise tisagenlecleucel extracellular domain and / or tisagenlecleucel transmembrane domain. In some embodiments, the payload polypeptide may comprise axicabutagenciloreucel or a fragment thereof.In some embodiments, the payload polypeptide may comprise axicabtagene siloreucel extracellular domain and / or axicabtagene siloreucel transmembrane domain. In some embodiments, the payload polypeptide may comprise lysocabtagene malareucel or a fragment thereof. In some embodiments, the payload polypeptide may comprise lysocabtagene malareucel extracellular domain and / or lysocabtagene malareucel transmembrane domain. In some embodiments, the payload polypeptide may comprise an A3B1 chimeric antigen receptor (A3B1 CAR) or a fragment thereof. In some embodiments, the payload polypeptide may comprise an A3B1 CAR extracellular domain and / or an A3B1 CAR transmembrane domain.

[0062] In some embodiments, the payload polypeptide comprises a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:72, or SEQ ID NO:79.

[0063] In some embodiments, the payload polypeptide is secreted fetal alkaline phosphatase (SEAP) or a fragment thereof; trastuzumab, trastuzumab scFv, or a fragment of trastuzumab; pertuzumab, pertuzumab scFV, or a fragment of pertuzumab; GB235, GB235 scFv, or fragment of GB235;adalimumab, adalimumab scFv, or fragment of adalimumab;pembrolizumab, pembrolizumab scFV, or fragment of pembrolizumab;aflibercept or fragment thereof;dupilumab, dupilumab scFv, or fragment of dupilumab;ustekinumab, ustekinumab scFv, or fragment of ustekinumab;nivolumab, nivolumab scFv, or fragment of nivolumab;bevacizumab, bevacizumab scFv, or fragment of bevacizumab;etaner sept or a fragment thereof; tisagenlecleucel or a fragment thereof (e.g., tisagenlecleucel extracellular domain and / or tisagenlecleucel transmembrane domain); axicabtageneciloreucel or a fragment thereof (e.g., axicabtageneciloreucel extracellular domain and / or axicabtageneciloreucel transmembrane domain); lysocabtagenemaraleucel or a fragment thereof (e.g., lysocabtagenemaraleucel extracellular domain and / or lysocabtagenemaraleucel transmembrane domain); or A3B1 chimeric antigen receptor (A3B1 CAR) or a fragment thereof (e.g., A3B1 CAR extracellular domain and / or A3B1 CAR transmembrane domain).

[0064] In some embodiments, a wild-type or modified α-factor prepro sequence, or a fragment thereof, is present at the N-terminus of the modified polypeptide. In some embodiments, a wild-type or modified α-factor prepro sequence, or a fragment thereof (e.g., the α-factor pre sequence or the α-factor pro sequence) is present at the C-terminus of the modified polypeptide.

[0065] In some embodiments, the modified polypeptide comprises a wild-type α-factor prepro sequence or a fragment thereof (e.g., an α-factor pre sequence or an α-factor pro sequence). In some embodiments, the wild-type α-factor prepro sequence comprises a sequence according to SEQ ID NO:2.

[0066] In some embodiments, the modified polypeptide comprises a wild-type α-factor presequence. In some embodiments, the wild-type α-factor presequence comprises a sequence according to SEQ ID NO:68.

[0067] In some embodiments, the modified polypeptide comprises a wild-type α-factor prosequence or a fragment thereof. In some embodiments, the wild-type α-factor prosequence comprises a sequence according to SEQ ID NO:69.

[0068] In some embodiments, the modified polypeptide comprises a modified α-factor prepro sequence or a fragment thereof (e.g., an α-factor pre sequence or an α-factor pro sequence). In some embodiments, the modified α-factor prepro sequence comprises a sequence according to SEQ ID NO:70.

[0069] In some embodiments the modified polypeptide comprises a modified α-factor prosequence or a fragment thereof. In some embodiments the modified α-factor prosequence comprises a sequence according to SEQ ID NO:71.

[0070] In some embodiments, the modified polypeptide comprises a modified α-factor preprosequence. In some embodiments, the modified α-factor preprosequence comprises a tripeptide motif (Surf4 motif) that has Surf4 binding affinity. In some embodiments, the modified α-factor preprosequence comprises at least two (e.g., two, three, four, or more) Surf4 motifs. In some embodiments, the Surf4 motif is MPL (SEQ ID NO: 58). In some embodiments, the Surf4 motif replaces the three most N-terminal residues of the α-factor propeptide sequence of the α-factor preprosequence. In some embodiments, the three most N-terminal residues of the α-factor prepeptide sequence of the α-factor preprosequence remain after cleavage of the α-factor propeptide sequence of the α-factor preprosequence. In some embodiments, the three most N-terminal residues of the α-factor propeptide sequence of the α-factor preprosequence are residues 20-22 of SEQ ID NO: 2.

[0071] In some embodiments, the modified α-factor prepro sequence comprises two or more, three or more, or four or more Surf4 motifs. In some embodiments, the Surf4 motifs are linked by a linker. In some embodiments, the linker comprises at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, or at least fifteen amino acids. In some embodiments, the linker is a serine-glycine linker. In some embodiments, the serine-glycine linker may comprise the sequence SGGGSGGSGS (SEQ ID NO: 66). In some embodiments, the linker is an alanine-alanine linker. In some embodiments, the serine-glycine linker may comprise the sequence AA (SEQ ID NO: 67).

[0072] In some embodiments, the modified α-factor prepro sequence does not contain a Ste13 cleavage site, hi some embodiments, the Ste13 cleavage site comprises EAEA (SEQ ID NO:56).

[0073] In some embodiments, the modified α-factor prepro sequence does not contain a Kex2 cleavage site. In some embodiments, the Kex2 cleavage site comprises KR (SEQ ID NO:57). In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence with a protease cleavage site for a human protease. In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence with a protease cleavage site for S1P, PCSK4, PCSK9, or Furin. As used herein, "substitute", "substitution", and variations thereof should not be understood to be limited to direct replacement (e.g., deletion of the original sequence and insertion of another sequence). Rather, "substitute", "substitution", or variations thereof encompass any situation in which the original sequence is no longer present after "substitution" and another sequence is present after "substitution". For example, amino acids of the original sequence may remain after "substitution", but the original sequence may not be present. A "substitution" can be achieved, for example, by mutation of one or more amino acids of the original sequence (eg, alteration of the nucleotide sequence encoding the original amino acid sequence).

[0074] In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence with one or more protease cleavage sites of S1P, PCSK4, PCSK9, Furin, or a combination thereof. In some embodiments, the one or more cleavage sites are the same. For example, in some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence with multiple (e.g., two, three, four, or more) S1P cleavage sites. In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence with multiple (e.g., two, three, four, or more) PCSK4 cleavage sites. In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence with multiple (e.g., two, three, four, or more) PCSK9 cleavage sites. In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced with multiple (e.g., two, three, four or more) Furin cleavage sites in the modified α-factor prepro sequence. In some embodiments, one or more of the cleavage sites are different. For example, in some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced with an S1P cleavage site and a PCSK4 cleavage site in the modified α-factor prepro sequence. In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced with two (2) S1P cleavage sites and a PCSK4 cleavage site in the modified α-factor prepro sequence. In some embodiments, the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in a modified α-factor prepro sequence that has two (2) Surf4 motifs joined by a glycine-serine linker, lacks the Ste13 cleavage site, and replaces the Kex2 cleavage site with three (3) S1P cleavage sites. As illustrated in these exemplary embodiments, any combination of S1P, PCSK4, PCSK9, and a furin cleavage site may be used as disclosed herein, and the disclosure should not be understood as limited to the specific embodiments presented above.Further support for combinations can be found, for example, in the Examples below.

[0075] The protease cleavage site of S1P may have a sequence according to SEQ ID NO: 55. In some embodiments, the protease cleavage site of PCSK4 has a sequence according to SEQ ID NO: 53. In some embodiments, the protease cleavage site of PCSK9 has a sequence according to SEQ ID NO: 54. In some embodiments, the protease cleavage site of Furin has a sequence according to SEQ ID NO: 52.

[0076] In some embodiments, the modified α-factor prepro sequence contains a sequence capable of binding to one or more receptors that transport proteins through one or more secretory pathways.

[0077] In some embodiments, the receptor that transports proteins through one or more secretory pathways is LMAN1. Proteins that have been reported to bind to LMAN1 include cathepsin C (CATHC) and coagulation factor VII (CFVII).

[0078] In some embodiments, the receptor that transports proteins through one or more secretory pathways is sortilin-1. Among the proteins that have been reported to bind to sortilin-1 are brain-derived neurotrophic factor (BDNF) and sortilin-1 propeptide.

[0079] In some embodiments, the modified α-factor prepro sequence comprises multiple (e.g., two, three, four, or more) LMAN1-binding domains. In some embodiments, the polypeptide comprising an LMAN1-binding domain is cathepsin C or coagulation factor VII (CFVII).

[0080] In some embodiments, the modified α-factor prepro sequence comprises multiple (e.g., two, three, four, or more) cathepsin C (CATHC) peptide sequences or fragments thereof. In some embodiments, the CATHC peptide comprises TPANCTYLDLLGTWVFQVGSSGSQRDVNCSVMG (SEQ ID NO: 95). In some embodiments, the CATHC peptide is inserted into the modified α-factor prepro sequence: (1) at the N-terminus of the propeptide after the 2x Surf4 motif with a flexible glycine-serine linker, (2) in the middle of the propeptide sequence, or (3) at the C-terminus immediately preceding the 3x S1P cleavage site.

[0081] In some embodiments, the modified α-factor preprosequence comprises multiple (e.g., two, three, four, or more) coagulation factor VII (CFVII) peptides containing glycosylation sites or fragments thereof. In some embodiments, the CFVII peptide comprises ILEKRNASKPQGR (SEQ ID NO: 96). In some embodiments, the CFVII peptide is inserted into the modified α-factor preprosequence: (1) at the propeptide N-terminus after the 2x Surf4 motif with a flexible glycine-serine linker, (2) in the middle of the propeptide sequence, or (3) at the C-terminus immediately preceding the 3x S1P cleavage site.

[0082] In some embodiments, the modified α-factor prepro sequence comprises multiple (e.g., two, three, four, or more) sortilin-1 binding domains. In some embodiments, the polypeptide that comprises a sortilin-1 binding domain is BDNF or sortilin-1.

[0083] In some embodiments, the modified α-factor prepro sequence comprises multiple (e.g., two, three, four, or more) sortilin 1 peptides or fragments thereof. In some embodiments, the sortilin 1 peptide comprises WSGPIGVSWGLR (SEQ ID NO:97). In some embodiments, the sortilin 1 peptide is inserted into the modified α-factor prepro sequence: (1) at the propeptide N-terminus after the 2x Surf4 motif with a flexible glycine-serine linker, (2) in the middle of the propeptide sequence, or (3) at the C-terminus immediately preceding the 3x S1P cleavage site.

[0084] In some embodiments, the modified α-factor prepro sequence comprises multiple (e.g., two, three, four, or more) brain-derived neurotrophic factor (BDNF) peptides or fragments thereof. In some embodiments, the BDNF peptide comprises ESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPL (SEQ ID NO: 98). In some embodiments, the BDNF peptide is inserted into the modified α-factor prepro sequence: (1) at the propeptide N-terminus after the 2x Surf4 motif with a flexible glycine-serine linker, (2) in the middle of the propeptide sequence, or (3) at the C-terminus immediately preceding the 3x S1P cleavage site.

[0085] In some embodiments, the modified α-factor prepro sequence comprises one or more tags, such as one or more Myc tags. In some embodiments, a modified α-factor prepro sequence comprising a 3X Myc tag is provided in SEQ ID NO: 106 or SEQ ID NO: 107.

[0086] In some embodiments, the modified α-factor prepro sequence contains one or more tripeptide motifs having Surf4 binding affinity (e.g., a motif according to SEQ ID NO: 58); contains one or more S1P cleavage sites (e.g., according to SEQ ID NO: 55); contains one or more PCSK4 cleavage sites (e.g., according to SEQ ID NO: 53); contains one or more PCSK9 cleavage sites (e.g., according to SEQ ID NO: 54); contains one or more Furin cleavage sites (e.g., according to SEQ ID NO: 52); does not contain a Ste13 cleavage site (e.g., according to SEQ ID NO: 56); does not contain a Kex2 cleavage site (e.g., according to SEQ ID NO: 57); or a combination thereof.

[0087] In some embodiments, the modified α-factor preprosequence has a sequence according to SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:71, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:109.

[0088] In some embodiments the modified polypeptide has a sequence according to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105.

[0089] In some embodiments, a wild-type or modified Arabidopsis root growth factor (GLV) sequence or a fragment thereof is present at the N-terminus of the modified polypeptide. In some embodiments, a wild-type or modified Arabidopsis root growth factor (GLV) sequence or a fragment thereof is present at the C-terminus of the modified polypeptide.

[0090] In some embodiments, the modified polypeptide comprises a modified Arabidopsis root growth factor (GLV) sequence or a fragment thereof, a modified alpha-factor prepro sequence or a fragment thereof, or both.

[0091] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:99.

[0092] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:82.

[0093] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:83.

[0094] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:84.

[0095] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:85.

[0096] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:86.

[0097] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:87.

[0098] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:88.

[0099] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:89.

[0100] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:90.

[0101] In some embodiments, the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:91.

[0102] As discussed above, the present disclosure provides polypeptides that are secreted at increased levels. In some embodiments, the modified polypeptides provided herein are characterized in that when evaluated in a cell, tissue, or subject, the modified polypeptide is secreted from a cell, tissue cell, or subject cell or tissue at a higher level than a comparable polypeptide. In some embodiments, the comparable polypeptide comprises the same payload polypeptide as the modified polypeptide and a signal peptide that is not a wild-type or modified α-factor prepro sequence.

[0103] In some embodiments, the secretion level is the amount of secreted protein (e.g., grams or moles of secreted protein, or concentration of secreted protein). In some embodiments, the secretion level is the secretion rate, grams or moles of secreted protein (per volume of medium, e.g., per volume of cell culture medium).

[0104] Polynucleotides The present disclosure provides a polynucleotide that encodes the modified polypeptide described herein.As will be recognized by those skilled in the art, multiple polynucleotides can encode the same polypeptide.Furthermore, the sequence of the polynucleotide that encodes the polypeptide can be determined by those skilled in the art using conventional methods.

[0105] In certain embodiments, a polynucleotide encoding a modified polypeptide described herein is a DNA polynucleotide.

[0106] In some embodiments, a polynucleotide encoding a modified polypeptide described herein is an RNA polynucleotide. In some embodiments, the RNA polynucleotide is an mRNA. In some embodiments, the mRNA comprises a cap and a poly(A) tail.

