Targeted viral transduction of b cells

Abstract

Aspects of the present disclosure are directed to methods for transducing a B cell by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope. Certain aspects relate to preventing, treating, and / or ameliorating and / or inducing an immune response against a disease, infection, or condition, by contacting a B cell with a nucleic acid encoding a pseudotyped retroviral envelope; methods for directing retroviral tropism to a B cell population and / or inducing B cell proliferation and / or differentiation, by contacting a B cell with a nucleic acid encoding a pseudotyped retroviral envelope; and methods of identifying an antibody directed against a targeting moiety by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope.

Description

TARGETED VIRAL TRANSDUCTION OF B CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 500,887, filed May 8, 2023, which is incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under AI095004, AI145044, AI149504, and AI108400 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] The application contains a Sequence Listing in compliance with ST.26 format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on February 27, 2024 is named UCLA.P0186WO Sequence Listing. xml and is 50,002 bytes in size.BACKGROUNDI. Field of the Disclosure

[0004] Aspects of this disclosure relate, generally, to at least the fields of molecular biology and medicine and, more specifically, to methods of targeted retroviral transduction of B cells.II. Background

[0005] Clinically effective gene therapy protocols for various disease conditions can provide for efficient and specific targeting of therapeutic genes to affected cells while maintaining stable transduction and long term expression. This can be accomplished by direct injection into the bloodstream followed by homing of the vector to the desired target cells or organs. Thus, there have been many attempts to develop targeted gene transduction systems based upon various viral vectors.

[0006] Binding of a retrovirus to target cells, the first step of retroviral infection, is mediated by retroviral proteins that can bind directly to receptors expressed on target cells. The affinity and specificity of receptor-binding viral proteins can determine the efficiency of replication in target cells and the tropism of the virus. Previous efforts have focused ontargeting therapeutic genes to cells affected by various disease conditions by inserting ligands, peptides, and / or single chain antibodies, for example, into the retroviral receptor binding envelope or bridging retrovirus and target cells using antibodies or ligands to alter and / or restrict the host range of lentiviral vectors. However, viral entry into immune response cells, e.g., B cells, would create new opportunities for antigen- specific gene therapy by viruses and viral vectors and would allow for generation of neutralizing antibodies for treatment or prevention of disease conditions.

[0007] There exists a need in the art for development of methods to selectively target key immune response cells in an antigen- specific manner through strategic pseudotyping of viral particles.SUMMARY

[0008] Aspects of the present disclosure address needs in the art by providing methods for selectively target key immune response cells, e.g., B cells, in an antigen- specific manner through strategic pseudotyping of lentiviral particles. Accordingly, provided herein, in some aspects, are methods for transducing a B cell by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope. Certain aspects relate to preventing, treating, and / or ameliorating and / or inducing an immune response against a disease, infection, or condition, by contacting a B cell with a nucleic acid encoding a pseudotyped retroviral envelope; methods for directing retroviral tropism to a B cell population and / or inducing B cell proliferation and / or differentiation, by contacting a B cell with a nucleic acid encoding a pseudotyped retroviral envelope; and methods of identifying an antibody directed against a targeting moiety by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope.

[0009] Aspects of the disclosure include methods for transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope; treating or preventing an infection by transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope; treating or preventing cancer by transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope; directing retroviral tropism to a B cell population by contacting B cells of the B cell population with a nucleic acid encoding a pseudotyped retroviral envelope; inducing B cell proliferation by contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope; inducingB cell differentiation by contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope; inducing B cell proliferation and differentiation by contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope; inducing an immune response against an infection, disease, or condition associated with a targeting moiety in a subject in need thereof by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudo typed retroviral envelope; inducing proliferation and / or differentiation of a B cell expressing a receptor to which a targeting moiety is directed by contacting the B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope; preventing, treating, and / or ameliorating an infection, disease, or condition associated with a targeting moiety in a subject in need thereof by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope; vaccinating a subject against an infection, disease, or condition associated with a targeting moiety by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope; generating neutralizing antibodies against an infection, disease, or condition associated with a targeting moiety by contacting a B cell with the targeting moiety and a nucleic acid encoding pseudotyped retroviral envelope; identifying an antibody against a virus by contacting a B cell with a nucleic acid encoding a pseudotyped retroviral envelope, identifying a B cell receptor that recognizes the pseudotyped retroviral envelope, and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody; identifying an antibody directed against a targeting moiety by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope, identifying a B cell receptor that recognizes the targeting moiety, and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody; producing an antibody, or an antigen-binding fragment thereof, by culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody under a condition where the antibody, or antigen-binding fragment thereof, is expressed by the host cell; preventing, treating, and / or ameliorating an infection or cancer by administering to a subject in need thereof a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, disclosed herein or a pharmaceutical composition disclosed herein; and manufacturing a medicament for preventing, treating, and / or ameliorating an infection or cancer by culturing a host cell comprising a vector comprising the nucleic acid sequence encoding herein under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell. Certain aspects of the disclosure may exclude one or more of the preceding elements and / or steps.

[0010] In some aspects, the infection is a viral, bacterial, fungal, or parasitic infection. In some aspects, the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

[0011] In some aspects, the B cell population comprises B cells that express B cell receptors specific for the pseudotyped retroviral envelope and / or a targeting moiety or to which the targeting moiety is directed. In some aspects, the B cell population comprises mature or differentiated B cells. In some aspects, the mature or differentiated B cells comprise plasma cells, germinal center-activated cells, and / or memory B cells. In some aspects, B cell proliferation and B cell differentiation are induced simultaneously. In some aspects, contacting the B cell with the targeting moiety and the nucleic acid encoding a pseudo typed retroviral envelope induces proliferation and / or differentiation of the B cell. In some aspects, the immune response comprises endogenous antibody production by the B cell.

[0012] In certain aspects, the B cell is contacted with the targeting moiety prior to contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. In certain aspects, the B cell is simultaneously contacted with the targeting moiety and the nucleic acid encoding the pseudotyped retroviral envelope. In certain aspects, the B cell is contacted with the targeting moiety after contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. In certain aspects, the B cell is contacted with the targeting moiety prior to and simultaneously with contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. In certain aspects, the B cell is contacted with the targeting moiety simultaneously with and after contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. In certain aspects, the B cell is contacted with the targeting moiety prior to, simultaneously with, and after contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope.

[0013] In certain aspects, cells of the subject are contacted with the targeting moiety and the nucleic acid encoding the pseudotyped retroviral envelope a single time. In certain aspects, B cells of the subject are contacted with the targeting moiety and / or the nucleic acid encoding the pseudotyped retroviral envelope a multiple times. In certain aspects, B cells of the subject are contacted with the nucleic acid encoding the pseudotyped retroviral envelope multiple times. In certain aspects, B cells of the subject are not contacted with the targeting moiety more than a single time.

[0014] In certain aspects, the B cell is transduced in vivo, and the nucleic acid encoding the pseudotyped retroviral envelope is administered intravenously. In certain aspects, the B cellis transduced ex vivo, and the B cell is a primary B cell or a B cell from a B cell line. In certain aspects, the B cell is transduced in vitro, and the B cell is a primary B cell or a B cell from a B cell line.

[0015] In some aspects, the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins. In other aspects, the pseudotyped retroviral envelope comprises lentiviral envelope proteins. In some aspects, the nucleic acid is comprised in a retroviral vector. In certain aspects, the retroviral vector is a gammaretroviral vector or oncoretroviral vector. In certain aspects, the retroviral vector is a lentiviral vector. In some aspects, the nucleic acid is comprised in a retroviral genome. In certain aspects, the retroviral genome is a gammaretroviral genome or oncoretroviral genome. In certain aspects, the retroviral genome is a lentiviral genome. In some aspects, the nucleic acid encoding the pseudotyped retroviral envelope or the retroviral vector or retroviral genome comprising the nucleic acid is comprised in a retrovirus. In certain aspects, the retrovirus is a gammaretrovirus or an oncoretrovirus. In certain aspects, the retrovirus is a lentivirus.

[0016] In some aspects, the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3. In some aspects, the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3. In some aspects, the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope. In some aspects, the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise substitutions or deletions at amino acid residues 226 and / or 227 of El ; substitutions or deletions at amino acid residues 68-71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60-64 of E3. In some aspects, the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted. In some aspects, the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG. In some aspects, the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted. In some aspects, the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA. In some aspects, the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted. In some aspects, the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA. In some aspects, the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted. In some aspects, the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted. In some aspects, the sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA. In some aspects, the mutatedSindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68- 71AAAA in E2, and the mutation KE159-160AA in E2. In some aspects, the mutated Sindbis envelope further comprises the mutation AK226-227SG in El. In some aspects, the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viral genome comprising the nucleic acid.

[0017] In some aspects, the pseudotyped retroviral envelope further comprises one or more flexible linkers. In some aspects, the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain. In some aspects, the detectable label comprises a FLAG peptide. In some aspects, the FLAG peptide comprises the amino acid sequence DYKDDDDK (SEQ ID NO: 12). In some aspects, the protein binding domain comprises an integrin-binding domain. In some aspects, the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21). In some aspects, the protein binding domain comprises an Fc-binding sequence. In some aspects, the Fc-binding sequence comprises a protein A ZZ amino acid sequence. In some aspects, the protein binding domain comprises a biotin-binding sequence. In some aspects, the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence. In some aspects, the one or more flexible linkers comprise two flexible linkers. In some aspects, a first of the one or more flexible linkers is inserted at amino acid 70 of E2. In some aspects, the detectable label and / or the protein binding domain is comprised between the two flexible linkers.

[0018] In some aspects, the pseudotyped retroviral envelope further comprises a targeting moiety. In some aspects, the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety. In some aspects, the nucleic acid is comprised in a viral vector. In other aspects, the nucleic acid is comprised in viral genome. In some aspects, the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety is comprised in a retrovirus. In certain aspects, the retrovirus is a gammaretrovirus or an oncoretrovirus. In certain aspects, the retrovirus is a lentivirus. In some aspects, the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof. In some aspects, the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof. In certain aspects, the pathogenicantigen comprises one or more viral envelope proteins. In some aspects, the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

[0019] In some aspects, the targeting moiety is conjugated to the pseudotyped retroviral envelope. In some aspects, the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope. In certain aspects, the targeting moiety is covalently linked to the pseudotyped retroviral envelope. In certain aspects, the targeting moiety is non- covalently linked to the pseudotyped retroviral envelope. In some aspects, the targeting moiety comprises an antibody. In some aspects, the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof. In some aspects, the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof. In certain aspects, the pathogenic antigen comprises one or more viral envelope proteins. In some aspects, the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

[0020] In some aspects, transduction is mediated by molecular mechanisms independent of interaction of the pseudotyped retroviral envelope with cognate retrovirus receptors. In some aspects, transduction is mediated by molecular mechanisms independent of interaction of the pseudotyped retroviral envelope with PtdSer receptors.

[0021] In some aspects, the nucleic acid encoding the pseudotyped retroviral envelope and, optionally, the targeting moiety, also encodes the heterologous gene. In some aspects, the heterologous gene is operably linked to a promoter. In certain aspects, the promoter is a tissuespecific promoter.

[0022] In some aspects, the targeting moiety and the nucleic acid encoding a pseudotyped retroviral envelope are comprised in a pharmaceutical composition. In some aspects, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients. In some aspects, the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older.