[0107] In some embodiments, the polynucleotide comprises one or more chemically modified nucleotides. In some embodiments, the chemically modified nucleotide can be N4-acetylcytidine. In some embodiments, the chemically modified nucleotide can be 5-hydroxymethyluridine. The present disclosure recognizes that the inclusion of chemically modified nucleotides (e.g., N4-acetylcytidine and / or 5-hydroxymethyluridine) in the polynucleotides described herein can enable the polynucleotide to evade the innate immune system, improve the viability of the cell into which the polynucleotide is introduced, and / or increase the expression of the payload in the cell into which the polynucleotide is introduced. The present disclosure recognizes that the inclusion of chemically modified nucleotides (e.g., N4-acetylcytidine and / or 5-hydroxymethyluridine) in the polynucleotides described herein can reduce the immunogenicity of the polynucleotide in a cell, tissue, or subject.

[0108] composition In addition to the embodiments discussed above, the present disclosure provides compositions. The compositions disclosed herein may include cells. In some embodiments, the cells may include modified polypeptides and / or polynucleotides described herein. In some embodiments, the cells are mammalian cells.

[0109] The compositions disclosed herein can include modified polypeptides and / or polynucleotides as described herein.

[0110] Pharmaceutical Compositions In some embodiments, the compositions comprising the modified polypeptides, polynucleotides, and / or cells described herein are pharmaceutical compositions.

[0111] In some embodiments, the pharmaceutical composition is or comprises an immunogenic composition, hi some embodiments, the pharmaceutical composition is or comprises a vaccine.

[0112] In some embodiments, the pharmaceutical composition is or comprises a gene therapy.

[0113] In some embodiments, the pharmaceutical composition is or comprises a chemotherapy.

[0114] In some embodiments, the pharmaceutical composition is or comprises a protein replacement therapy.

[0115] In some embodiments, the pharmaceutical composition is or comprises an immunotherapy.

[0116] In some embodiments, the pharmaceutical composition is or comprises a cell modifying therapy.

[0117] In some embodiments, pharmaceutical compositions may include pharma- ceutically acceptable carriers or excipients, which as used herein include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Suitable pharma-ceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, glycerol, sugars such as mannitol, sucrose, and the like, dextrose, fatty acid esters, and the like, and combinations thereof.

[0118] The pharmaceutical composition may be mixed with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances, etc.) as necessary, which do not adversely react with the active compound or interfere with its activity. In certain embodiments, a water-soluble carrier suitable for intravenous administration is used. In some embodiments, the pharmaceutical composition may be sterilized.

[0119] Suitable pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.The pharmaceutical composition can be a liquid solution, suspension, or emulsion.

[0120] The pharmaceutical composition can be formulated according to the usual procedures as a pharmaceutical composition suitable for administration to humans. The formulation of the pharmaceutical composition must suit the method of administration. For example, in some embodiments, a composition for intravenous administration is usually a solution in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed in unit dosage form, for example as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampule or sachette indicating the quantity of active agent. If the pharmaceutical composition is to be administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline, or dextrose / water. If the pharmaceutical composition is to be administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0121] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any type of animal or cell, in vitro or ex vivo. Modifications of pharmaceutical compositions suitable for administration to humans to provide compositions suitable for administration to a variety of animals or cells, in vitro or ex vivo, are well understood and an ordinarily skilled professional, e.g., a veterinary pharmacologist, can design and / or perform such modifications with only routine experimentation, if any.

[0122] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the step of combining the active ingredient with a diluent or other excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into the desired single- or multi-dosage unit.

[0123] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition described herein.

[0124] Methods of using the compositions disclosed herein The disclosure provides, inter alia, methods of using the modified polypeptides, polynucleotides, and / or cells described herein.

[0125] In some embodiments, methods are provided for administering modified polypeptides, polynucleotides, and / or cells described herein to a cell, tissue, or subject. In some embodiments, the cell is a mammalian cell. In some embodiments, the tissue is a mammalian tissue. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0126] In some embodiments, provided herein are methods of vaccination comprising administering to a cell, tissue, or subject a modified polypeptide, polynucleotide, and / or cell described herein.

[0127] Disclosed herein in some embodiments are gene therapy methods comprising administering to a cell, tissue, or subject a modified polypeptide, polynucleotide, and / or cell described herein. In some embodiments, the gene therapy method comprises delivery of one or more components of the gene therapy, such as a guide RNA and / or a Cas polypeptide.

[0128] In some embodiments, provided herein are methods for stimulating an immune response comprising administering to a cell, tissue, or subject a modified polypeptide, polynucleotide, and / or cell described herein.

[0129] Also provided herein, in some embodiments, are methods of modifying cell therapy comprising administering to a cell, tissue, or subject the modified polypeptides, polynucleotides, and / or cells described herein.

[0130] In some embodiments, provided herein are immunotherapies comprising administering to a cell, tissue, or subject a modified polypeptide, polynucleotide, and / or cell described herein, hi some embodiments, the immunotherapy comprises administration of antibody therapy and / or immune checkpoint therapy.

[0131] Disclosed herein in some embodiments is a protein replacement therapy comprising administering to a cell, tissue, or subject a modified polypeptide, polynucleotide, and / or cell described herein, hi some embodiments, the protein replacement therapy comprises delivery of an enzyme replacement therapy.

[0132] In some embodiments, provided herein are chemotherapy methods comprising administering to a cell, tissue, or subject a modified polypeptide, polynucleotide, and / or cell described herein.

[0133] In some embodiments, the methods provided herein comprise determining the level of a variant polypeptide that is secreted from a cell, a cell of a tissue, or a cell or tissue of a subject.

[0134] In some embodiments, the method further comprises comparing the level of the modified polypeptide secreted from the cells, cells of the tissue, or cells or tissues of the subject to a reference level, in some embodiments, the reference level is the level of a reference polypeptide secreted from a comparable cell, tissue, or subject, where the reference polypeptide comprises the same payload polypeptide as the modified polypeptide and a signal peptide that is not the wild-type or modified α-factor prepro sequence.

[0135] In some embodiments, the methods or uses disclosed herein further comprise determining the efficacy of the modified polypeptides, polynucleotides, and / or cells described herein in a cell, tissue, or subject to which the modified polypeptides, polynucleotides, and / or cells described herein have been administered.

[0136] In some embodiments, determining efficacy includes determining an antibody or cellular response in a cell, tissue, or subject. In some embodiments, a cell, tissue, or subject to which a modified polypeptide, polynucleotide, and / or cell described herein has been administered exhibits an increased antibody or cellular response compared to a reference. In some embodiments, the reference is an antibody or cellular response of a cell, tissue, or subject to which a comparable modified polypeptide, polynucleotide, and / or cell described herein has been administered. In some embodiments, a comparable modified polypeptide described herein comprises a signal polypeptide that is not a wild-type or modified α-factor prepro sequence.

[0137] In some embodiments, the methods or uses disclosed herein comprise administering to a cell, tissue, or subject at least two times the modified polypeptides, polynucleotides, and / or cells comprising same described herein, in some embodiments, the methods disclosed herein comprise administering to a cell, tissue, or subject two, three, four, five, six, seven, eight, nine, or ten times the modified polypeptides, polynucleotides, and / or cells described herein.

[0138] In some embodiments, the methods or uses disclosed herein comprise administering to a cell, tissue, or subject multiple doses of the modified polypeptides, polynucleotides, and / or cells described herein, in some embodiments, a second or subsequent dose of the modified polypeptides, polynucleotides, and / or cells described herein has substantially similar efficacy in the cell, tissue, or subject compared to a first dose of the modified polypeptides, polynucleotides, and / or cells described herein.

[0139] In some embodiments of any of the methods or uses disclosed herein, the composition is administered via any route of administration: intramuscular, intravenous, subcutaneous, intrathecal, intradermal, intraocular, intranasal, sublingual, or oral.

[0140] In some embodiments of any of the methods or uses disclosed herein, the cell is a mammalian cell.

[0141] In some embodiments of any of the methods or uses disclosed herein, the tissue is mammalian tissue.

[0142] In some embodiments of any of the methods or uses disclosed herein, the subject is a mammal. In some embodiments, the mammal is a human.

[0143] The disclosure also provides a method of producing a modified polypeptide comprising expressing a polynucleotide described herein from a cell. In some embodiments, the cell is a mammalian cell.

[0144] kit Another aspect of the disclosure further provides a pharmaceutical pack or kit. In some embodiments, the kit may include modified polypeptides, polynucleotides, and / or cells described herein. In some embodiments, the kit may be used in any applicable method, such as the methods described herein. EXAMPLES

[0145] Example 1: Cloning Method This example provides exemplary methods for generating constructs and encoded polypeptides for improving polypeptide secretion from mammalian, insect, and plant cells.

[0146] Example 1.1: Receptor Binding Domain Constructs DNA gblocks encoding the wild-type receptor binding domain (WT RBD) of SARS-CoV-2 and two other variants of the SARS-CoV-2 receptor binding domain (variant_1 and variant_2) were ordered and individually cloned into the NanoLuc protein fusion vector, pNLF1-C (Promega), using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). The master mix contained enzymes and buffers for rapid and efficient Gibson assembly of two or more DNA pieces. More specifically, the pNLF1-C vector was first linearized by double digestion with the restriction enzymes EcoRI-HF and Nhel-HF. The digested vector was then purified using a QIAquick Gel Extraction Kit (Qiagen) and concentrated using a Zymo DNA Clean and Concentrator-5 (Zymoresearch). In separate reactions, gblock constructs corresponding to WT RBD, variant_1, and variant_2 were inserted into the linearized vector by mixing 50 ng of purified vector, 2-fold molar excess of insert, 10 μl of master mix, and an appropriate amount of ultrapure water to create 20 μl reactions in 1.5 ml tubes. The reactions were then incubated at 50° C. for 15 min. The resulting constructs are called pNLF1C_RBD_WT, pNLF1C_RBD_variant_1, and pNLF1C_RBD_variant_2.

[0147] The ligation reaction was then diluted 1:4 and 2 μl of the diluted reaction was then used to transform One Shot TOP10 Chemically Competent E. coli cells (ThermoFisher Scientific) by heat shock at 42°C for 30 seconds. The cells were then plated on ampicillin agar plates and incubated at 37°C for 24 hours. The next day, colonies were screened for uptake of the chimeric vector using colony PCR. First, colonies were suspended in 100 μl water and 1 μl of the mixture was mixed with 1 μl of 10 μM forward and reverse primers, 10 μl of 2x Platinum SuperFi PCR Master Mix (ThermoFisher Scientific), and an appropriate amount of water to create a 20 μl reaction in a 1.5 ml tube. PCR was then performed using a Mastercycler X50 (Eppendorf) with appropriate cycling parameters based on the primer properties and the recommendations provided by the PCR enzyme vendor. PCR products of positive colonies were sent to Genewiz for sequencing to confirm insertion of the construct. Meanwhile, positive colonies were grown for 18 hours in a solution of LB and ampicillin with shaking in a 37°C incubator. After 18 hours, chimeric plasmids were extracted from one positive colony per construct by miniprepping using a NucleoSpin Plasmid Mini Kit (Macherey-Nagel). Plasmids were sent for resequencing to confirm that the correct construct was inserted and that no mutations were introduced during replication in E. coli.

[0148] Example 1.2: Receptor binding domain constructs containing prepro sequences The α-factor prepro sequence (e.g., α-factor signal peptide and propeptide sequences) was added to variant_1 and variant_2 of pNLF1-C using restriction-free cloning with gBlock Megaprimer (IDT) encoding a prepro sequence with 50 bp of homology on either end of both pNLF1C_RBD_variant_1 and pNLF1C_RBD_variant_2. The homology arms were designed to insert the prepro sequence between the Kozak sequence and the RBD variant to replace the spike secretion peptide. 27.2 μL water, 10 μL 5x Phusion HF buffer, 1 uL 10 mM dNTPs, 20 ng pNLF1C_RBD_variant_1 or pNLF1C_RBD_variant_2 in 1 uL water, 10 μL prepro megaprimer at 10 ng / μL, and 0.8 uL Phusion DNA polymerase (New England BioLabs) were mixed in a PCR tube on ice. The tubes were placed in a thermal cycler and the following PCR program was run: 95°C for 30 seconds. 25 cycles of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 5 minutes. 72°C for 7 minutes. 10 μL of these reactions were mixed with 34 μL water, 5 μL 10x CutSmart buffer, and 1 μL DpnI (20 units / μL) (New England BioLabs) in a PCR tube on ice.

[0149] The tubes were placed in a thermal cycler and incubated at 37°C for 1 hour to digest the template DNA. The reactions were diluted 1:4 in water, transformed into TOP10 cells (ThermoFisher Scientific) and plated on LB agar plates containing ampicillin (Teknova) as described above. The next day, colony PCR was performed as described above using NLuc_Nterm_cPCR_F primers (SEQ ID NO:59) and NLuc_Nterm_cPCR_R primers (SEQ ID NO:60) to identify colonies containing the pre-pro insert. PCR products were sequenced on Genewiz using NLuc_seq primer (SEQ ID NO:61). Colonies containing the insert were grown overnight in LB medium containing ampicillin and mini-prepped as described in Example 1.1 above.

[0150] The prepro sequence was added to the WT RBD sequence using PCR. Water (31.5 μL), 5x Phusion HF buffer (10 μL), 10 mM dNTPs (1 μL), 10 μM NLuc_RBD_F primer (SEQ ID NO: 62) (2.5 μL), 10 μM NLuc_RBD_R primer (SEQ ID NO: 63) (2.5 μL), 5 ng / μL RBD_WT_NLuc gBlock (1 μL), 5 ng / μL prepro Mega primer gBlock (1 μL), and Phusion DNA polymerase (New England BioLabs) (0.5 μL) were mixed in a PCR tube on ice. The tube was transferred to a thermal cycler preheated to 98°C and the following PCR program was run: 98°C for 30 seconds; 98°C for 7 seconds followed by 30 cycles of 72°C for 15 seconds; 72°C for 7 minutes and 30 seconds. The PCR reaction was purified using DNA Clean and Concentrator-5 (Zymo Research). The purified PCR product was cloned into pNLF1-C using NEBuilder HiFi DNA Assembly Master Mix as described above.

[0151] Example 1.3: Optimized prepro sequence To optimize the α-factor prepro sequence, a new vector was constructed consisting of variant_1 of pNLF1-C with an upstream cloning portion for the prepro sequence optimization library. pNLF1-C was digested with NheI and EcoRI as described in Example 1.1 above. gBlock (IDT) containing the Kozak sequence, NheI cleavage site, 6 bp spacer, EcoRI cleavage site, and variant_1 was cloned into the digested vector using NEBuilder HiFi DNA Assembly Master Mix as described in Example 1.1 above. The homology arms of gBlock were designed to remove the NheI and EcoRI restriction sites from the pNLF1-C plasmid, so that the cleavage site within gBlock is unique in the new vector, called pSP. The vector was digested with NheI and EcoRI as described in Example 1.1 above. Prepro sequence variations were ordered from IDT as eBlocks, gBlocks, or primers based on length and contained 20 bp homology arms with pSP to allow in-frame cloning with variant_1. These sequences were cloned into digested pSP using NEBuilder HiFi DNA Assembly Master Mix as described in Example 1.1 above. eBlocks were kept in 2-fold molar excess over pSP and primers were kept at 45 nM. Colony PCR was performed using NLuc_Nterm_cPCR_F (SEQ ID NO: 59) and NLuc_Nterm_cPCR_R primers (SEQ ID NO: 60).

[0152] Example 1.4: Additional Constructs As described above in Example 1.1, influenza H1N1 / PR8 hemagglutinin extracellular domain (HA) containing the native secretory peptide or prepro, human secreted fetal alkaline phosphatase (SEAP) containing the native secretory peptide or prepro, trastuzumab scFv containing the SARS-CoV-2 spike secretory peptide (Ssp) or prepro, pertuzumab scFv containing Ssp or prepro, and GB235 scFv containing Ssp or prepro. scFv, adalimumab scFv with potent immunoglobulin secreting peptide or PPA, pembrolizumab scFv with potent immunoglobulin secreting peptide or PPA, aflibercept with vascular endothelial growth factor receptor secreting peptide or PPA, dupilumab scFv with potent immunoglobulin secreting peptide or PPA, ustekinumab scFv with potent immunoglobulin secreting peptide or PPA, nivolumab scFv with potent immunoglobulin secreting peptide or PPA, bevacizumab scFv with potent immunoglobulin secreting peptide or PPA, and etanercept with tumor necrosis factor receptor secreting peptide or PPA were cloned into pNLF1-C.

[0153] Example 1.5: Chimeric Antigen Receptor Constructs As described above in Example 1.1, DNA gBlocks encoding tisagenlecleucel extracellular and transmembrane domains with CD8a secretory peptide or prepro, axicabtageneciloreucel extracellular and transmembrane domains with interleukin 2 secretory peptide or prepro, and lysocabtagenemaraleucel with immunoglobulin kappa secretory peptide or prepro were cloned into pcDNA3.3-TOPO (ThermoFisher Scientific) digested with XbaI and AgeI.

[0154] Example 1.6: Constructs for expression in insect and plant cells Sequences encoding SARS-CoV-2 receptor binding domain variant_1 fused to NanoLuciferase and with adipokinetic hormone (AKH) signal peptide or alpha-factor signal peptide and propeptide were codon-optimized for Spodoptera frugiperda and ordered as DNA gBlocks (IDT). These sequences were cloned into pIEx-4 insect cell expression vector (Millipore Sigma) digested with NcoI and NotI restriction enzymes as described in Example 1.1 above.

[0155] Sequences encoding NanoLuciferase and variant_1 fused to Arabidopsis root growth factor GLV1, GLV2, GLV3, GLV4, GLV5, GLV6, GLV7, GLV8, GLV9, GLV10, or GLV11 signal peptide and propeptide; α-factor signal peptide and propeptide; Arabidopsis root growth factor signal peptide and α-factor propeptide; or α-factor signal peptide and Arabidopsis root growth factor propeptide were codon-optimized for Arabidopsis thaliana and ordered as DNA gBlocks (IDT). These sequences were cloned into pRI 101-AN plant cell expression vector (Takara) digested with NdeI and EcoRI restriction enzymes as described in Example 1.1 above.

[0156] Example 2: Secretion Screening This example provides an exemplary method for detecting the secretion of a polypeptide from a mammalian cell.

[0157] HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) containing fetal bovine serum (FBS), penicillin, and streptomycin in a C170i CellXpert CO2 cell culture incubator (Eppendorf) at 37 °C and 5% CO2. When cells reached approximately 90% confluence, they were harvested using trypsin, spun at 200g for 5 min, and 2x10 6 Resuspend at 1x10 cells / ml and then 5 Next, 100 μl (1x10 4 Cells) were added to wells of a white-walled round-bottom 96-well plate. The cells were incubated for 24 hours. After 24 hours, the cells were transfected with the appropriate amount of chimeric plasmid DNA using Lipofectamine 3000 transfection reagent (ThermoFisher Scientific) and the accompanying protocol. After transfection, the cells were incubated for 72 hours.

[0158] After approximately 72 hours, the supernatants of the cell solutions in the 96-well plates were screened for luciferase activity using Intracellular TE Nano-Glo Substrate / Inhibitor (Promega). First, the cells were removed from the incubator and allowed to equilibrate at room temperature for 15 minutes. Next, a 3x substrate solution was prepared in Opti-MEM reduced serum medium (ThermoFisher Scientific) according to the Promega protocol. The supernatants were then transferred to a 96-well PCR plate and spun at 200g for 5 minutes. Next, a 10 μl aliquot of the spun down supernatant was transferred to a white 96-well plate and diluted in 90 μl of phosphate-buffered saline (PBS). Finally, 50 μl of the 3x substrate solution was added to the wells and the plate was incubated at room temperature for 2–3 minutes. Luminescence was measured within 10 minutes of adding the substrate solution using a GloMax Discover System (Promega) with an integration time of 0.1 seconds.

[0159] Example 3: ACE2 binding measurement This example provides an exemplary method for measuring SARS-CoV-2 spike RBD polypeptide binding to ACE2. SARS-CoV-2 spike polypeptides (e.g., those containing RBD) are known to bind to ACE2 expressed on the surface of mammalian cells. Thus, an ACE2 binding assay can indicate the amount of SARS-CoV-2 spike polypeptide that is secreted from a cell (e.g., a mammalian cell).

[0160] ACE2 protein (Sino Biological 10108-H08H) was diluted to 1 μg / mL in PBS. 100 μL was added to the wells of a clear flat-bottom Immuno MaxiSorp 96-well plate. The plate was covered and incubated overnight at 4° C. The plate was washed with PBS containing 0.05% Tween®-20 (PBS-T) (3 washes with 300 μL / well each). 300 μL of SuperBlock+0.05% Tween®-20 (Thermo Fisher Scientific 37516) in PBS was added to each well, the plate was covered and incubated at room temperature for 1 hour, and the wells were washed again.

[0161] 72 hours after transfection of HEK293T cells, the supernatant was collected as described in Example 2 above. 50 μL of SuperBlock and 50 μL of the supernatant were added to the wells of the ACE2-coated plate. The plate was covered and incubated at room temperature for 2 hours, then the wells were washed again. 100 μL of PBS was added to each well. 3x complete NanoLuc substrate solution (Promega) was added to each well, and luminescence was measured as described in Example 2 above.

[0162] Example 4: Increasing secretion of recombinant polypeptides from mammalian cells by prepro sequences This example demonstrates that inclusion of a prepro sequence can increase secretion of a recombinant polypeptide from mammalian cells.

[0163] Variant forms of the SARS-CoV-2 spike RBD antigen are being developed as antigens to focus immune responses on epitopes of interest and neutralization of variants. However, introducing mutations into variants can have significant effects on expression, and these modified antigens are often expressed at significantly reduced levels compared to the wild-type sequence, for example, after administration of the encoding RNA. In two RBD variants (variant_1 and variant_2), replacement of the native spike secretory peptide with the α-factor signal peptide and propeptide sequence (prepro) increased protein secretion by 10-70 fold at various doses in HEK293T human cells (Figure 1). Secreted proteins maintained proper folding, as measured by a 5- to 20-fold increase in ACE2 binding (Figure 1). Notably, in the case of variant_2, both secretion and ACE2 binding were nearly restored to wild-type RBD levels.

[0164] We tested the performance of the prepro sequence in a small library of model secreted proteins. The library consisted of wild-type SARS-CoV-2 spike RBD, influenza H1N1 / PR8 hemagglutinin extracellular domain (HA), human secreted fetal alkaline phosphatase (SEAP), and three single-chain antibody fragments engineered from Her2-targeting monoclonal antibodies targeting different epitopes: trastuzumab (Herceptin), pertuzumab (Perjeta), and GB235 (Shu et al., incorporated herein by reference in its entirety). Replacement of the native secreted peptide (the SARS-CoV-2 spike secreted peptide was used for the scFv) with the prepro sequence increased secretion from HEK293T cells by 2.5- to 7.5-fold at various doses (Figure 2).

[0165] We next investigated the performance of prepro sequences in eight of the top-selling biological drugs from 2020 (Buntz et al., incorporated herein by reference in its entirety). These molecules consist of chimeric fusion proteins consisting of a single-chain antibody and a cellular receptor fused to an immunoglobulin domain. For biologics where the native secretory peptide was not publicly available, we instead selected potent secretory peptides from human antibody sequences that have been experimentally validated (Haryadi et al., incorporated herein by reference in its entirety). In all eight biologics tested, the prepro sequence enhanced secretion over native / potent human antibody secretory peptides (Figure 3). Effect sizes ranged from 1.2-40 fold.

[0166] Example 5: Optimized prepro sequences increase secretion Based on the mechanism of enhanced secretion in yeast cells, the prepro sequence was optimized for activity in human cells. Normally, the function of the propeptide region of α-factor is to enable export from the endoplasmic reticulum (ER) and transport to the Golgi apparatus, where it is cleaved by proteases to generate the mature α-factor peptide to be secreted. Specifically, the propeptide sequence binds to the ER membrane protein Erv29p, which is involved in packaging proteins into COPII vesicles for secretion from the cell (Otte et al., incorporated herein by reference in its entirety). The human homolog of Erv29p is Surf4, which establishes distinct steady-state concentrations of soluble proteins in the ER through interactions with an amino-terminal tripeptide exposed after cleavage of the signal peptide (Yin et al., incorporated herein by reference in its entirety). The affinity of the tripeptide motif for Surf4 determines the steady-state concentration of the cargo protein, with a subset of all possible tripeptides establishing a continuum of concentrations, with the highest affinity motif promoting the greatest secretion at the lowest ER concentrations (Yin et al., incorporated herein by reference in its entirety). Mutating the amino-terminal residues of the α-factor propeptide sequence to a tripeptide motif with high Surf4-binding affinity increased secretion of SARS-CoV-2 spike RBD variant antigens with altered prepro sequences (Figure 4).

[0167] Furthermore, upon entry into the ER, the propeptide region is cleaved by proteases to generate the mature protein. Specifically, the α-factor propeptide is cleaved by the yeast proteases Kex2 and Ste13 (Fitzgerald et al., incorporated herein by reference in its entirety). Mutation of the Kex2 cleavage site to the cleavage sites of the human proteases S1P, PCSK9, Furin, and PCSK4, and deletion of the Ste13 cleavage site, enhanced secretion of the variant RBD antigen over the wild-type prepro sequence (Figure 4).

[0168] The greatest enhancement was obtained when the humanized protease site was combined with the Ste13 deletion, suggesting that these secretion-enhancing mutations could be combined to further enhance secretion and fully optimize the sequence for function in human cells.

[0169] Hits that individually enhanced secretion in the first screen were combined for a second round of sequence optimization. Specifically, sequences tested included (1) a combination of a tripeptide Surf4 binding motif with a humanized protease cleavage site, (2) a combination of different humanized protease cleavage sites, (3) repeats of the Surf4 binding motif with various spacer structures, and (4) repeats of individual humanized protease cleavage sites. All sequences tested contained a deletion of the Ste13 cleavage site. In general, combinations of individual secretion-enhancing mutations further enhanced secretion additively (Figure 5).

[0170] Hits from the second round of screening were combined for a third round of sequence optimization. All of these sequences lacked the Ste13 cleavage site and contained 2x Surf4 motifs with a flexible glycine-serine linker. Although all combinations tested enhanced secretion compared to the wild-type prepro sequence (FIG. 6), the greatest enhancement observed came from the combination of 2x Surf4 motifs with a flexible glycine-serine linker, deletion of the Ste13 cleavage site, and mutation of the Kex2 cleavage site to a 3x S1P cleavage site. The human protease S1P (membrane-bound transcription factor site 1 protease) processes protein and peptide precursors that are transported through the secretory pathway in the ER and Golgi apparatus (Garten et al., incorporated herein by reference in its entirety). Together, these modifications to the wild-type prepro sequence enhanced secretion 10-fold.

[0171] This modified prepro sequence (hereafter referred to as prepro_2) was further optimized for activity in human cells by inserting an additional peptide sequence that intentionally binds to a receptor that transports proteins through the secretory pathway. LMAN1 / ERGIC53 is an integral protein of the endoplasmic reticulum (ER)-Golgi intermediate compartment, which transports glycosylated proteins from the ER to the Golgi (Nichols et al.). Confirmed cargoes that bind to LMAN1 include protease cathepsin C (CATHC) and coagulation factor proteins. Peptide sequences from CATHC and coagulation factor VII (CFVII) that contain glycosylation sites enhanced protein secretion when inserted into the modified prepro_2 sequence (Figure 7). The peptide sequences were inserted into one of three locations in the prepro_2 sequence: (1) the propeptide N-terminus following the 2x Surf4 motif with a flexible glycine-serine linker, (2) in the middle of the propeptide sequence, or (3) at the C-terminus immediately preceding the 3x S1P cleavage site.

[0172] Another membrane-bound receptor that transports cargo through the secretory pathway is sortilin 1 (SORT1), which resides in the Golgi apparatus and serves to transport cargo proteins into secretory vesicles (Petersen et al.). SORT1 has been shown to bind and transport multiple protein cargoes, including brain-derived neurotrophic factor (BDNF) (Chen et al.). In addition, SORT1 binds to its own propeptide, which is cleaved upon reaching the Golgi apparatus. Peptide sequences derived from BDNF and SORT1 enhanced protein secretion when inserted into prepro_2 at the same three positions as above (Figure 7).

[0173] These data suggest that independent sequences and domains can be combined within a single polypeptide sequence to target proteins to multiple independent secretory pathway receptors and secretion mechanisms. Combining the LMAN1-binding domain and the SORT1-binding domain in a modified sequence did not appear to further enhance secretion. Without wishing to be bound by any particular theory, this data suggests that separate sequences containing the LMAN1-binding domain and the SORT1-binding domain, respectively, in the prepro_2 context can be co-delivered to cells such that two different secretory receptors / pathways occur in parallel.

[0174] Example 6: Measurement of chimeric antigen receptor expression This example provides an exemplary method for detecting the display of anti-CD19 chimeric antigen receptor (CAR) T cell therapy receptor polypeptides on the surface of mammalian cells.

[0175] Although membrane-bound proteins at the cell surface follow similar trafficking pathways as secreted proteins, it was unclear whether prepro sequences function similarly to enhance cell surface display of transmembrane proteins. Clinically approved CAR-T cell therapy receptors can serve as model systems to measure the effect of prepro sequences on the display of cell membrane proteins. Specifically, both expression and folding of anti-CD19 CAR-T receptors on the surface of HEK293T cells can be measured using fluorescently labeled CD19 ligand and quantified on a cell-by-cell basis using flow cytometry.