[0023] Disclosed herein are the following aspects 1-235. Aspect 1 is a method of transducing a B cell with a heterologous gene, comprising contacting the B cell with a nucleicacid encoding a pseudotyped retroviral envelope. Aspect 2 is a method of treating or preventing an infection, comprising transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 3 is the method of aspect 2, wherein the infection is a viral, bacterial, fungal, or parasitic infection. Aspect 4 is a method of treating or preventing cancer, comprising transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudo typed retroviral envelope. Aspect 5 is the method of Aspect 4, wherein the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 6 is a method of directing retroviral tropism to a B cell population, comprising contacting B cells of the B cell population with a nucleic acid encoding a pseudotyped retroviral envelope. Aspect7 is the method of Aspect 6, wherein the B cell population comprises B cells that express B cell receptors specific for the pseudotyped retroviral envelope and / or a targeting moiety. Aspect8 is the method of Aspect 6 or 7, wherein the B cell population comprises mature or differentiated B cells. Aspect 9 is the method of Aspect 8, wherein the mature or differentiated B cells comprise plasma cells, germinal center-activated cells, and / or memory B cells. Aspect 10 is a method of inducing B cell proliferation, comprising contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 11 is a method of inducing B cell differentiation, comprising contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 12 is a method of inducing B cell proliferation and differentiation, comprising contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 13 is the method of Aspect 12, wherein B cell proliferation and B cell differentiation are induced simultaneously. Aspect 14 is the method of any one of Aspects 1-13, wherein the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins. Aspect 15 is the method of any one of Aspects 1-13, wherein the pseudotyped retroviral envelope comprises lentiviral envelope proteins. Aspect 16 is the method of any one of Aspects 1-15, wherein the nucleic acid is comprised in a viral vector. Aspect 17 is the method of Aspect 16, wherein the viral vector is a gammaretroviral vector or oncoretroviral vector. Aspect 18 is the method of Aspect 16, wherein the viral vector is a lentiviral vector. Aspect 19 is the method of any one of Aspects 1-15, wherein the nucleic acid is comprised in a viral genome. Aspect 20 is the method of Aspect 19, wherein the viral genome is a gammaretroviral genome or oncoretroviral genome. Aspect 20 is the method of Aspect 19, wherein the viral genome is a lentiviral genome. Aspect 22 is the method of any one of Aspects 1-21, wherein the nucleic acid encoding the pseudotyped retroviral envelope or the viral vectoror viral genome comprising the nucleic acid is comprised in a retrovirus. Aspect 23 is the method of Aspect 22, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus. Aspect 24 is the method of Aspect 22, wherein the retrovirus is a lentivirus. Aspect 25 is the method of any one of Aspects 1-24, wherein the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3, wherein the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3, and wherein the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope. Aspect 26 is the method of Aspect 25, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise: substitutions or deletions at amino acid residues 226 and / or 227 of El; substitutions or deletions at amino acid residues 68-71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60-64 of E3. Aspect 27 is the method of Aspect 25 or 26, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted. Aspect 28 is the method of Aspect 27, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG. Aspect 29 is the method of any one of Aspects 25-28, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted. Aspect 30 is the method of Aspect 29, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA. Aspect 31 is the method of any one of Aspects 25-30, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted. Aspect 32 is the method of Aspect 31, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA. Aspect 33 is the method of any one of Aspects 25-32, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted. Aspect 34 is the method of Aspect 33, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted. Aspect 35 is the method of any one of Aspects 25-32, wherein the sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA. Aspect 36 is the method of any one of Aspects 25- 35, wherein the mutated Sindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68-71AAAA in E2, and the mutation KE159-160AA in E2. Aspect 37 is the method of Aspect 36, wherein the mutated Sindbis envelope further comprises the mutation AK226-227SG in El. Aspect 38 is the method of any one of Aspects 25-37, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viralgenome comprising the nucleic acid. Aspect 39 is the method of any one of Aspects 1-38, wherein the pseudotyped retroviral envelope further comprises one or more flexible linkers. Aspect 40 is the method of any one of Aspects 1-39, wherein the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain. Aspect 41 is the method of Aspect 40, wherein the detectable label comprises a FLAG peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12). Aspect 42 is the method of Aspect 40 or 41, wherein the protein binding domain comprises an integrin-binding domain. Aspect 43 is the method of Aspect 42, wherein the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21). Aspect 44 is the method of Aspect 40 or Aspect 41, wherein the protein binding domain comprises an Fc-binding sequence. ). Aspect 45 is the method of Aspect 44, wherein the Fc-binding sequence comprises a protein A ZZ amino acid sequence. ). Aspect 46 is the method of Aspect 40 or 41, wherein the protein binding domain comprises a biotin-binding sequence. ). Aspect 47 is the method of Aspect 46, wherein the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence. Aspect 48 is the method of any one of Aspects 39-47, wherein the one or more flexible linkers comprise two flexible linkers. Aspect 49 is the method of Aspect 46, wherein a first of the one or more flexible linkers is inserted at amino acid 70 of E2. Aspect 50 is the method of Aspect 48 or 49, wherein the detectable label and / or the protein binding domain is comprised between the two flexible linkers. Aspect 51 is the method of any one of Aspects 1-50, wherein the pseudotyped retroviral envelope further comprises a targeting moiety. Aspect 52 is the method of any one of Aspects 1-51, wherein the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety. Aspect 53 is the method of Aspect 52, wherein the nucleic acid is comprised in a viral vector. Aspect 54 is the method of Aspect 52, wherein the nucleic acid is comprised in viral genome. Aspect 55 is the method of any one of Aspects 1-55, wherein B cells express B cell receptors specific for the pseudotyped retroviral envelope, a targeting moiety, or a combination thereof. Aspect 56 is the method of any one of Aspects 1-55, wherein the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety is comprised in a retrovirus. Aspect 57 is the method of Aspect 56, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus. Aspect 58 is the method of Aspect 56, wherein the retrovirus is a lentivirus. Aspect 59 is the method of any one of Aspects 51-58, wherein the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer- associated antigens, or one or more auto- or self-antigens, or a combination thereof. Aspect 60is the method of Aspect 59, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof. Aspect 61 is the method of Aspect 59 or 60, wherein the pathogenic antigen comprises one or more viral envelope proteins. Aspect 62 is the method of any one of Aspects 56-61, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 63 is the method of Aspect 51, wherein the targeting moiety is conjugated to the pseudotyped retroviral envelope. Aspect 64 is the method of Aspect 63, wherein the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope. Aspect 65 is the method of Aspect 63 or 64, wherein the targeting moiety is covalently linked to the pseudotyped retroviral envelope. Aspect 66 is the method of Aspect 63 or 64, wherein the targeting moiety is non-covalently linked to the pseudotyped retroviral envelope. Aspect 67 is the method of any one of Aspects 63-66, wherein the targeting moiety comprises an antibody. Aspect 68 is the method of Aspect 67, wherein the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer- associated antigens, or one or more auto- or self-antigens, or a combination thereof. Aspect 69 is the method of Aspect 68, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof. Aspect 70 is the method of Aspect 68 or 69, wherein the pathogenic antigen comprises one or more viral envelope proteins. Aspect 71 is the method of any one of Aspects 68-70, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 72 is the method of any one of Aspects 1-71, wherein transduction is mediated by molecular mechanisms independent of interaction of the pseudotyped retroviral envelope with cognate retrovirus receptors. Aspect 73 is the method of any one of Aspects 1-72, wherein transduction is mediated by molecular mechanisms independent of interaction of the pseudotyped retroviral envelope with PtdSer receptors. Aspect 74 is the method of any one of Aspects 1-73, wherein the nucleic acid encoding the pseudotyped retroviral envelope and, optionally, the targeting moiety, also encodes the heterologous gene. Aspect 75 is the method of Aspect 74, wherein the heterologous gene is operably linked to a promoter. Aspect 76 is the method of Aspect 75, wherein the promoter is a tissue- specific promoter. Aspect 77 is the method of any one of Aspects 1-76, wherein the B cell is transduced in vivo. Aspect 78 is the method of Aspect 77, wherein the nucleic acid encoding the pseudotyped retroviral envelope is administered intravenously. Aspect 79 is themethod of any one of Aspects 1-76, wherein the B cell is transduced ex vivo. Aspect 80 is the method of Aspect 79, wherein the B cell is a primary B cell. Aspect 81 is the method of Aspect 79, wherein the B cell is a B cell from a B cell line. Aspect 82 is the method of any one of Aspects 1-76, wherein the B cell is transduced in vitro. Aspect 82 is the method of any one of Aspects 82, wherein the B cell is a primary B cell. Aspect 84 is the method of any one of Aspects 82, wherein the B cell is a B cell from a B cell line. Aspect 84 is a method of inducing an immune response against an infection, disease, or condition associated with a targeting moiety in a subject in need thereof, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 86 is the method of any one of Aspects 85, wherein the B cell expresses a receptor to which the targeting moiety is directed. Aspect 87 is the method of Aspect 85 or 86, wherein the B cell expresses receptors specific for the pseudotyped retroviral envelope, the targeting moiety, or a combination thereof. Aspect 88 is the method of any one of Aspects 1-86, wherein contacting the B cell with the targeting moiety and the nucleic acid encoding a pseudotyped retroviral envelope induces proliferation and / or differentiation of the B cell. Aspect 89 is the method of any one of Aspects 85-88, wherein the immune response comprises endogenous antibody production by the B cell. Aspect 90 is a method of inducing proliferation and / or differentiation of a B cell expressing a receptor to which a targeting moiety is directed, comprising contacting the B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 91 is the method of Aspect 90, wherein the B cell is contacted with the targeting moiety prior to contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. Aspect 92 is the method of Aspect 90, wherein the B cell is simultaneously contacted with the targeting moiety and the nucleic acid encoding the pseudotyped retroviral envelope. Aspect 93 is the method of Aspect 90, wherein the B cell is contacted with the targeting moiety after contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. Aspect 94 is the method of Aspect 90, wherein the B cell is contacted with the targeting moiety prior to and simultaneously with contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. Aspect 95 is the method of Aspect 90, wherein the B cell is contacted with the targeting moiety simultaneously with and after contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. Aspect 96 is the method of Aspect 90, wherein the B cell is contacted with the targeting moiety prior to, simultaneously with, and after contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope. Aspect 97 is a method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with a targeting moiety in a subject in need thereof, comprisingcontacting a B cell with the targeting moiety and a nucleic acid encoding a pseudo typed retroviral envelope. Aspect 98 is a method of vaccinating a subject against an infection, disease, or condition associated with a targeting moiety, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope. Aspect 99 is a method of generating neutralizing antibodies against an infection, disease, or condition associated with a targeting moiety, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding pseudotyped retroviral envelope. Aspect 100 is the method of any one of Aspects 85-99, wherein the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins. Aspect 101 is the method of any one of Aspects 85-99, wherein the pseudotyped retroviral envelope comprises lentiviral envelope proteins. Aspect 102 is the method of any one of Aspects 85-101, wherein the nucleic acid is comprised in a viral vector. Aspect 103 is the method of Aspect 102, wherein the viral vector is a gammaretroviral vector or oncoretroviral vector. Aspect 104 is the method of Aspect 102, wherein the viral vector is a lentiviral vector. Aspect 105 is the method of any one of Aspects 85-101, wherein the nucleic acid is comprised in a viral genome. Aspect 106 is the method of Aspect 105, wherein the viral genome is a gammaretroviral genome or oncoretroviral genome. Aspect 107 is the method of Aspect 105, wherein the viral genome is a lentiviral genome. Aspect 108 is the method of any one of Aspects 85-107, wherein the nucleic acid encoding the pseudotyped retroviral envelope or the viral vector or viral genome comprising the nucleic acid is comprised in a retrovirus. Aspect 109 is the method of any one of Aspects 108, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus. Aspect 110 is the method of any one of Aspects 108, wherein the retrovirus is a lentivirus. Aspect 111 is the method of any one of Aspects 85-110, wherein the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3, wherein the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3, and wherein the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope. Aspect 112 is the method of Aspect 111, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise: substitutions or deletions at amino acid residues 226 and / or 227 of El; substitutions or deletions at amino acid residues 68-71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60-64 of E3. Aspect 113 is the method of Aspect 111 or 112, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted. Aspect 114 is the method of Aspect 113, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG. Aspect115 is the method of any one of Aspects 111-114, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted. Aspect 116 is the method of Aspect 115, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA. Aspect 117 is the method of any one of Aspects 111-116, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted. Aspect 118 is the method of Aspect 117, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA. Aspect 119 is the method of any one of Aspects 111-118, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted. Aspect 120 is the method of Aspect 119, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted. Aspect 121 is the method of any one of Aspects 111-118, wherein the sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA. Aspect 122 is the method of any one of Aspects 111-121, wherein the mutated Sindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68-71AAAA in E2, and the mutation KE159- 160AA in E2. Aspect 123 is the method of Aspect 122, wherein