[0176] Example 6.1: Expression of chimeric antigen receptors in HEK293T cells HEK293T cells are cultured, harvested, seeded and treated with a plasmid encoding the CAR protein formulated in Lipofectamine 3000 as described in Example 2 above. After approximately 72 hours, cells are harvested using enzyme-free cell dissociation buffer (ThermoFisher Scientific) and spun down in a black V-bottom plate at 300xg for 10 minutes. The supernatant is discarded and the cells are placed on ice. The cells are resuspended in eBioscience flow cytometry staining buffer (ThermoFisher Scientific) containing 20μg / mL biotinylated CD19 (BPS Bioscience) and incubated on ice for 30 minutes. The cells are spun down, the supernatant is discarded, and the cells are resuspended in eBioscience flow cytometry staining buffer containing 10μg / mL phycoerythrin (PE)-conjugated streptavidin and incubated on ice for 30 minutes. The cells are spun down, the supernatant is discarded, and the cells are resuspended in eBioscience flow cytometry staining buffer. Cells are diluted to approximately 1e5 cells / mL in eBioscience flow cytometry staining buffer and strained through a 35 μm mesh (StemCell) immediately prior to flow cytometry analysis.

[0177] Binding of CD19 to the cell surface is quantified using flow cytometry. CD19 signals are measured using the BL2 channel (488 nm excitation laser, 574 / 26 nm filter emission) of an Attune CytPix flow cytometer (ThermoFisher Scientific).

[0178] Example 6.2: Expression of chimeric antigen receptors in primary human T cells Approximately 2e7 frozen human peripheral blood pan-T cells (STEMCELL) are thawed and transferred to a 50ml tube and complete T cell media (50ml ImmunoCult-XF T cell growth media (STEMCELL) and 50ul 0.1mg / ml CHO-expressed recombinant human IL-2 (STEMCELL)) is added dropwise to the tube while stirring. The cells are pelleted by spinning the tube at 300g for 10 minutes. The cells are washed by aspirating off the supernatant and resuspending in 20ml complete T cell media. The cells can be pelleted again, the supernatant removed, and the cells resuspended in 5ml complete T cell media as above. The cell suspension is adjusted to 1e6 cells / ml. 7ml of this cell suspension (7e6 cells) is transferred to a T25 flask. ImmunoCult Human CD3 / CD28 / CD2 T cell activator (STEMCELL) 20ul / 1e6 cells are added to the cells in the flask. The cells are then cultured for 72 hours at 37° C., 95% RH, 5% CO2. Two days after activation, the cell concentration in the flask is maintained at 1e6 cells / ml by supplementing the medium with fresh complete T cell medium.

[0179] Lipid nanoparticles (LNPs) are formulated using the protocol provided with the Spark (PRECISION NANOSYSTEMS), Genvoy-ILMT cell kit. Briefly, 1 mg / ml mRNA encoding tisagenlecleucel with CD8 signal peptide, prepro, or prepro_2, Genvoy lipid mix, and aqueous diluent formulation buffer are mixed in a 1:2:3 ratio using the set 3 protocol on the Spark instrument (PRECISION NANOSYSTEMS). The LNPs are then characterized for payload concentration, diameter, and encapsulation efficiency using a modified Ribogreen assay as described in the kit protocol above.

[0180] Remove the activated T cells from the incubator and transfer to a 15ml tube. Pellet the cells by spinning at 300g for 10 minutes and suspend at 0.5e6 cells / ml in 1ug / ml of supernatant supplement. Then transfer 125ul of the 0.5e6 cells / ml suspension to a clear flat bottom 96 well plate. Add LNP to each well. The plate is then incubated at 37°C, 95% RH, 5% CO2 for 48 hours.

[0181] To quantify CAR expression, remove cells from incubator and pellet by spinning at 300g for 10 minutes. Wash cells by removing supernatant, resuspending in 200ul of 1x PBS, pelleting and removing supernatant. Stain cells with 100ul of a 1:1000 dilution of LIVE / DEAD fixable Violet dead cell stain (ThermoFisher Scientific) for 30 minutes. Remove viability stain by pelleting cells and removing supernatant. Next, stain cells with 100ul of a 1:50 dilution of APC-labeled human CD19 (ACROBIOSYSTEMS) in eBioscience flow cytometry staining (FACS) buffer (ThermoFisher Scientific) for 1 hour. Remove staining reagent by pelleting cells and removing supernatant. Wash cells 3 times with ice-cold FACS buffer and suspend in FACS buffer. Quantify CAR expression surrogately via binding of CD19 to the cell surface using flow cytometry. The VL1 channel (405 nm excitation laser, 440 / 50 nm filter emission) is used to measure cell viability and the RL1 channel (637 nm excitation laser, 670 / 14 nm filter emission) is used to measure CD19 signal on an Attune CytPix flow cytometer (ThermoFisher Scientific).

[0182] To test the activity of surface-expressed anti-CD19 CAR on primary human T cells, the killing activity is measured on CD19-positive Nalm6 cells stably expressing GFP and firefly luciferase (Fluc) (Nalm6-FLuc / EGFP) (Imanis). Nalm6-FLuc / EGFP cells are cultured in RPMI 1640 medium (STEMCELL) containing 10% FBS, 1% Penn-Strep, 10 mM HEPES, 1 mg / ml G418, and 1 ug / ml puromycin. Cells are maintained by passaging every 3 days to keep the cell concentration at 1e6 cells / ml.

[0183] To test the killing effect, 1e5 Nalm6-FLuc / EGFP cells are co-cultured with various amounts of CAR-T cells in flat-bottom opaque white 96-well plates to achieve the following effector-target ratios: 0:1, 2:1, 1:1, 1:2, 1:4, 1:8, and 1:16. Plates are incubated at 37° C., 95% RH, 5% CO2 for 18 hours.

[0184] After 18 hours, the cell plates are removed from the incubator and allowed to equilibrate to room temperature. After equilibration, 100 ul of Bright-Glo luciferase substrate (Promega) is added to each plate well and incubated at room temperature for 2 minutes. Luminescence is measured using a GloMax Discover microplate reader (Promega). Luminescence data is then used to calculate specific cell lysis.

[0185] Different cell types have different secretory requirements and therefore display unique expression patterns of secretory pathway genes and proteins (Feizi et al.). Therefore, a sequence optimized for protein secretion in one cell type may not be the optimal sequence for protein secretion in a different cell type. Combining a modified pro sequence (pro2) with a signal peptide sequence known to cause high protein secretion in primary T cells (e.g., IgK light chain, CD8, and IL2 signal peptide sequences) or common mammalian cells (e.g., SARS-CoV-2 spike signal peptide sequence) can enhance protein cell surface display on primary human T cells more than the modified prepro sequence alone and can be screened for expression, folding, and activity as described above.

[0186] CAR T cell therapy receptor polypeptide sequences are highly optimized for protein expression and folding in mammalian cells and for target antigen binding; indeed, any polypeptide sequence that has been successfully developed for biomanufacturing or clinical use is highly optimized for these purposes. In many cases, it may be ideal to utilize a polypeptide sequence that is not highly optimized. For example, omitting the directed evolution and / or affinity maturation steps in antibody single chain fragment development may save significant time in the biotherapeutic development process. Furthermore, treatment with CAR T cell therapy is often accompanied by several severe side effects, such as cytokine storm or off-target toxicity (Rafiq et al.), that arise from the high affinity of the receptor for the target cancer cell antigen; utilizing low affinity receptors may mitigate these effects, but often at the cost of reduced protein expression. Prepro sequences may be fused to these non-optimal protein sequences to enhance expression and thereby rescue the deleterious effects. For example, the anti-CD19 single chain fragment A3B1 (Castella et al.) shows lower expression and affinity than clinically approved CAR-T cell therapy single chain fragments and can serve as a model system to measure the effect of prepro sequences on the expression and cell surface display of suboptimal protein sequences. Specifically, the expression, folding, and activity of the anti-CD19 single chain fragment A3B1 on the surface of primary human T cells can be measured as described above.

[0187] Example 7: Expression in additional biomanufacturing cell lines This example provides exemplary methods for detecting the secretion of polypeptides from additional mammalian, insect, and plant cells that are routinely used to produce biotherapeutics.

[0188] Example 7.1: Exemplary protocols for biomanufacturing in mammalian and insect cell lines CHO-K1 Chinese Hamster Ovary cells (ATCC) are grown in F12K medium (ATCC) supplemented with 10% FBS and penicillin and streptomycin in a C170i CellXpert CO2 cell culture incubator (Eppendorf) at 37° C. and 5% CO2. Cells are harvested, plated, and treated with mammalian pNLF1C-based plasmids formulated in Lipofectamine 3000 as described in Example 2 above.

[0189] Insect Sf9 cells (ThermoFisher Scientific) are grown in Grace's insect medium (ThermoFisher Scientific) supplemented with 10% FBS and penicillin and streptomycin in a non-humidified incubator at 27° C. Cells are harvested and plated as described in Example 2 above. Cells are treated with pIEx-4 insect cell expression vector-based chimeric plasmids formulated in Lipofectamine 3000 as described in Example 2 above.

[0190] Example 7.2: Bioproduction and expression in CHO-K1 and Sf9 cell lines CHO-K1 Chinese Hamster Ovary cells (ATCC) were grown in F12K medium (ATCC) supplemented with 10% FBS and penicillin and streptomycin in a C170i CellXpert CO2 cell culture incubator (Eppendorf) at 37° C. and 5% CO2. Cells were harvested, plated, and treated with mammalian pNLF1C-based plasmids formulated in Lipofectamine 3000 as described in Example 2 above.

[0191] Insect Sf9 cells (ThermoFisher Scientific) were grown in Grace's insect medium (ThermoFisher Scientific) supplemented with 10% FBS and penicillin and streptomycin in a non-humidified incubator at 27° C. When the cells reached approximately 90% confluence, they were harvested by pipetting, centrifuged at 200 g for 5 min, and resuspended in 4×10 cells in Sf-900 III serum-free medium (ThermoFisher Scientific). 5 The diluted cell solution was then added to 2 ml (8 x 10 5 Cells) were added to wells of a 6-well plate. Cells were allowed to attach for 15 minutes. Cells were then transfected with pIEx-4 insect cell expression vector-based chimeric plasmids using CellFectin II reagent (ThermoFisher Scientific) and the accompanying protocol. 72 hours after transfection, supernatants were collected and luminescence was measured as described in Example 2 above.

[0192] As shown in Figures 8A-8B, replacement of the native spike signal peptide (Ssp) with the α-factor signal peptide and propeptide sequence (prepro) enhanced protein secretion of RBD variant_1 and variant_2 by 1.1- to 15.6-fold over a dose range of 1-100 ng in CHO-K1 Chinese hamster ovary cells (Figure 8A). Replacement of the cell-type specific adipokinetic hormone signal peptide with α-factor prepro enhanced secretion of variant_1 by 3.7- to 5.6-fold over a dose range of 100-1,000 ng in Sf9 Spodoptera frugiperda cells (Figure 8B).

[0193] Example 8: Expression in plant cell lines This example provides an exemplary method for detecting the secretion of a polypeptide from plant cells that is routinely used to produce biotherapeutics.

[0194] Plant T87 cells (Ohio State University Arabidopsis Biological Resource Center) are grown in NT-1 medium in a non-humidified incubator at 24° C. with orbital shaking at 120 rpm. Cells are grown in suspension to approximately 90% confluence and then transfected with an appropriate amount of pRI 101-AN plant cell expression vector-based chimeric plasmid DNA using either electroporation or Agrobacterium-mediated transfection. Electroporation is performed using a Bio-Rad Gene Pulser II in a 4 mm wide cuvette with the following program: for poring pulses, 375 V / cm, 10 ms, 5 times, and 50 ms intervals; for transfer pulses, 50 V / cm, 50 ms, 20 times, and 50 ms intervals. After electroporation, cells are seeded in 96-well plates. Agrobacterium-mediated transfection is performed using LBA4404 cells (Takara). LBA4404 cells are electroporated with 1 ng of pRI 101-AN-based chimeric plasmid DNA using a Bio-Rad Gene Pulser II in a 1 mm wide cuvette with the following program (25 μF, 200 ohms, 2 kV, single pulse). Cells will be recovered in SOC medium with shaking at 100 rpm at 30° C. for 1 hour, then plated on agar medium containing kanamycin and incubated at 30° C. for 48 hours. Colonies will then be selected and cultured in LB medium containing kanamycin with shaking at 30° C. For transfection of T87 plant cells, LBA4404 cells transformed with the appropriate plasmid are added to T87 cells seeded in a 96-well plate. After transfection, cells are incubated for 72 hours. Supernatants are then collected and luminescence measured as described in Example 2 above.

[0195] Example 9: Prepro enhances immune response to vaccination This example provides an exemplary method for measuring immune responses in vivo after vaccination with SARS-CoV-2 antigenic polypeptides, including IgG and IgA induction, pseudotype virus neutralization. It also contains data showing that prepro and prepro_2 enhance serum IgG and mucosal IgA responses and pseudotype virus neutralization upon SARS-CoV-2 spike RBD vaccination in mice. Finally, it contains proposed experiments to enhance IgA responses by blocking propeptide cleavage and data supporting this strategy in vitro.

[0196] Example 9.1: Vaccination Methods Constructs were synthesized as IDT gBlocks. DNA templates were amplified with T7-AGG_fwd and 120pA_rev primers using Herculase II polymerase (Agilent) at an annealing temperature of 50°C. The resulting PCR products were cleaned up using a DNA Clean & Concentrator-25 kit (Zymo Research). 19.9 μL of transcription mix consisting of 1x HiScribe T7 High Yield Buffer (NEB), 7.5 mM of each NTP (ac4-CTP, GTP, ATP, and UTP), 7.5 mM CleanCap AG (TriLink Biotech), 2 M betaine (ThermoSci), 20 mM MgCl2, and 0.1 μL / μL HiScribe T7 polymerase mix was added to 2.1 uL of DNA solution consisting of 200 ng of T7 template in nuclease-free H2O. Transcription was carried out at 50°C for 1 hour. Transcribed RNA was purified using a 500 μg volume of the Monarch RNA Cleanup Kit, treated with 10 U of DNAse I (New England Biolabs) and 100 U of Calf Intestinal Alkaline Phosphatase (Promega) in DNase I buffer for 5 min at 37° C., and purified again using a 500 μg volume of the Monarch column. Concentration was measured using a NanoDrop spectrophotometer.

[0197] Formulations of mRNA in lipid nanoparticles (mRNA-LNPs) were prepared using a NanoAssemlr Ignite microfluidic mixer (Precision Nanosystems). GenVoy-ILM lipid mixture (Precision Nanosystems) was diluted to 12.5 mM in absolute ethanol and mixed with aqueous mRNA solution (0.14 mg / mL) in PNI buffer (Precision Nanosystems) using the manufacturer's recommended formulation parameters. The formulation was immediately diluted 30:1 with phosphate-buffered saline (pH 7.4) and concentrated using an Amicon centrifugal filter (MilliporeSigma UFC901008). Formulations were stored at 4 °C and used for in vivo studies within 14 days.