the mutated Sindbis envelope further comprises the mutation AK226-227SG in El. Aspect 124 is the method of any one of Aspects 111-123, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viral genome comprising the nucleic acid. Aspect 125 is the method of any one of Aspects 85-124, wherein the pseudotyped retroviral envelope further comprises one or more flexible linkers. Aspect 126 is the method of any one of Aspects 85-125, wherein the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain. Aspect 127 is the method of Aspect 126, wherein the detectable label comprises a FLAG peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12). Aspect 128 is the method of Aspect 126 or 127, wherein the protein binding domain comprises an integrin-binding domain. Aspect 129 is the method of Aspect 128, wherein the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21). Aspect 130 is the method of Aspect 126 or 127, wherein the protein binding domain comprises an Fc-binding sequence. Aspect 131 is the method of Aspect 130, wherein the Fc-binding sequence comprises a protein A ZZ amino acid sequence. Aspect 132 is the method of Aspect 126 or 127, wherein the protein binding domain comprises a biotin-binding sequence. Aspect 133 is the method of Aspect 132, wherein the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence. Aspect 134 is the method of any one ofAspects 125-133, wherein the one or more flexible linkers comprise two flexible linkers. Aspect 135 is the method of Aspect 132, wherein a first of the one or more flexible linkers is inserted at amino acid 70 of E2. Aspect 136 is the method of Aspect 134 or 135, wherein the detectable label and / or the protein binding domain is comprised between the two flexible linkers. Aspect 137 is the method of any one of Aspects 85-136, wherein the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof. Aspect 138 is the method of Aspect 137, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof. Aspect 139 is the method of Aspect 137 or 138, wherein the one or more pathogenic antigens comprise one or more viral envelope proteins. Aspect 140 is the method of any one of Aspects 137-139, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 141 is the method of any one of Aspects 85-140, wherein the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety. Aspect 142 is the method of Aspect 141, wherein the nucleic acid is comprised in a viral vector. Aspect 143 is the method of Aspect 141, wherein the nucleic acid is comprised in viral genome. Aspect 144 is the method of any one of Aspects 41-143, wherein the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope the targeting moiety is comprised in a retrovirus. Aspect 145 is the method of Aspect 144, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus. Aspect 146 is the method of Aspect 144, wherein the retrovirus is a lentivirus. Aspect 147 is the method of any one of Aspects 85-140, wherein the targeting moiety is conjugated to the pseudotyped retroviral envelope. Aspect 148 is the method of Aspect 147, wherein the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope. Aspect 149 is the method of Aspect 147 or 148, wherein the targeting moiety is covalently linked to the pseudotyped retroviral envelope. Aspect 150 is the method of Aspect 147 or 148, wherein the targeting moiety is non-covalently linked to the pseudotyped retroviral envelope. Aspect 151 is the method of any one of Aspects 147-150, wherein the targeting moiety comprises an antibody. Aspect 152 is the method of Aspect 151, wherein the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof. Aspect 153 is the method of Aspect 152, wherein the infectious agent is a viral, bacterial, fungal, or parasitic agent, or a combination thereof.Aspect 154 is the method of Aspect 152 or 153, wherein the one or more antigenic molecules comprise one or more viral envelope proteins. Aspect 155 is the method of any one of Aspects 152-154, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 156 is the method of any one of Aspects 85-155, wherein B cells of the subject are contacted with the targeting moiety and the nucleic acid encoding the pseudotyped retroviral envelope a single time. Aspect 157 is the method of any one of Aspects 85-156, wherein B cells of the subject are contacted with the targeting moiety and / or the nucleic acid encoding the pseudotyped retroviral envelope a multiple times. Aspect 158 is the method of any one of Aspects 85-157, where B cells of the subject are contacted with the nucleic acid encoding the pseudotyped retroviral envelope multiple times. Aspect 159 is the method of any one of Aspects 85-158, wherein B cells of the subject are not contacted with the targeting moiety more than a single time. Aspect 160 is the method of any one of Aspects 85-159, wherein the targeting moiety and the nucleic acid encoding a pseudotyped retroviral envelope are comprised in a pharmaceutical composition. Aspect 161 is the method of any one of Aspects 85-156, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients. Aspect 162 is the method of any one of Aspects 85-161, wherein the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older. Aspect 163 is a method of identifying an antibody against a virus, comprising: contacting a B cell with a nucleic acid encoding a pseudotyped retroviral envelope; identifying a B cell receptor that recognizes the pseudotyped retroviral envelope; and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody. Aspect 164 is a method of identifying an antibody directed against a targeting moiety, comprising: contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope; identifying a B cell receptor that recognizes the targeting moiety; and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody. Aspect 165 is the method of Aspect 163 or 164, wherein the B cell expresses B cell receptors specific for the pseudotyped retroviral envelope, a targeting moiety, or a combination thereof. Aspect 166 is the method of any one of Aspects 163-165, wherein the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins. Aspect 167 is the method of any one of Aspects 163-165, wherein the pseudotyped retroviral envelope compriseslentiviral envelope proteins. Aspect 168 is the method of Aspect 167, wherein the nucleic acid is comprised in a viral vector. Aspect 169 is the method of Aspect 168, wherein the viral vector is a gammaretroviral vector or oncoretroviral vector. Aspect 170 is the method of Aspect 168, wherein the viral vector is a lentiviral vector. Aspect 171 is the method of Aspect 167, wherein the nucleic acid is comprised in a viral genome. Aspect 172 is the method of Aspect 171, wherein the viral genome is a gammaretroviral genome or oncoretroviral genome. Aspect 173 is the method of Aspect 171, wherein the viral genome is a lentiviral genome. Aspect 174 is the method of any one of Aspects 163-173, wherein the nucleic acid encoding the pseudotyped retroviral envelope or the viral vector or viral genome comprising the nucleic acid is comprised in a retrovirus. Aspect 175 is the method of Aspect 174, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus. Aspect 176 is the method of Aspect 174, wherein the retrovirus is a lentivirus. Aspect 177 is the method of any one of Aspects 164-176, wherein the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3, wherein the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3, and wherein the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope. Aspect 178 is the method of Aspect 177, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise: substitutions or deletions at amino acid residues 226 and / or 227 of El; substitutions or deletions at amino acid residues 68- 71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60- 64 of E3. Aspect 179 is the method of Aspect 177 or 178, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted. Aspect 180 is the method of Aspect 179, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG. Aspect 181 is the method of any one of Aspects 177-180, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted. Aspect 182 is the method of Aspect 181, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA. Aspect 183 is the method of any one of Aspects 177-182, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted. Aspect 184 is the method of Aspect 183, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA. Aspect 185 is the method of any one of Aspects 177-184, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted. Aspect 186 is the method of Aspect 185, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted. Aspect 187 is the method of any one of Aspects 177-184, whereinthe sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA. Aspect 188 is the method of any one of Aspects 177-187, wherein the mutated Sindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68-71AAAA in E2, and the mutation KE159-160AA in E2. Aspect 189 is the method of Aspect 188, wherein the mutated Sindbis envelope further comprises the mutation AK226- 227SG in El. Aspect 190 is the method of any one of Aspects 177-189, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viral genome comprising the nucleic acid. Aspect 191 is the method of any one of Aspects 163-190, wherein the pseudotyped retroviral envelope further comprises one or more flexible linkers. Aspect 192 is the method of any one of Aspects 163-191, wherein the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain. Aspect 193 is the method of Aspect 192, wherein the detectable label comprises a FLAG peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12). Aspect 194 is the method of Aspect 192 or 193, wherein the protein binding domain comprises an integrin-binding domain. Aspect 195 is the method of Aspect 194, wherein the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21). Aspect 196 is the method of Aspect 192 or 193, wherein the protein binding domain comprises an Fc-binding sequence. Aspect 197 is the method of Aspect 196, wherein the Fc-binding sequence comprises a protein A ZZ amino acid sequence. Aspect 198 is the method of Aspect 192 or 193, wherein the protein binding domain comprises a biotin-binding sequence. Aspect 199 is the method of Aspect 198, wherein the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence. Aspect 200 is the method of any one of Aspects 191-199, wherein the one or more flexible linkers comprise two flexible linkers. Aspect 201 is the method of Aspect 198, wherein a first of the one or more flexible linkers is inserted at amino acid 70 of E2. Aspect 202 is the method of Aspect 200 or 201, wherein the detectable label and / or the protein binding domain is comprised between the two flexible linkers. Aspect 203 is the method of any one of Aspects 164-202, wherein the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or selfantigens, or a combination thereof. Aspect 204 is the method of Aspect 203, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof. Aspect 205 is the method of Aspect 203 or 204, wherein the one or more pathogenic antigens comprise one or more viral envelope proteins. Aspect 206 is themethod of any one of Aspects 203-205, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 207 is the method of any one of Aspects 164-206, wherein the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety. Aspect 208 is the method of Aspect 207, wherein the nucleic acid is comprised in a viral vector. Aspect 209 is the method of Aspect 207, wherein the nucleic acid is comprised in viral genome. Aspect 210 is the method of any one of Aspects 207-209, wherein the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope the targeting moiety is comprised in a retrovirus. Aspect 211 is the method of Aspect 210, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus. Aspect 212 is the method of Aspect 210, wherein the retrovirus is a lentivirus. Aspect 213 is the method of any one of Aspects 164-206, wherein the targeting moiety is conjugated to the pseudotyped retroviral envelope. Aspect 214 is the method of Aspect 213, wherein the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope. Aspect 215 is the method of Aspect 213 or 214, wherein the targeting moiety is covalently linked to the pseudotyped retroviral envelope. Aspect 216 is the method of Aspect 213 or 214, wherein the targeting moiety is non-covalently linked to the pseudotyped retroviral envelope. Aspect 217 is the method of any one of Aspects 213-216, wherein the targeting moiety comprises an antibody. Aspect 218 is the method of Aspect 217, wherein the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof. Aspect 219 is the method of Aspect 218, wherein the infectious agent is a viral, bacterial, fungal, or parasitic agent, or a combination thereof. Aspect 220 is the method of Aspect 218 or 219, wherein the one or more antigenic molecules comprise one or more viral envelope proteins. Aspect 221 is the method of any one of Aspects aims 218-220, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof. Aspect 222 is the method of any one of Aspects 163-221, further comprising producing the antibody, or an antigen-binding fragment thereof, by culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell. Aspect 223 is a pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of Aspect 222, and apharmaceutically acceptable carrier or excipient. Aspect 224 is a method of preventing, treating, and / or ameliorating an infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, of Aspect 222 or the pharmaceutical composition of Aspect 223. Aspect 225 is a method of manufacturing a medicament for preventing, treating, and / or ameliorating an infection, the method comprising culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody of Aspect 222 under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell. Aspect 226 is the method of Aspect 224 or 225, wherein the infection is a viral, bacterial, fungal, or parasitic infection. Aspect 227 is a method of preventing, treating, and / or ameliorating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, of Aspect 222 or the pharmaceutical composition of Aspect 223. Aspect 228 is a method of manufacturing a medicament for preventing, treating, and / or ameliorating cancer, the method comprising culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody of Aspect 222 under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell. Aspect 229 is the method of Aspect 227 or 228, wherein the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer. Aspect 230 is the antibody, or antigen binding fragment thereof, of Aspect 222, for use in the treatment of at least one sign and / or symptom of an infection, disease, or condition. Aspect 231 is the antibody, or antigen binding fragment thereof, of Aspect 230, wherein the infection, disease, or condition is viral, bacterial, fungal, or parasitic infection. Aspect 232 is the antibody, or antigen binding fragment thereof, of Aspect 230, wherein the infection, disease, or condition is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer. Aspect 233 is a kit for the treatment of an infection, disease, or condition, comprising a therapeutically effective amount of the antibody, or antigen binding fragment thereof, of Aspect 222. Aspect 234 is the kit of Aspect 233, wherein the infection, disease, or condition is viral, bacterial, fungal, or parasitic infection. Aspect 235 is the kit of Aspect 233, wherein the infection, disease, or condition is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer. Aspect 236 is the method of any one of Aspects 1-221, wherein the B cell is contacted in vivo. Aspect 237 is the method of any one of Aspects.1-221and Aspect 236, wherein the targeting moiety, the nucleic acid encoding a pseudotyped retroviral envelope, or a comination thereof is administered to a subject. Aspect 238 is the method of any one of Aspects 1-221, Aspect 237, or Aspect 237, wherein the targeting moiety, the nucleic acid encoding a pseudotyped retroviral envelope, or a comination thereof is administered intravenously. Aspect 239 is the method of any one of Aspects 1-221, wherein the B cell is transduced ex vivo. Aspect 240 is the method of Aspect 239, wherein the B cell is a primary B cell. Aspect 240 is the method of Aspect 239, wherein the B cell is a B cell from a B cell line. Aspect 241 is the method of any one of Aspects 1-221, wherein the B cell is transduced in vitro. Aspect 242 is the method of Aspect 241, wherein the B cell is a primary B cell. Aspect 243 is the method of Aspect 242, wherein the B cell is a B cell from a B cell line.

[0024] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0025] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0027] FIG. 1 shows a schematic representation of chimeric Sindbis virus envelope proteins. The Sindbis virus envelope protein is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate the E3, E2, 6K, and El proteins. El and E2 are incorporated into the viral envelope. Both 2.2 and 2.2 ILFlaglL were derived from the wild type Sindbis virus envelope protein with four mutations (shown as red lines) to reduce their binding to original receptors, while maintaining fusion activity. In addition, 2.2 1L1L has two flexible linkers at amino acid (a.a.) 70 of the E2 protein; 2.2 ILFlaglL contains the Flag peptide (DYKDDDDK; SEQ ID NO: 12) between the two flexible linkers of 2.2 1L1L; and4CRGD contains the integrin-binding peptide (RGD-4C) (CDCRGDCFC; SEQ ID NO:21) between the two flexible linkers of 2.2 1L1L.

[0028] FIGS. 2A-2H show robust transduction of splenic B cells by intravenous administration of lentiviral vectors. FIG. 2A. Whole-body imaging of LumiScarlet lentiviral transgene after intravenous injection of lentiviral vectors pseudotyped with either VSV-g, Sindbis, or 2.2 ILFlaglL. The amounts of the vectors injected were adjusted by the HIV p24 CA protein (3 pg p24 / mouse). Representative images of each experimental group are shown. FIG. 2B. Transgene expression in liver and spleen. Averages of relative luminescence units per 1 mg of protein are shown. Error bars represent standard deviations (SD). n=3 for VSV-g and Sindbis, and n=6 for 2.2 ILFlaglL. FIG. 2C. Average percentages of cell populations in the Scarlet-expressing cells analyzed by flow cytometry. Numbers are shown as average ± SD. Averages of percentages of transduced cells in whole B cell (FIG. 2D) or B cell subpopulations in the spleen after intravenous injection of (FIG. 2E) VSV-g; (FIG. 2F) Sindbis; or (FIG. 2G) 2.2 ILFlaglL pseudotype. Significance was calculated using a two-sample two-sided unpaired student t-test (**, p<0.01). FIG. 2H. Percentages of transduced cells in whole B cell or B cell subpopulations in the spleen after intravenous injection of 2.2 ILFlaglL pseudotypes produced in the presence (n=l) or absence (n=3) of Saquinavir. The transduction data of Saquinavir- treated 2.2 ILFlaglL pseudotype is the average and SD of three mice. (D) Significance was calculated using a two-sample two-sided unpaired student t-test ( * * , p<0.01). Fo: follicular B cells, Mz: marginal zone B cells, Sho-L Plsm: short-lived plasma cells, Lo-L Plsm: long- lived plasma cells, GCA: germinal center activated B cells, Mem: memory B cells. Error bars represent SD.