[0198] All animal experiments were performed in accordance with the guidelines set forth by the Charles River Accelerator Development Lab (CRADL) and approved by the Animal Care and Use Committee of CRADL. Female BALB / C mice (7-9 weeks old) were purchased from Charles River Laboratories and housed at CRADL. Mice were acclimated for at least 3 days prior to the start of the study. On day 1, mice were injected with 50 μL of mRNA-LNP formulation (0.4 μg of mRNA) in the right quadriceps. On day 21, mice were injected with 50 μL of mRNA-LNP formulation in the left quadriceps for booster immunization. On day 27, mice were euthanized, at which point blood was collected by intracardiac puncture. Blood was allowed to clot for 30 min at room temperature, after which serum was separated from the blood in MiniCollect serum separator tubes (Greiner Bio-One 450472) by centrifugation at 1200 x g for 10 min at 4 °C. Fresh serum was stored at 4°C and used to assess immunogenicity by ELISA, the remainder was aliquoted and frozen at -80°C.

[0199] Immediately after the death of the animals and IC blood collection, a vaginal mucosa wash was performed. Using a p20 pipette, 20 μl of sterile PBS was introduced into the vagina. The liquid was pipetted back and forth in the organ 8 times, after which the sample volume was transferred to a 1.5 mL tube. This process was repeated a total of 3 times per animal, using a sterile p20 tip each time, resulting in a final pooled collection volume of 60 μl per animal. The vaginal wash samples were centrifuged at 300×g for 7 min, and the supernatant was collected and stored at 4°C overnight for use in the IgA ELISA assay the following day.

[0200] Example 9.2: Antibody Titer Assay A serum IgG ELISA protocol was adapted from that previously established by Amanat, et al. (Nat Med 26:1033-1036, 2020). 96-well Immulon 4 HBX plates (Thermo Fisher Scientific) were coated with 50 μl per well of SARS-CoV-2 (2019-nCoV) spike S1+S2 ECD-His recombinant protein (Sino Biological #40589-V08B1) in PBS (2 μg / ml) and incubated overnight at 4 °C. Plates were then washed three times with 300 μl of 0.1% Tween® 20 in PBS (PBS-T) using an automated plate washer (BioTek) and blocked with 200 μl / well of SuperBlock PBS blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature. Serial dilutions of serum samples were prepared in a 1:3 dilution of Superblock in PBS. 100 μl of each serial dilution was added to the plate and incubated for 2 h at room temperature. The wells were then washed three times with PBS-T as described above. 50 μl of a 1:3,000 dilution of goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, AP127P) in a 1:3 dilution of Superblock in PBS was added to all wells and incubated for 1 h at room temperature. Again, the plate was washed three times with PBS-T. 100 μl of SIGMAFAST OPD (Sigma-Aldrich) solution was added to each well and incubated for 10 min at room temperature. The reaction was stopped by adding 50 μl of 2 N hydrochloric acid per well. Optical density was measured at 490 nm using a GloMax Discover (Promega) plate reader. The endpoint titer was determined by taking the last dilution before the signal fell to less than one standard deviation above the mean of the signals of untreated control sera at the same dilution. If the signal did not fall below this threshold, the last dilution was taken as the titer. If no signal above the threshold was detected, the value from the dilution series before the least diluted sample was used was used.

[0201] For IgA ELISA, 96-well Immulon 4 HBX plates (Thermo Fisher Scientific) were coated with 50 μl per well of SARS-CoV-2 (2019-nCoV) spike S1+S2 ECD-His recombinant protein (Sino Biological #40589-V08B1) in PBS (4 μg / ml) and incubated overnight at 4 °C. Plates were then washed three times with 0.1% Tween® 20 in PBS (PBS-T) (300 μl) using an automated plate washer (BioTek) and blocked with 3% nonfat milk in PBS-T (200 μl) per well for 1 h at room temperature. Serial dilutions of vaginal wash samples were prepared in 1% nonfat milk in PBS-T. 75 μl of each serial dilution was added to the plate and incubated for 2 h at room temperature. Wells were then washed three times using PBS-T as described above. 50 μl of a 1:1,000 dilution of goat anti-mouse IgA horseradish peroxidase conjugated secondary antibody (abcam ab97235) in 1% milk PBS-T was added to all wells and incubated for 1 hour at room temperature. The plate was again washed 3 times with PBS-T. 100 μl of SIGMAFAST OPD (Sigma-Aldrich) solution was added to each well and incubated for 10 minutes at room temperature. The reaction was stopped by adding 50 μl of 2N hydrochloric acid per well. Optical density was measured at 490 nm using a GloMax Discover (Promega) plate reader. The endpoint antibody titer was determined by taking the last dilution before the signal fell below one standard deviation above the mean of the signal of the untreated control serum at the same dilution. If the signal did not fall below this threshold, the last dilution was taken as the antibody titer. If no signal above the threshold was detected, the value from the dilution series before the least diluted sample was used was used.

[0202] Example 9.3: SARS-CoV-2 pseudovirus neutralization assay Human ACE2-overexpressing HEK cells (Integral Molecular) used for viral transduction experiments were maintained in high glucose GlutaMAX-containing DMEM (ThermoFisher Scientific 10564) supplemented with 1 μg / mL puromycin, 10% heat-inactivated fetal bovine serum, and 100 U / mL penicillin / streptomycin. Serum dilutions were mixed with spike-pseudotyped (either D61G Wuhan or Omicron) Renilla luciferase-encoded reporter viral particles constructed using a second-generation lentiviral system (Integral Molecular) in puromycin-free medium to a total volume of 100 μl. Virus and serum were incubated for 1 h at 37 °C in 96-well cell culture plates. Then, 20,000 freshly harvested HEK cells were added in 50 μL of puromycin-free medium and allowed to transduce for 3 days. Cells were stored at -20 °C before analysis. Luciferase was measured using the Renilla-Glo Luciferase Assay System (Promega). The N50 antibody titer was determined by taking the last dilution before the signal fell below 50% of that of the untreated control serum at the same dilution.

[0203] Example 9.4: Prepro sequences enhance humoral IgG and IgA responses to vaccination BALB / c mice were vaccinated and boosted with either the native spike signal peptide, prepro, or prepro_2 with ac4C-modified mRNA encoding the receptor binding domain (RBD) of the SARS-CoV-2 Wuhan strain as described above. Both prepro and prepro_2 showed a marked improvement over the strong spike signal peptide in both total serum IgG antibody titers and vaginal mucosal IgA antibody titers (Figure 9). Concomitant with improved secretion, prepro_2 showed approximately 4-fold higher IgA antibody titers than prepro.

[0204] When RBD was fused to the immunostimulatory Sbi(III-IV) protein domain, preproleader generated mucosal IgA antibody titers 2.3-fold higher than prepro_2 (FIG. 10). Since the efficiency of procleavage of prepro is expected to be lower than human-adapted prepro_2, this observation suggests that retention of the proleader sequence in secreted antigens may be immunostimulatory. Without wishing to be bound by a particular theory, a possible mechanism of action may include SURF4-mediated uptake of soluble pre-RBD-Sbi(III-IV) by antigen-presenting cells, vascular epithelial cells, or other cell types capable of stimulating a humoral immune response. In some embodiments, preproleaders lacking or attenuated Ste13 and / or Kex2 cleavage sites and prepro_2 leaders lacking or attenuated S1P cleavage sites may be immunostimulatory. This observation suggests that in some embodiments, vaccination of mammals with prepro(ΔSte13 ΔKex2) or prepro_2(ΔS1P) fused to an antigen, with or without further fusion to Sbi(III-IV) or other immunostimulatory adjuvant domains, can generate stronger mucosal IgA responses than when a mammalian signal peptide (e.g., a strong mammalian signal peptide) is used.

[0205] Example 9.5: Prepro_2 Cleavage To examine whether the prepro_2 sequence is cleaved from the protein before secretion, both total and uncleaved protein secretion was measured in HEK293T cells.

[0206] HEK293T cells were cultured and transfected as described above in Example 2. Cells were transfected with a chimeric plasmid encoding SARS-CoV-2 spike receptor binding domain variant_1 fused to NanoLuciferase with prepro_2 containing a 3x MYC protein tag to allow detection of the propeptide sequence of the secreted protein in the supernatant via MYC ELISA.

[0207] Total protein secretion was measured as described in Example 2 above. Secretion of uncleaved protein was measured by MYC ELISA. Capture anti-MYC tag antibody (Abcam ab32) was diluted to 8 μg / mL in PBS. 100 μL was added to the wells of a clear flat-bottom Immuno MaxiSorp 96-well plate. The plate was covered and incubated overnight at 4° C. The plate was washed with PBS containing 0.05% Tween®-20 (PBS-T) (3 washes with 300 μL / well each). 300 μL of SuperBlock+0.05% Tween®-20 (Thermo Fisher Scientific 37516) in PBS was added to each well, the plate was covered, incubated at room temperature for 1 hour, and the wells were washed again. 72 hours after transfection of HEK293T cells, the supernatant was collected as described in Example 2 above. 50 μL of SuperBlock and 50 μL of supernatant were added to the wells of the anti-MYC antibody-coated plate. The plate was covered and incubated at room temperature for 2 hours, then the wells were washed again. The detection anti-MYC tag antibody (abcam 1326) conjugated to HRP was diluted to 400 ng / mL in SuperBlock and 100 μL was added per well. The plate was covered and incubated at room temperature for 1 hour, then the wells were washed again. SIGMAFAST OPD chromogen (Millipore Sigma) was prepared according to the manufacturer's protocol and 100 μL was added to each well. After 10 minutes, the reaction was stopped with 50 μL of 2 M HCl (Millipore Sigma). The absorbance at 490 nm was measured using the GloMax Discover System (Promega). To account for differences in protein secretion between constructs, the uncleaved protein secretion measurements (MYC ELISA signal) were normalized to the total protein secretion measurements (luminescence signal).

[0208] Uncleaved protein was detected in the supernatant of HEK293T cells transfected with a chimeric plasmid containing 3x MYC-tagged prepro_2 (Figure 11). The amount of uncleaved protein detected in the supernatant containing prepro_2 was less than that detected with prepro_2 in which the protease cleavage site was removed (Figure 11). This suggests that the propeptide of prepro_2 is partially cleaved from the secreted protein. Without wishing to be bound by a particular theory, this observation suggests that the prepro_2 propeptide is present in the secreted polypeptide and may affect the antibody response to vaccination in vivo.

[0209] Example 9.6: Prepro sequences enhance SARS-CoV-2 pseudotype virus neutralization and antibody titers by vaccination with RBD fused to Sbi(III-IV) with prepro sequences BALB / C mice were vaccinated with ac4C-modified mRNA encoding the receptor-binding domain (RBD) of the SARS-CoV-2 Wuhan or Omicron strain fused to the immunostimulatory Sbi(III-IV) protein domain, with either the native spike signal peptide or prepro, as described above, except mice were administered 10 μg of mRNA and euthanized on day 35.

[0210] Pseudotype virus neutralization assays demonstrated that prepro enhanced N50 antibody titers of both virus strains (Figure 12). Consistent with the increased neutralization, prepro enhanced both mucosal IgA and serum IgG antibody titers (Figure 13).

[0211] This data indicates that the presence of a prepro sequence on an antigen increases the effectiveness and / or efficiency of vaccination and may also enhance the immune response generated by vaccination.

[0212] Exemplary embodiments Embodiment 1. A modified polypeptide comprising: (i) a payload polypeptide, (ii) a wild-type or modified α-factor prepro sequence, or a fragment thereof. The modified polypeptide, wherein the wild-type or modified α-factor prepro sequence, or a fragment thereof, is operably linked to the payload polypeptide.

[0213] Embodiment 2 The modified polypeptide of embodiment 1, wherein the payload polypeptide is a therapeutic polypeptide.

[0214] Embodiment 3. The modified polypeptide of embodiment 1 or 2, wherein the payload polypeptide comprises one or more antigens.

[0215] Embodiment 4 The modified polypeptide of embodiment 3, wherein the one or more antigens are one or more viral antigens.

[0216] Embodiment 5. The modified polypeptide of embodiment 4, wherein the one or more viral antigens are one or more SARS-CoV-2 antigens.

[0217] Embodiment 6. The modified polypeptide of embodiment 5, wherein the one or more viral antigens comprises a SARS-CoV-2 receptor binding domain or a fragment thereof.

[0218] Embodiment 7. The modified polypeptide of embodiment 5 or 6, wherein the one or more viral antigens comprises a SARS-CoV-2 spike protein or a fragment thereof.

[0219] Embodiment 8 The modified polypeptide of embodiment 4, wherein the one or more viral antigens are one or more influenza antigens.

[0220] Embodiment 9 The modified polypeptide of embodiment 8, wherein said one or more influenza antigens are one or more influenza A antigens.

[0221] Embodiment 10 The modified polypeptide of embodiment 3, wherein the one or more antigens are one or more bacterial antigens.

[0222] Embodiment 11. The modified polypeptide of embodiment 3, wherein the one or more antigens are one or more cancer antigens.

[0223] Embodiment 12 The modified polypeptide of embodiment 3, wherein said one or more antigens are one or more archaeal antigens.

[0224] Embodiment 13 The modified polypeptide of embodiment 1 or 2, wherein the payload polypeptide comprises an antibody or a fragment thereof.

[0225] Embodiment 14 The modified polypeptide of embodiment 1 or 2, wherein the payload polypeptide comprises a fusion protein comprising an extracellular receptor and an immunoglobulin constant region.

[0226] Embodiment 15. The modified polypeptide of embodiment 1 or 2, wherein the payload polypeptide comprises a fusion protein comprising a receptor agonist and an immunoglobulin constant region.

[0227] Embodiment 16 The modified polypeptide of embodiment 1 or 2, wherein the payload polypeptide comprises a fusion protein comprising a receptor antagonist and an immunoglobulin constant region.

[0228] Embodiment 17. The modified polypeptide of any one of embodiments 1 to 16, wherein the wild-type or modified α-factor prepro sequence, or a fragment thereof, is present at the N-terminus of the modified polypeptide.

[0229] Embodiment 18. The modified polypeptide of any one of embodiments 1 to 16, wherein the wild-type or modified α-factor prepro sequence, or a fragment thereof, is present at the C-terminus of the modified polypeptide.

[0230] Embodiment 19. The modified polypeptide of any one of embodiments 1 to 18, wherein the modified polypeptide comprises a wild-type α-factor prepro sequence or a fragment thereof.

[0231] Embodiment 20 The modified polypeptide of embodiment 19, wherein the wild-type α-factor prepro sequence comprises the sequence set forth in SEQ ID NO:2, SEQ ID NO:68, or SEQ ID NO:69.