[0029] FIGS. 3A-3O shows cell number and histological analysis of murine spleen cells transduced with 2.2 ILFlaglL or VSV-g pseudotypes. Histological analysis of murine spleen cells transduced with (FIG. 3A) VSV-g or (FIG. 3B) 2.2 ILFlaglL pseudotype. Clockwise, H&E, CD3e, B220, GFP, embedded scale bar, 200um. The serial sections are stained by Hematoxylin and Eosin, Hematoxylin and anti-GFP, anti-B220, or anti-CD3 antibody. FIG. 3C. Gating strategy for flow cytometric identification of Tl, T2, T3, marginal zone (Mz), B-l, and follicular (Fo) B cells. FIG. 3D. Gating strategy for flow cytometric identification of short-lived plasma (Sho-L plsm), long-lived plasma (Lo-L plsm), germinal center-activated B cells (GCA) and memory B cells (Mem). Absolute cell numbers of a subpopulation of splenic B cells with or without lentiviral vector administration. (FIG. 3E) Total B cells; (FIG. 3F) Tl B cells; (FIG. 3G) T2 B cells; (FIG. 3H) T3 B cells; (FIG. 31)follicular B cells, (FIG. 3 J) marginal zone B cells; (FIG. 3K B-l B cells; (FIG. 3L)short-lived plasma cells; (FIG. 3M) long-lived plasma cells; (FIG. 3N) germinal center activated B cells; and (FIG. 30) memory B cells.

[0030] FIGS. 4A-4I show properties of viral receptors mediating binding of pseudotyped lentiviral vectors. Transduction of (FIG. 4A) mouse B cell line Sp2 / 0; (FIG. 4B) human B cell line Ramos; and (FIG. 4C, 4D) two human primary B cells from two donors. FIG. 4E. Expression of Axl and TIM-1 on splenic B cells transduced with 2.2 ILFlaglL- pseudotyped lentiviral vectors. FIG. 4F. Schematic figures of hypothesis of transduction of B cells with lentiviral vectors expressing GFP and Sclt pseudotyped with different types of envelope proteins, respectively, when both envelope proteins use the same receptor (left panel) or different receptors expressed on different cells (right panel). FIG. 4G. Expression of GFP and Scarlet in splenic B cell and its subpopulations when these transgenes are delivered by lentiviral vectors pseudotyped with the same or different envelope proteins. Averages and SDs derived from three replicates as well as representative flow cytometry profiles are shown for each experimental condition. n=3 for each experimental group. FIG. 4H. Binding of Scarlet- labeled MEV pseudovirion pseudotyped with VSV-g [VSV-g (MEV) Src Sclt] or 2.2 ILFlaglL [2.2 ILFlaglL (MLV) Src Sclt] to splenic B cells (C57BL6) transduced with lentiviral vectors pseudotyped with VSV-g (GFP+) or 2.2 ILFlaglL (Ametrine+). Averages and SDs derived from 3 replicates, as well as representative flow profiles are shown for each experimental condition. Significance was calculated using a two-sample two-sided unpaired student t-test (**, p<0.01). FIG. 41. Binding of MLV pseudovirion pseudotyped with VSV-g to splenic B cells transduced with VSV-g-pseudotyped lentiviral vector and binding of MLV pseudovirion pseudotyped with 2.2 ILFlaglL to splenic B cells transduced with 2.2 ILFlaglL-pseudo typed lentiviral vector in the absence or presence of goat anti-mouse Ig F(ab) domain or control goat antibody. Averages and SDs derived from three replicates are shown. Significance was calculated using a two-sample two-sided unpaired student t-test (**, p<0.01).

[0031] FIGS. 5A-5F show properties of viral receptors mediating lentiviral transduction of B cells. Transduction of (FIG. 5A) P3X63; (FIG. 5B) Y3-Ag 1.2.3; (FIG. 5C) Raji; and (FIG. 5D) Daudi cells. FIG. 5E. Binding of MLV pseudovirion pseudotyped with VSV-g [VSV-g (MLV) Src Sclt] or 2.2 ILFlaglL [2.2 ILFlaglL (MLV) Src Sclt] to splenic B cells (Balb / C) transduced with lentiviral vectors pseudotyped with VSV-g (GFP+) or 2.2 ILFlaglL (Ametrine+). The numbers represent averages and SD, and a representative flow profile is shown for each experimental condition. n=3 for each experimental group. FIG. 5F. Binding of MLV pseudovirion pseudotyped with EboZ [EboZ (MLV) Src Sclt] to splenic Bcells (C57BL6) transduced with lentiviral vectors pseudotyped with EboZ (GFP+) or 2.2 ILFlaglL (Ametrine+). The numbers represent averages and SD, and a representative flow profile is shown for each experimental condition. n=3 for each experimental group.

[0032] FIGS. 6A-6D show that BCR mediates envelope protein- specific transduction of pseudotypes. FIG. 6A. Sorting strategy for single-cell analysis of a splenic B cell population transduced with VSV-g or 2.2 IFFlaglE pseudotype, and untransduced with any pseudotype, and splenic B cells of a mouse with no vector injection. Heavy chain (FIG. 6B) and (FIG. 6C) light chain V region usage of B cells transduced with VSV-g pseudotype, 2.2 IFFlaglE pseudotype, untransduced splenic B cells, or splenic B cells with no vector injection. FIG. 6D. Enhancement of 2.2 IFFlaglE or VSV-g pseudotype transduction of CD79 293T cells by ectopic expression of BCRs isolated from splenic B cells transduced with VSV-g or 2.2 IFFlaglE pseudotype. Averages and SDs derived from three replicates are shown. Transduction efficiencies of untransfected CD79 293T cells with 2.2 IFFlaglE and VSV-g pseudotypes are 1.32+0.03 and 5.4+0.6%, respectively.

[0033] FIGS. 7A-7C show binding specificities of BCRs of splenic B cells transduced with 2.2 IFFlaglE or VSV-g pseudotype. Binding of recombinant antibodies derived from BCR sequences obtained from splenic B cells transduced with (FIG. 7A) 2.2 IFFlaglE or (FIG. 7B) VSV-g pseudotype to B16F10 or 293T cells transfected with empty vector (control), VSV-g, or 2.2 IFFlaglE expression vector. FIG. 7C. Binding of Sclt-labeled HIV pseudovirion pseudotyped with VSV-g or 2.2 IFFlaglE to 293T cells expressing control (antiHIV- 1 3BNC117), 2.2 08, 2.2 10, or VSV 04 BCR.

[0034] FIGS. 8A-8K show roles of BCR signaling in BCR-mediated lentiviral transduction. FIG. 8A. Transduction of Ramos cells ectopically expressing ScFv BCRs with VSV-g (top panel) or 2.2 IFFlaglE (bottom panel) pseudotype. N=3, and averages are shown with SD (error bar). Significance was calculated by comparing transduction efficiencies of Ramos cells to the cells expressing ScFv BCR, using a two-sample two-sided unpaired student t-test (*, p<0.05; **, p<0.01). FIG. 8B. Transduction of human primary B cells ectopically expressing ScFv BCRs with 2.2 IFFlaglE (left panel) or VSV-g (right panel) pseudotype. N=3, and averages are shown with SD (error bar). Significance was calculated by comparing transduction efficiencies of human primary B cell to the cells expressing ScFv BCR using a two-sample two-sided unpaired student t-test ( * , p<0.05; * * , p<0.01). FIG. 8C. Recombinant antibodies derived from lentivirally transduced B cells neutralize viruses containing the same envelope protein as the transducing lentiviral vectors. Blocking ofinfection of 293T with vesicular stomatitis virus by recombinant antibodies. Significance was calculated by comparing transduction efficiencies without blocker to those with the blockers using a two-sample two-sided unpaired student t-test (*, p<0.05; **, p<0.01). FIG. 8D. Singlecell gene expression analysis to compare subpopulations of splenic B cells of naive, untransduced, and those transduced with VSV or 2.2 ILFlaglL pseudotype. Tra: Transitional B cells GC: germinal center B cells, Mz: marginal zone B cells, Fo: follicular B cells. FIG. 8E. Heatmap of expression of genes mediating various B cell functions in naive, untransduced, and those transduced with VSV or 2.2 ILFlaglL pseudotype. FIG. 8F. Transduction of human primary B cells ectopically expressing ScFv BCRs with 2.2 ILFlaglL (left panel) or VSV-g (right panel) pseudotype. N=3, and averages are shown with SD (error bar). Significance was calculated by comparing transduction efficiencies of human primary B cell to the cells expressing ScFv BCR, using a two-sample two-sided unpaired student t-test (*, p<0.05; **, p<0.01). Blocking of transduction of 2.2 05 ScFV BCR Ramos with (FIG. 8G) 2.2 ILFlaglL pseudotype (FIG. 8H) VSV 04 or (FIG. 81) 05 ScFV BCR Ramos and Ramos with (FIG. 8J) VSV-g pseudotype by recombinant antibodies. Significance was calculated by comparing transduction efficiencies without blocker to those with the blockers using a two-sample two- sided unpaired student t-test (*, p<0.05; **, p<0.01). FIG. 8K. Blocking of infection of 293T with vesicular stomatitis virus by recombinant antibodies. Significance was calculated by comparing transduction efficiencies without blocker to those with the blockers using a two- sample two-sided unpaired student t-test (*, p<0.05; **, p<0.01).