[0232] Embodiment 21 The modified polypeptide of any one of embodiments 1 to 18, wherein the modified polypeptide comprises a modified α-factor prepro sequence or a fragment thereof.

[0233] Embodiment 22 The modified polypeptide of embodiment 21, wherein the modified α-factor prepro sequence comprises a tripeptide motif that has Surf4 binding affinity.

[0234] Embodiment 23 The modified polypeptide of embodiment 22, wherein the tripeptide motif is MPL (SEQ ID NO:58).

[0235] Embodiment 24 The modified polypeptide of any one of the preceding embodiments, wherein the modified α-factor prepro sequence does not contain a Ste13 cleavage site.

[0236] Embodiment 25. The modified polypeptide of embodiment 24, wherein the Ste13 cleavage site comprises EAEA (SEQ ID NO:56).

[0237] Embodiment 26 The modified polypeptide of any one of the preceding embodiments, wherein the modified α-factor prepro sequence does not contain a Kex2 cleavage site.

[0238] Embodiment 27 The modified polypeptide of embodiment 26, wherein the Kex2 cleavage site comprises KR (SEQ ID NO:57).

[0239] Embodiment 28. The modified polypeptide of embodiment 26 or 27, wherein the Kex2 cleavage site found in the wild-type α-factor prepro sequence has been replaced in the modified α-factor prepro sequence by a protease cleavage site for a human protease.

[0240] Embodiment 29. The modified polypeptide of embodiment 28, wherein the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence by an S1P, PCSK4, PCSK9, or Furin protease cleavage site.

[0241] Embodiment 30. The modified polypeptide of embodiment 29, wherein said protease cleavage site of S1P has a sequence according to SEQ ID NO:55.

[0242] Embodiment 31 The modified polypeptide of embodiment 29, wherein said protease cleavage site of PCSK4 has a sequence according to SEQ ID NO:53.

[0243] Embodiment 32 The modified polypeptide of embodiment 29, wherein said protease cleavage site of PCSK9 has a sequence according to SEQ ID NO:54.

[0244] Embodiment 33 The modified polypeptide of embodiment 29, wherein said protease cleavage site of Furin has a sequence according to SEQ ID NO:52.

[0245] Embodiment 34 The modified polypeptide of any one of the preceding embodiments, wherein the modified α-factor preprosequence comprises a CATHC peptide sequence or a fragment thereof.

[0246] Embodiment 35 The modified polypeptide of embodiment 34, wherein the CATHC peptide has a sequence according to SEQ ID NO:95.

[0247] Embodiment 36 The modified polypeptide of any one of the preceding embodiments, wherein the modified α-factor preprosequence comprises a CFVII peptide sequence or a fragment thereof.

[0248] Embodiment 37 The modified polypeptide of embodiment 36, wherein the CFVII peptide has a sequence according to SEQ ID NO:96.

[0249] Embodiment 38 The modified polypeptide of any one of the preceding embodiments, wherein the modified alpha-factor preprosequence comprises a sortilin 1 peptide sequence or a fragment thereof.

[0250] Embodiment 39. The modified polypeptide of embodiment 38, wherein the sortilin-1 peptide has a sequence according to SEQ ID NO:97.

[0251] Embodiment 40 The modified polypeptide of any one of the preceding embodiments, wherein the modified alpha-factor preprosequence comprises a BDNF peptide sequence or a fragment thereof.

[0252] Embodiment 41 The modified polypeptide of embodiment 40, wherein the BDNF peptide has a sequence according to SEQ ID NO:98.

[0253] Embodiment 42. The modified polypeptide of any one of embodiments 1 to 18 and 21, wherein the modified α-factor preprosequence has a sequence according to SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:71, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:109.

[0254] Embodiment 43. The modified polypeptide of any one of embodiments 1, 2, and 17-42, wherein the payload polypeptide comprises a sequence according to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:72, or SEQ ID NO:79.

[0255] Embodiment 44. The modified polypeptide of embodiment 1, wherein the modified polypeptide has a sequence according to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, or SEQ ID NO:105.

[0256] Embodiment 45. A modified polypeptide according to any one of the preceding embodiments, characterized in that when assessed in a cell, tissue, or subject, the modified polypeptide is secreted from the cell, cells of the tissue, or cells or tissues of the subject at a level higher than a comparable polypeptide.

[0257] Embodiment 46 The modified polypeptide of embodiment 45, wherein the comparable polypeptide comprises the same payload polypeptide as said modified polypeptide and a signal peptide which is not the wild-type or modified α-factor prepro sequence.

[0258] Embodiment 47. A polynucleotide encoding a modified polypeptide according to any one of embodiments 1 to 46.

[0259] Embodiment 48. The polynucleotide of embodiment 47, wherein the polynucleotide is DNA.

[0260] Embodiment 49. The polynucleotide of embodiment 40, wherein the polynucleotide is RNA.

[0261] Embodiment 50. The polynucleotide of embodiment 49, wherein the RNA is mRNA.

[0262] Embodiment 51. The polynucleotide of embodiment 50, wherein the mRNA comprises a cap and a poly(A) tail.

[0263] Embodiment 52. The polynucleotide of any one of embodiments 49 to 51, wherein the polynucleotide comprises one or more N4-acetylcytidine residues.

[0264] Embodiment 53. The polynucleotide of any one of embodiments 49 to 52, wherein the polynucleotide comprises one or more 5-hydroxymethyluridine residues.

[0265] Embodiment 54. A cell comprising a modified polypeptide according to any one of embodiments 1 to 46 or a polynucleotide according to any one of embodiments 47 to 53.

[0266] Embodiment 55. The cell of embodiment 54, wherein the cell is a mammalian cell.

[0267] Embodiment 56. A composition comprising a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, or a cell according to embodiment 54 or 55.

[0268] Embodiment 57. The composition of embodiment 56, wherein the composition is a pharmaceutical composition.

[0269] Embodiment 58. The composition of embodiment 57, wherein the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0270] Embodiment 59. The composition of embodiment 57 or 58, wherein the pharmaceutical composition is or comprises an immunogenic composition.

[0271] The composition according to any one of embodiments 57 to 59, wherein the pharmaceutical composition is or comprises a vaccine.

[0272] Embodiment 61. The composition of embodiment 57 or 58, wherein the pharmaceutical composition is or comprises a gene therapy.

[0273] Embodiment 62. The composition of embodiment 57 or 58, wherein the pharmaceutical composition is or comprises a chemotherapy.

[0274] Embodiment 63. The composition of embodiment 57 or 58, wherein the pharmaceutical composition is or comprises a protein replacement therapy.

[0275] Embodiment 64. The composition of embodiment 57 or 58, wherein the pharmaceutical composition is or comprises an immunotherapy.

[0276] Embodiment 65. The composition of embodiment 57 or 58, wherein the pharmaceutical composition is or comprises a cell modifying therapy.

[0277] Embodiment 66. A method comprising administering to a cell, tissue, or subject a modified polypeptide described in any one of embodiments 1 to 36, a polynucleotide described in any one of embodiments 47 to 53, a cell described in embodiments 54 or 55, or a composition described in any one of embodiments 56 to 65.

[0278] Embodiment 67. The method of embodiment 66, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0279] Embodiment 68. The method of embodiment 66 or 67, wherein the subject is a human.

[0280] Embodiment 69. A method comprising administering a polynucleotide described in any one of embodiments 47 to 53 to a cell, tissue, or subject.

[0281] Embodiment 70 The method of embodiment 69, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0282] Embodiment 71 The method of embodiment 69 or 70, wherein the subject is a human.

[0283] Embodiment 72. The method of any one of embodiments 69 to 71, comprising determining the level of modified polypeptide secreted from said cell, cells of said tissue, or cells or tissues of said subject.

[0284] Embodiment 73 The method of embodiment 72, further comprising comparing the level of the modified polypeptide secreted from said cell, cells of said tissue, or cells or tissues of said subject with a reference level.

[0285] Embodiment 74. The method of embodiment 73, wherein the reference level is the level of a reference polypeptide secreted from a comparable cell, tissue, or subject, wherein the reference polypeptide comprises the same payload polypeptide as the modified polypeptide and a signal peptide that is not a wild-type or modified α-factor prepro sequence.

[0286] Embodiment 75. The method of any one of embodiments 66 to 74, wherein the method is a method of stimulating an immune response.

[0287] Embodiment 76. The method according to any one of embodiments 66 to 74, wherein the method is a vaccination method.

[0288] Embodiment 77. The method of any one of embodiments 66 to 74, wherein the method is a gene therapy.

[0289] Embodiment 78. The method according to any one of embodiments 66 to 74, wherein the method is a cell therapy modification method.

[0290] Embodiment 79. The method of any one of embodiments 66 to 74, wherein the method is an immunotherapy.

[0291] Embodiment 80. The method of embodiment 79, wherein the immunotherapy comprises delivery of an antibody therapy and / or an immune checkpoint therapy.

[0292] Embodiment 81 The method of any one of embodiments 66 to 74, wherein the method is a protein replacement therapy.

[0293] Embodiment 82 The method of embodiment 81, wherein said protein replacement therapy comprises delivery of enzyme replacement therapy.

[0294] Embodiment 83. The method of any one of embodiments 66 to 74, wherein the method is chemotherapy.

[0295] Embodiment 84. A method for producing a modified polypeptide, comprising expressing from a cell the polynucleotide described in any one of embodiments 47 to 53.

[0296] Embodiment 85 The method of embodiment 84, wherein the cell is a mammalian cell.

[0297] Embodiment 86. Use of a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 for stimulating an immune response.

[0298] Embodiment 87. Use of a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 as a vaccine.

[0299] Embodiment 88. Use of a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 as immunotherapy.

[0300] Embodiment 89. Use of a polynucleotide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 as a gene therapy.

[0301] Embodiment 90. Use of a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 as a protein replacement therapy.

[0302] Embodiment 91. Use of a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 as a cell modification therapy.

[0303] Embodiment 92. Use of a modified polypeptide according to any one of embodiments 1 to 36, a polynucleotide according to any one of embodiments 47 to 53, a cell according to embodiments 54 or 55, or a composition according to any one of embodiments 56 to 65 as chemotherapy.

[0304] Embodiment 93. The use according to any one of embodiments 86 to 92, wherein the polypeptide, polynucleotide, cell or composition is administered to a cell, tissue or subject.

[0305] Embodiment 94. The use of embodiment 93, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

[0306] Embodiment 95. The use according to embodiment 94, wherein the mammal is a human.

[0307] Embodiment 96. A method of making a polynucleotide comprising adding a nucleotide sequence encoding a wild-type or modified α-factor prepro sequence to a nucleotide sequence encoding a payload polypeptide.

[0308] Embodiment 97. The modified polypeptide of any one of embodiments 1 to 41, wherein the modified polypeptide further comprises an Arabidopsis root growth factor (GLV) secretory peptide sequence or a fragment thereof.

[0309] Embodiment 98. The modified polypeptide of embodiment 97, wherein the Arabidopsis root growth factor (GLV) sequence comprises SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, or SEQ ID NO:99.

[0310] Embodiment 99. A modified polypeptide comprising: (i) a payload polypeptide, (ii) a wild-type or modified Arabidopsis root growth factor (GLV) secretory peptide sequence or a fragment thereof, The modified polypeptide, wherein the wild-type or modified Arabidopsis root growth factor (GLV) secretory peptide sequence, or a fragment thereof, is operably linked to a payload polypeptide.

[0311] Embodiment 100. The modified polypeptide of embodiment 99, wherein the modified polypeptide further comprises an α-factor prepro sequence or a fragment thereof.

[0312] Embodiment 101. The modified polypeptide of any one of embodiments 34 to 41, wherein the CATHC, CFVII, sortilin-1, or BDNF peptide is inserted into the modified alpha-factor prepro sequence at the propeptide N-terminus after a 2xSurf4 motif with a flexible glycine-serine linker.

[0313] Embodiment 102. The modified polypeptide of any one of embodiments 34 to 41, wherein the CATHC, CFVII, sortilin-1, or BDNF peptide is inserted into the modified alpha-factor prepro sequence in the middle of the propeptide sequence.

[0314] Embodiment 103. The modified polypeptide of any one of embodiments 34-41, wherein the CATHC, CFVII, sortilin-1, or BDNF peptide is inserted into the modified alpha-factor preprosequence at the C-terminus, immediately prior to the 3x S1P cleavage site.

[0315] Exemplary Sequences

[0316] Exemplary SARS-CoV-2 spike secreted peptide amino acid sequence (SEQ ID NO:1): MFVFLVLLPLVSSAA An exemplary prepro amino acid sequence (SEQ ID NO:2): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAA

[0317] An exemplary pre-amino acid sequence (SEQ ID NO:68): MRFPSIFTAVLFAASSALA

[0318] Exemplary proamino acid sequence (SEQ ID NO:69): APVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAA

[0319] Exemplary prepro_2 amino acid sequence = a prepro sequence with 2x Surf4 motif replacements with a flexible glycine-serine linker, 3x S1P cleavage sites, and lacking a Ste13 cleavage site ("2x_Surf4_SG_3x_S1P_delSTE13") (SEQ ID NO: 70) MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAA

[0320] Exemplary Pro_2 amino acid sequence (SEQ ID NO:71): MPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAA

[0321] Exemplary SARS-CoV-2 Wild-Type Receptor Binding Domain (WT-RBD) Amino Acid Sequence (SEQ ID NO:3): MFVFLVLLPLVSSAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADY NYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0322] Exemplary amino acid sequence of WT-RBD with prepro sequence (SEQ ID NO:4): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0323] Exemplary variant_1 amino acid sequence (SEQ ID NO:5): MFVFLVLLPLVSSAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASAAAAACYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADY NYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0324] Exemplary amino acid sequence of variant_1 including the prepro sequence (SEQ ID NO:6): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASAAAAACYGVS PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0325] Exemplary variant_2 amino acid sequence (SEQ ID NO:7):

[0326] MFVFLVLLPLVSSAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADY NYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAAAAACGP

[0327] Exemplary amino acid sequence of variant_2 with prepro sequence (SEQ ID NO:8): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAAAAACGP

[0328] An exemplary influenza H1N1 / PR8 hemagglutinin extracellular domain (HA) amino acid sequence (SEQ ID NO:9): MKANLLVLLCALAAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFE IFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNL IAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVN TVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQ

[0329] Exemplary amino acid sequence of HA with the prepro sequence (SEQ ID NO:10): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAG WLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHPPNSKEQQNLYQNENAYVSVVTSNYNRRFT PEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGY AADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQ

[0330] An exemplary human secreted fetal alkaline phosphatase (SEAP) amino acid sequence (SEQ ID NO:11): MLGPCMLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVK GNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGK NLVQEWLAKRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTAD HSHVFSFGYPLRGSSIFFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD

[0331] An exemplary amino acid sequence of SEAP including the prepro sequence (SEQ ID NO:12): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAA RILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDI DVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIER AGQLTSEEDTLSLVTADHSHVFSFGYPLRGSSIFGLAPKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTTD

[0332] Exemplary Trastuzumab scFv amino acid sequence (SEQ ID NO: 13): MFVFLVLLPLVSSAAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0333] Exemplary amino acid sequence of Trastuzumab scFv with prepro sequence (SEQ ID NO: 14): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR

[0334] Exemplary Pertuzumab scFv amino acid sequence (SEQ ID NO: 15): MFVFLVLLPLVSSAAEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGT LVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKR

[0335] Exemplary amino acid sequence of Pertuzumab scFv with prepro sequence (SEQ ID NO: 16): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRS KNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKR

[0336] Exemplary GB235 scFv amino acid sequence (SEQ ID NO:17): MFVFLVLLPLVSSAAEVQLVQSGAEVKKPGAPVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCARGYYLSRGDFWGQGTL VTVSSGGGGSGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKR

[0337] Exemplary amino acid sequence of GB235 scFv with prepro sequence (SEQ ID NO:18): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVQSGAEVKKPGAPVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTS ISTAYMELSSLRSEDTAVYYCARGYYLSRGDFWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKR

[0338] Exemplary Adalimumab scFv amino acid sequence (SEQ ID NO:19): MEFGLSWVFLVALFRGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG QGTLVTVSSGGGGSGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR

[0339] Exemplary amino acid sequence of Adalimumab scFv with prepro sequence (SEQ ID NO:20): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAK NSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR

[0340] Exemplary pembrolizumab scFv amino acid sequence (SEQ ID NO:21): MDWTWRFLFVVAAATGVQSQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIKR

[0341] Exemplary amino acid sequence of pembrolizumab scFv with prepro sequence (SEQ ID NO:22): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTT AYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIKR

[0342] Exemplary Aflibercept Fragment Amino Acid Sequence (SEQ ID NO:23): MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYK TNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK

[0343] Exemplary amino acid sequence of an aflibercept fragment including the prepro sequence (SEQ ID NO:24): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAASDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRII WDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK

[0344] Exemplary dupilumab scFv amino acid sequence (SEQ ID NO:25): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIKR

[0345] Exemplary amino acid sequence of dupilumab scFv with prepro sequence (SEQ ID NO:26): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTT AYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIKR

[0346] Exemplary ustekinumab scFv amino acid sequence (SEQ ID NO:27): MELGLSWIFLLAILKGVQCEVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSPVDSDIRYSPSFQGQVTMSVDKSITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQ GTLVTVSSGGGGSGGGGSGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKLEIKR

[0347] Exemplary amino acid sequence of ustekinumab scFv with prepro sequence (SEQ ID NO:28): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWLGWVRQMPGKGLDWIGIMSPVDSDIRYSPSFQGQVTMSVDKS ITTAYLQWNSLKASDTAMYYCARRRPGQGYFDFWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPYTFGQGTKLEIKR

[0348] Exemplary Nivolumab scFv amino acid sequence (SEQ ID NO:29): MKHLWFFLLLVAAPRWVLSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTL VTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR

[0349] Exemplary amino acid sequence of nivolumab scFv with prepro sequence (SEQ ID NO:30): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISR DNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKR

[0350] Exemplary bevacizumab scFv amino acid sequence (SEQ ID NO:31): MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSGGGGSGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR

[0351] Exemplary amino acid sequence of bevacizumab scFv with prepro sequence (SEQ ID NO:32): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKS TAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR

[0352] Exemplary Etanercept Fragment Amino Acid Sequence (SEQ ID NO:33): MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL SKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD

[0353] Exemplary amino acid sequence of an etanercept fragment with a prepro sequence (SEQ ID NO:34): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECL SCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD

[0354] An exemplary amino acid sequence of tisagenlecleucel extracellular and transmembrane domains (SEQ ID NO:35): MALPVTALLLPLALLLHAARPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSS LSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0355] An exemplary amino acid sequence of the tisagenlecleucel extracellular domain and transmembrane domain with prepro sequence (SEQ ID NO:36): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSSGGGGSGGGGSGGGSDIQMTQTTSSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0356] Exemplary tisagenlecleucel amino acid sequence (SEQ ID NO:72): MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0357] An exemplary amino acid sequence of tisagenlecleucel with a prepro sequence (SEQ ID NO:73): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSR LTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0358] An exemplary amino acid sequence of tisagenlecleucel having the prepro_2 sequence (SEQ ID NO:74): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAAEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISK YLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTY YNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0359] An exemplary amino acid sequence of tisagenlecleucel with an IgK light chain signal peptide sequence and a pro_2 sequence (SEQ ID NO:75): MDMRVPAQLLGLLLLWLSGARCMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAAEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQD ISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0360] An exemplary amino acid sequence of tisagenlecleucel with a CD8 signal peptide sequence and a pro_2 sequence (SEQ ID NO:76): MALPVTALLLPLALLLHAARPMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAAEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDI SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0361] An exemplary amino acid sequence of tisagenlecleucel with an IL2 signal peptide sequence and a pro_2 sequence (SEQ ID NO:77): MYRMQLLSCIALSLALVTNSMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAAEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDIS KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0362] An exemplary amino acid sequence of tisagenlecleucel with the SARS-CoV-2 spike signal peptide sequence and the pro_2 sequence (SEQ ID NO:78): MFVFLVLLPLVSSMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAAEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0363] Exemplary amino acid sequence of A3B1 CAR (SEQ ID NO:79): MALPVTALLLPLALLLHAARPEQKLISEEDLTGNIVLTQSPASLAVSLGQRATISCRASESVDNFGNSFMHWYQQKSGQPPRLLIYIASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNN EDPLTFGAGTKLELKGGGGGSGGGGSGGGGSHSQIQLQQSGAELVRPGSSVKISCKASGFAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTKYNVKFRGKATLTADESSSTAYIQLTSLTSEDSGVYFCAR KRITAVITTVFDVWGAGTTVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0364] Exemplary amino acid sequence of A3B1 CAR with prepro sequence (SEQ ID NO:80): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAAEQKLISEEDLTGNIVLTQSPASLAVSLGQRATISCRASESVDNFGNSFMHWYQQKS GQPPRLLIYIASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDPLTFGAGTKLELKGGGGSGGGGSGGGSHSQIQLQQSGAELVRPGSSVKISCKASGFAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTKYNVK FRGKATLTADESSSTAYIQLTSLTSEDSGVYFCARKRITAVITTVFDVWGAGTTVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0365] Exemplary amino acid sequence of A3B1 CAR with prepro_2 sequence (SEQ ID NO:81): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAAEQKLISEEDLTGNIVLTQSPASLAVSLGQRATISCRASESVDN FGNSFMHWYQQKSGQPPRLLIYIASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCHQNNEDPLTFGAGTKLELKGGGGSGGGGSGGGGSHSQIQLQQSGAELVRPGSSVKISCKASGFAFSSYWMNWVKQRPGQGLEWIGQIYPGD GDTKYNVKFRGKATLTADESSSTAYIQLTSLTSEDSGVYFCARKRITAVITTVFDVWGAGTTVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0366] An exemplary amino acid sequence of an Axicabtageneciloleucel extracellular and transmembrane domain (SEQ ID NO:37): MYRMQLLSCIALSLALVTNSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGSEVKLQESGPG LVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0367] Exemplary amino acid sequence of axicabutagensiloleucel extracellular and transmembrane domain with prepro sequence (SEQ ID NO:38): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYT FGGGTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0368] An exemplary amino acid sequence of Lysocabtagenemaraleucel extracellular and transmembrane domains (SEQ ID NO:39): MDMRVPAQLLGLLLLWLRGARCDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQES GPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPSPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0369] Exemplary amino acid sequence of Lysocabtagenemaraleucel extracellular and transmembrane domain with prepro sequence (SEQ ID NO:40): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP YTFGGGTKLEITGGGSGGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPSPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0370] Exemplary amino acid sequence of variant_1 nanoluciferase fusion with adipokinetic hormone signal peptide sequence (SEQ ID NO:41): MYKLTVFLMFIAFVIIAEAAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASAAAAACYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY RVVVLSFELLHAPATVCGPSGGGSGGSGSVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0371] Exemplary amino acid sequence of variant_1 nanoluciferase fusion with Arabidopsis root growth factor GLV1 signal peptide and propeptide sequence (SEQ ID NO:42): MSCSLRSGLVIVFCFILLLLSSNVGCASAARRLRSHKHHHHKVASLDVFNGGERRRALGGVETGEEVVVMAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYN SASAAAACYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNG VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPSGGGSGGSGSVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDI HVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0372] Exemplary amino acid sequence of variant_1 nanoluciferase fusion with prepro sequence (SEQ ID NO:43): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAANITNLCPFGEVFNATRFASVYAWNRK RISNCVADYSVLYNSASAAAAACYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIY QAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPSGGGSGGSGSVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGL KIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0373] Exemplary amino acid sequence of Arabidopsis root growth factor GLV1 secreted peptide sequence (SEQ ID NO:99): MSCSLRSGLVIVFCFILLLLSSNVGCASAARRLRSHKHHHHKVASLDVFNGGERRRALGGVETGEEVVVM

[0374] Exemplary amino acid sequence of Arabidopsis root growth factor GLV2 secreted peptide sequence (SEQ ID NO:82): MAIRVSHKSFLVALLLILFISSPTQARSLREVVRNRTLLVVEKSQESRKIRHEGGGSDVDGLMDM

[0375] Exemplary amino acid sequence of Arabidopsis root growth factor GLV3 secreted peptide sequence (SEQ ID NO:83): MMRFTIIVIAFLLIIQSLEEEHILVYAHEGGEAGHKSLDYQGDQDSSTLHPKELFDAPRKVRFGRTTRAEKEQVTAMNNDSWSFKISGEHKQTNILADHDTTKNTFCKKMMIIVNDLTSLPTLEPSTSSTNDMEKLARLLRD

[0376] Exemplary amino acid sequence of Arabidopsis root growth factor GLV4 secreted peptide sequence (SEQ ID NO:84): MEMKKWSYANLITLALLFLFFIILLLAFQGGSRDDDHQHVHVAIRTKDISMGRKLKSLKPINPTKKNGFEYPDQGSHDVQEREVYVELR

[0377] Exemplary amino acid sequence of Arabidopsis root growth factor GLV5 secreted peptide sequence (SEQ ID NO:85): MTNITSSFLCLLILLLFCLSFGYSLHGDKDEVLSVDVGSNAKVMKHLDGDDAMKKAQVRGRSGQEFSKETTKMMMMKKTTKKETNVEEEDDLVAYTA

[0378] Exemplary amino acid sequence of Arabidopsis root growth factor GLV6 secreted peptide sequence (SEQ ID NO:86): MKLIRVTLFLCALAILLLVTPTSSLQLKHPYSSPSQGLSKKIVTKMATRKLMIISSEYVMTSTSSHEGSSEQLRVTSSGKSKDEEKKLSEEEEEKKALAKYLSM

[0379] Exemplary amino acid sequence of Arabidopsis root growth factor GLV7 secreted peptide sequence (SEQ ID NO:87): MTTLSKILCVLIILLLCFSFRYSLHEDGNQQSSRDFVSTAKAIKYGDVMKKMIRGRKLMMASGEKEEAETKMKRGNRETERNSSKSVEEDGLVAYTA

[0380] Exemplary amino acid sequence of Arabidopsis root growth factor GLV8 secreted peptide sequence (SEQ ID NO:88): MKKWSYAKLMTSALLLVFLSIILLAFHGGSRGDNHLYDHVAIGTKDILMGRKLKDLKPKTESLKMINPKKKNGFEYSDQVSSDLSRQEVFVDMMAR

[0381] Exemplary amino acid sequence of Arabidopsis root growth factor GLV9 secreted peptide sequence (SEQ ID NO:89): MKKTSLKLMTLVLGFCFVIYLLQGPRGGSRNGDLLIARKLISLEPIETKNAARSLKDSISTDLEEEVDRLMEH

[0382] Exemplary amino acid sequence of Arabidopsis root growth factor GLV10 secreted peptide sequence (SEQ ID NO:90): MSSIHVASMILLLFLFLHHSDSRHLDNVHITASRFSLVKDQNVVSSSTSKEPVKVSRFVPGPLKHHHRRPLLFA

[0383] Exemplary amino acid sequence of Arabidopsis root growth factor GLV11 secreted peptide sequence (SEQ ID NO:91): MVSIRVICYLLVFSVLQVHAKVSNANFNSQAPQMKNSEGLGASNGTQIAKKHAEDVIENRKTLKHVNVKVEANEKNGLEIESKEMVKKRKNKKRLTKTESLTA

[0384] Exemplary amino acid sequence of WT-RBD with prepro_2 sequence (SEQ ID NO:100): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRLRRLLAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASF STFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP

[0385] Exemplary amino acid sequence of the SARS-CoV-2 Wuhan strain receptor binding domain (RBD) fused to Staphylococcus aureus binder of IgG protein subunits III and IV (SbiIII-IV) (SEQ ID NO:101): MFVFLVLLPLVSSAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPGGGGSGGGGGSGGGGSIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD

[0386] Exemplary amino acid sequence of the SARS-CoV-2 Wuhan strain receptor binding domain (RBD) fused to Staphylococcus aureus binder of IgG protein subunits III and IV with prepro sequence (SbiIII-IV) (SEQ ID NO:102): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNL DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPGGGGSGGGSGGGGSIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD

[0387] Exemplary amino acid sequence of the SARS-CoV-2 Wuhan strain receptor binding domain (RBD) fused to Staphylococcus aureus binder of IgG protein subunits III and IV with prepro_2 sequence (SbiIII-IV) (SEQ ID NO:103): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLRLLAANITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPGGGGSGGGS GGGGSIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD

[0388] Exemplary amino acid sequence of the SARS-CoV-2 Omicron (BA.1) strain receptor binding domain (RBD) fused to Staphylococcus aureus binding agent of IgG protein subunits III and IV (SbiIII-IV) (SEQ ID NO:104): MFVFLVLLPLVSSAANITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNK PCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPGGGGSGGGGGSGGGGSIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD Exemplary amino acid sequence of the SARS-CoV-2 Omicron (BA.1) strain receptor binding domain (RBD) fused to Staphylococcus aureus binder of IgG protein subunits III and IV with prepro sequence (SbiIII-IV) (SEQ ID NO:105): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEANITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKL DSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPGGGGSGGGGSIENADKAIKDFQDNKAPHDKSAAYEANSKLPKDLRDKNNRFVEKVSIEKAIVRHDERVKSANDAISKLNEKDSIENRRLAQREVNKAPMDVKEHLQKQLD

[0389] Modified prepro sequence

[0390] An exemplary prepro sequence with a furin cleavage site ("furin") (SEQ ID NO:44): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDRKRKRKRREAEAAA

[0391] An exemplary prepro sequence with a Furin cleavage site and lacking a Ste13 cleavage site ("Furin_delSTE13") (SEQ ID NO:45): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDRKRKRKRRAA

[0392] An exemplary prepro sequence having a PCSK4 cleavage site ("PCSK4") (SEQ ID NO:46): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKRKREAEAAA

[0393] An exemplary prepro sequence having a PCSK4 cleavage site and lacking a Ste13 cleavage site ("PCSK4_delSTE13") (SEQ ID NO:47): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKRKRAA

[0394] An exemplary prepro sequence with an S1P cleavage site and lacking a Ste13 cleavage site ("S1P_delSTE13") (SEQ ID NO:48): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLAA

[0395] An exemplary prepro sequence having a PCSK9 cleavage site and lacking the Ste13 cleavage site ("PCSK9_delSTE13") (SEQ ID NO:49): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDVFAQSIPAA

[0396] An exemplary prepro sequence lacking the Ste13 cleavage site ("delSTE13") (SEQ ID NO:50): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKRAA

[0397] Exemplary prepro sequences with Surf4 motif substitutions ("Surf4_motif") (SEQ ID NO:51): MRFPSIFTAVLFAASSALAMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREAEAAA

[0398] An exemplary prepro_2 sequence having a cathepsin C peptide at the N-terminus of the propeptide ("CATHC_1") (SEQ ID NO:92): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLTPANCTYLDLLGTWVFQVGSSGSQRDVNCSVMGNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLL

[0399] An exemplary prepro_2 sequence with a cathepsin C peptide in the middle of the propeptide ("CATHC_2") (SEQ ID NO:93): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVATPANCTYLDLLGTWVFQVGSSGSQRDVNCSVMGVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLL

[0400] An exemplary prepro_2 sequence having a cathepsin C-peptide at the C-terminus of the propeptide ("CATHC_3") (SEQ ID NO:94): MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSTPANCTYLDLLGTWVFQVGSSGSQRDVNCSVMGRRLLRRLLRRLL

[0401] Exemplary amino acid sequence of prepro_2 containing 3x MYC tag (SEQ ID NO:106):

[0402] MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLEQKLISEEDLGGGEQKLISEEDLGGGEQKLISEEDLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSRRLLRRLLRRLLAA

[0403] Exemplary amino acid sequence of prepro_2 containing a 3x MYC tag, lacking the 3x S1P protease cleavage site (SEQ ID NO:107):

[0404] MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLEQKLISEEDLGGGEQKLISEEDLGGGEQKLISEEDLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSAA

[0405] Exemplary amino acid sequence of prepro_2 lacking the 3x S1P protease cleavage site (SEQ ID NO:108):

[0406] MRFPSIFTAVLFAASSALAMPLSGGGSGGSGSMPLNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSAA

[0407] An exemplary amino acid sequence of prepro lacking a Ste13 cleavage site and lacking a Kex2 cleavage site (SEQ ID NO:109):

[0408] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDAA

[0409] Specific optimization related sequences

[0410] Furin cleavage site (SEQ ID NO:52): RKRKRKRR

[0411] PCSK4 cleavage site (SEQ ID NO:53): KRKR

[0412] PCSK9 cleavage site (SEQ ID NO:54): VFAQSIP

[0413] S1P cleavage site (SEQ ID NO:55): R.R.L.L.

[0414] Ste13 cleavage site (SEQ ID NO:56): EEA

[0415] Kex2 cleavage site (SEQ ID NO:57): KR

[0416] Surf4 tripeptide motif (SEQ ID NO:58): MPL

[0417] Cathepsin C peptide (SEQ ID NO:95) TPANCTYLDLLGTWVFQVGSGSGSQRDVNCSVMG

[0418] Coagulation factor VII peptide (SEQ ID NO: 96) ILEKRNASKPQGR

[0419] Sortilin 1 peptide (SEQ ID NO:97) WSGPIGVSWGLR

[0420] Brain-derived neurotrophic factor peptide (SEQ ID NO: 98) ESVNGPKAGSRGLTSLADTFEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPL

[0421] Primer sequences

[0422] NLuc_Nterm_cPCR_F (SEQ ID NO:59): CGAGACAGAGAAGACTCTTGCGTTTCTGATAGG

[0423] NLuc_Nterm_cPCR_R (SEQ ID NO: 60): CACAATTGCTGATTCGTTTTCGATTCCACGCATATACG

[0424] NLuc_seq (SEQ ID NO: 61): GGTCTTACTGACATCCACTTTG

[0425] NLuc_RBD_F (SEQ ID NO: 62): TAATACGACTCACTATAGGGGCCGCCACCATGAGATTTCC

[0426] NLuc_RBD_R (SEQ ID NO: 63): GATATCCGCGGTAGGAATTCAGGACCACAAACTGTCG

[0427] NLuc_colony_forward (SEQ ID NO: 64): CGAGACAGAGAAGACTCTTGCGTTTCTGATAGG

[0428] NLuc_colony_reverse (SEQ ID NO: 65): GAGAGCTCGCCTGCAGGAATTGG

[0429] T7-AGG_fwd (SEQ ID NO: 110): gaattTAATACGACTCACTATAAGGcttgttctttttgcagaagc

[0430] 120pA_rev (SEQ ID NO: 111): TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTT

[0431] References Each of the references included below is incorporated herein by reference in its entirety.

[0432] Amant, F. et a., “A serological assay to detect SARS-CoV-2 seroconversion in humans,” Nature Medicine, 26:1033-1036, 2020.

[0433] Buntz, B., “50 of 2020's best-selling pharmaceuticals,” Drug Discovery & Development, https: / / www.drugdiscoverytrends.com / 50-of-2020s-best-selling-pharmaceuticals / , 2021.

[0434] Castella, M., et al., “Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions,” Molecular Therapy Methods & Clinical Development, 12:134-144, 2019.

[0435] Chen, Z. et al., “Sortilin Controls Intracellular Sorting of Brain-Derived Neurotrophic Factor to the Regulated Secretory Pathway,” The Journal of Neuroscience, 25(26):6156-6166, 2005.

[0436] Feizi,A.,et al.,“Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome,”npj Systems Biology and Applications,3:22,2017.

[0437] Fitzgerald,I.,et al.,“Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting,”Microbial Cell Factories,13(125),2014.

[0438] Garten,W.,Characterization of Proprotein Convertases and Their Involvement in Virus Propagation,”Activation of Viruses by Host Proteases,Springer International Publishing:205-248,2018.

[0439] Haryadi,R.,et al.,“Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells,”PLoS ONE,10(2):e0116878,2015.

[0440] Naider,F.,et al.,“The alpha-factor mating pheromone of Saccharomyces cerevisiae:a model for studying the interaction of peptide hormones and G protein-coupled receptors,”Peptides,5(9):1441-63,2004.

[0441] Nichols,W.et al.,“Mutations in the ER-Golgi Intermediate Compartment Protein ERGIC-53 Cause Combined Deficiency of Coagulation Factors V and VIII,”Cell,93(1):61-70,1998.

[0442] Oka,C.,et al.,“Human Lysozyme Secretion Increased by Alpha-factor Pro-sequence in Pichia pastoris,”Bioscience,Biotechnology,and Biochemistry,63(11):1977-1983,1999.

[0443] Otte,S.,et al.,“Sorting signals can direct receptor-mediated export of soluble proteins into COPII vesicles,”Nature Cell Biology,6,1189-1194,2004.

[0444] Petersen,C.et al.,“Propeptide cleavage conditions sortilin / neurotensin receptor-3 for ligand binding,”The EMBO Journal,18:595-604,1999.

[0445] Rafiq, S., et al., “Engineering strategies to overcome the current roadblocks in CAR T cell therapy,” Nature Reviews Clinical Oncology, 17:147 - 167, 2020.

[0446] Shu, M., et al., “A novel anti - HER2 antibody GB235 reverses Trastuzumab resistance in HER2 - expressing tumor cells in vitro and in vivo,” Scientific Reports, 10:2986, 2020.

[0447] Yin, Y., et al., “Surf4(Erv29p) binds amino - terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum,” PLoS Bio, 16(8):e2005140, 2018.

[0448] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. It should be understood that the invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims that are introduced into another claim that is dependent on the same base claim (or any other related claim), unless otherwise specified or unless a contradiction or inconsistency would be obvious to one of ordinary skill in the art. It should also be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, regardless of whether a specific exclusion is described in the specification. The scope of the invention is not limited to the above description, but is instead set forth in the following claims.

Claims

1. A modified polypeptide comprising: (i) a payload polypeptide, and (ii) the modified polypeptide comprising a wild-type or modified α-factor prepro sequence or a fragment thereof, wherein the wild-type or modified α-factor prepro sequence or a fragment thereof is operably linked to the payload polypeptide.

2. The modified polypeptide of claim 1 , wherein the payload polypeptide is a therapeutic polypeptide.

3. The modified polypeptide of claim 1 , wherein the payload polypeptide comprises a therapeutic polypeptide and / or one or more antigens.

4. The modified polypeptide of claim 3 , wherein the one or more antigens include one or more viral antigens, one or more bacterial antigens, one or more cancer antigens, and / or one or more archaeal antigens.

5. 5. The modified polypeptide of claim 4, wherein the one or more viral antigens comprise one or more SARS-CoV-2 antigens and / or one or more influenza antigens.

6. The modified polypeptide of claim 1 , wherein the payload polypeptide comprises an antibody or a fragment thereof.

7. 2. The modified polypeptide of claim 1, wherein the payload polypeptide comprises a fusion protein comprising (i) an extracellular receptor, a receptor agonist, or a receptor antagonist, and (ii) an immunoglobulin constant region.

8. The modified polypeptide of claim 1 , wherein the wild-type or modified α-factor prepro sequence or a fragment thereof is present at the N-terminus of the modified polypeptide.

9. The modified polypeptide of claim 1 , wherein the wild-type or modified α-factor prepro sequence or a fragment thereof is present at the C-terminus of the modified polypeptide.

10. The modified polypeptide of claim 1, wherein the modified polypeptide comprises a modified α-factor prepro sequence or a fragment thereof.

11. The modified polypeptide of claim 10, wherein the modified α-factor prepro sequence comprises a tripeptide motif that has Surf4 binding affinity.

12. The modified polypeptide of claim 11, wherein the tripeptide motif has the amino acid sequence MPL (SEQ ID NO: 58).

13. The modified polypeptide of claim 1 , wherein the modified α-factor prepro sequence does not contain a Ste13 cleavage site.

14. A modified polypeptide described in claim 13, wherein the Ste13 cleavage site comprises the amino acid sequence EAEA (sequence number 56).

15. The modified polypeptide of claim 1 , wherein the modified α-factor prepro sequence does not contain a Kex2 cleavage site.

16. The modified polypeptide of claim 15, wherein the Kex2 cleavage site comprises the amino acid sequence KR.

17. 16. The modified polypeptide of claim 15, wherein the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence by a protease cleavage site for a human protease.

18. 18. The modified polypeptide of claim 17, wherein the Kex2 cleavage site found in the wild-type α-factor prepro sequence is replaced in the modified α-factor prepro sequence by a protease cleavage site for S1P, PCSK4, PCSK9, or Furin.

19. (a) the protease cleavage site of S1P has a sequence according to SEQ ID NO: 55; (b) the protease cleavage site of PCSK4 has a sequence according to SEQ ID NO: 53; (c) the protease cleavage site of PCSK9 has a sequence according to SEQ ID NO: 54; or (d) the protease cleavage site of Furin has the sequence according to SEQ ID NO:

52.

20. the modified α-factor prepro sequence is (a) comprises a CATHC peptide sequence or a fragment thereof, optionally wherein the CATHC peptide has a sequence according to SEQ ID NO: 95; (b) comprising a CFVII peptide sequence or a fragment thereof, optionally wherein the CFVII peptide has a sequence according to SEQ ID NO: 96; (c) comprising a sortilin 1 peptide sequence or a fragment thereof, optionally wherein said sortilin-1 peptide has a sequence according to SEQ ID NO: 97; and / or (d) comprising a BDNF peptide sequence or a fragment thereof, optionally wherein the BDNF peptide has a sequence according to SEQ ID NO: 98; The modified polypeptide of claim 1.

21. The modified polypeptide of claim 1, wherein the modified polypeptide further comprises an Arabidopsis root growth factor (GLV) secretory peptide sequence or a fragment thereof.

22. 2. The modified polypeptide of claim 1, wherein, when evaluated in a cell, tissue, or subject, the modified polypeptide is secreted from the cell, cells of the tissue, or cells or tissues of the subject at a higher level than a comparable polypeptide, and optionally, the comparable polypeptide comprises the same payload polypeptide as the modified polypeptide and a signal peptide that is not a wild-type or modified alpha-factor prepro sequence.

23. A polynucleotide encoding the modified polypeptide of claim 1.

24. 24. The polynucleotide of claim 23, wherein the polynucleotide is DNA or RNA.

25. 25. The polynucleotide of claim 24, wherein the polynucleotide comprises (i) one or more N4-acetylcytidine nucleosides, and / or (ii) one or more 5-hydroxymethyluridine nucleosides.

26. A cell comprising a modified polypeptide according to any one of claims 1 to 22 or a polynucleotide according to any one of claims 23 to 25.

27. A composition comprising a modified polypeptide according to any one of claims 1 to 22 or a polynucleotide according to any one of claims 23 to 25.

28. 28. The composition of claim 27, wherein the composition is a pharmaceutical composition.

29. The pharmaceutical composition comprises: (a) an immunogenic composition; (b) a vaccine; (c) gene therapy; (d) chemotherapy; (e) immunotherapy, (f) cell-modifying therapy; or (g) any combination thereof 29. The composition of claim 28, which is or comprises:

30. 26. A composition comprising a modified polypeptide of any one of claims 1 to 22 or a polynucleotide of any one of claims 23 to 25, for use in delivering said composition to a cell, tissue, or subject.

31. 31. The composition of claim 30, wherein the cell is a mammalian cell, the tissue is a mammalian tissue, or the subject is a mammal.

32. The use, (a) stimulating an immune response; (b) vaccination; (c) gene therapy; (d) cell therapy modifications; (e) immunotherapy, (f) protein replacement therapy; (g) chemical treatment; (h) antibody treatment, (i) immunomodulatory, or (j) any combination thereof The composition of claim 30 for:

33. A method for producing a modified polypeptide, comprising expressing the polynucleotide of any one of claims 23 to 25 from a cell.

34. 34. The method of claim 33, wherein the cell is a mammalian cell.

35. 31. The composition of claim 30, wherein the composition is administered to a cell, tissue, or subject.

36. A method for making a polynucleotide comprising adding a nucleotide sequence encoding a wild-type or modified α-factor prepro sequence to a nucleotide sequence encoding a payload polypeptide.

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