[0035] FIGS. 9A-9C. FIG. 9A. Flow cytometric analysis of phosphorylation of ERK and Akt after binding of VSV-g (blue line), 2.2 ILFlaglL (red line), or control medium to Ramos cells or Ramos cells expressing ScFv BCR. FIG. 9B. Effects of BCR signaling blockers on BCR-mediated transduction. FIG. 9C shows blocking virus-induced BCR signaling by Ibrutinib and Idelalisib. Akt and ERK phosphorylation status after binding of VSV-g pseudotype to VSV 04 BCR-expressing Ramos cells in the presence or absence of Ibrutinib or Idelalisib.

[0036] FIGS. 10A-10B show comparison of expression levels of endogenous IgGl BCR and lentivirally transduced ScFv BCR on human primary B cells. The cells were stained with human IgGl antibody conjugated with APC. FIG. 10A. Control (anti-HIV-1 3BNC117), 2.205, VSV 04, VSV 05, and VSV 06 ScFv BCR. FIG. 10B. control (anti-HIV-1 3BNC117), anti-ZIKV ScFv BCR (ZKA190 and ZKA230).

[0037] FIGS. 11A-11E show anti-ZIKV BCR mediates ZIKV infection. Infection of (FIG. 11A) Ramos or (FIG. 11B) Sp2 / 0 cells with or without ectopic expression of control(anti-HIV-1 3BNC117) 56 or anti-ZIKV ScFv BCR with replication-competent ZIKV. N=3 and averages are shown with SD (error bar). FIG. 11C. Infection of primary human B cells from two donors with or without ectopic expression of control or anti-ZIKV ScFv BCR with ZIKV replicon. N=3 and averages are shown with SD (error bar). FIG. 11D. Gating strategy to analyze in vivo ZIKV infection of splenic B cells expressing control or anti-ZIKV ScFv BCR. FIG. HE. Percentages of ZIKV-infected splenic B cells expressing control or anti-ZIKV ScFv BCR with ZIKV replicon. Averages are shown with SD (error bar). Significance was calculated using a two-sample two-sided unpaired student t-test (**, p<0.01).

[0038] FIGS. 12A-12E show anti-SARS-CoV-2 BCRs mediate SARS-CoV-2 entry. FIG. 12A. Binding of Sclt-labeled lentiviral vector pseudotyped with S protein to 293T cells with or without ectopic expression of TIM-1, ACE2, control (anti-HIV-1 3BNC117), or anti- SARS-CoV-2 BCR (REGN10933 and CB6) at multiple MOIs (pg p24 / ml). FIG. 12B. Fusion / entry of P-lactamase containing lentiviral vector pseudotyped with S protein with or without ectopic expression of TIM-1, ACE2, control, or anti-SARS-CoV-2 BCR. Using flow cytometry, entry of virus is quantitated by cleavage of CCF4-AM in the cytoplasm of target cells. N=3 and averages are shown with SD (error bar). FIG. 12C. Transduction of 293T cells with or without ectopic expression of TIM-1, ACE2, control, or anti-SARS-CoV-2 BCR with lentiviral vector pseudotyped with S protein. FIG. 12D. Transduction of Sp2 / 0 cells with or without ectopic expression of ACE2, control (anti-HIV-1 3BNC117), or anti-SARS-CoV-2 ScFv BCR with lentiviral vector pseudotyped with S protein in the absence and or presence of Nevirapine (10 pM). N=3 and averages are shown with SD (error bar). FIG. 12E. Infection of Sp2 / 0 cells with or without ectopic expression of ACE2, control, or anti-SARS-CoV-2 ScFv BCR with live or heat-inactivated SARS-CoV-2. The copy numbers of SARS-CoV-2 N1 (left panel) or N2 (right panel) per 100 copies of GAPDH are shown with SD (error bar). N=3.

[0039] FIGS. 13A-13B show binding and fusion of S protein pseudotyped lentiviral vector. FIG. 13A. Flow cytometric profiles of S protein pseudotyped lentiviral vector binding to cells ectopically expressing TIM-1, ACE2, or ScFv BCRs [control or anti-SARS-CoV-2 (REGN and CB6)]. FIG. 13B. Representative flow cytometric profiles of virus fusion / entry assays detected by elimination of FRET induced by cleavage of CCF4-AM with virionincorporated P-lactamase.

[0040] FIGS. 14A-14C show that lentiviral vectors pseudotyped with the modified Sindbis virus envelope proteins (E2 71 eMA Sindbis) can transduce B-cells expressing BCRs against conjugated antigens.

[0041] FIGS. 15A-15D show that antiviral BCRs mediate transduction of redirected lentiviral vectors. FIG. 15A. Enhancement of 4CRGD pseudotype transduction of CD79293T cells by ectopic expression of BCRs isolated from splenic B cells transduced with 2.2 ILFlaglL or VSV-g pseudotype. Averages and SDs derived from three replicates are shown. Transduction efficiencies of untransfected CD79 293T cells with 4CRGD pseudotypes is 0.99+0.2%. Significance was calculated by comparison to transduction efficiencies of untransfected CD79 293T using a two-sample two-sided unpaired student t-test (**, p<0.01). FIG. 15B. Transduction of splenic B cells (C57BL6) with lentiviral vectors pseudotyped with VSV-g (GFP+) or 4CRGD (Ametrine+). Averages and SDs of percent transduced cells derived from 3 replicates are shown. FIG. 15C. Binding of Scarlet-labeled MEV pseudovirion pseudotyped with VSV-g [VSV-g (MEV) Src Sclt] or 2.2 ILFlaglL [2.2 ILFlaglL (MLV) Src Sclt] to splenic B cells transduced with lentiviral vectors pseudotyped with VSV-g (GFP+) or 4CRGD (Ametrine+). Averages and SDs derived from 3 replicates. Significance was calculated using a two-sample two-sided unpaired student t-test (**, p<0.01). FIG. 15D. Binding of MLV pseudovirion pseudotyped with 2.2 ILFlaglL to splenic B cells transduced with 4CRGD-pseudotyped lentiviral vector in the absence or presence of goat anti-mouse Ig F(ab) domain or control goat antibody. Averages and SDs derived from three replicates are shown. Significance was calculated using a two-sample two-sided unpaired student t-test (**, p<0.01).DETAILED DESCRIPTION

[0042] The present disclosure is based, at least in part, on the surprising discovery that B cell receptors (BCR; cell membrane-associated antibody proteins) represent a highly efficient mechanism for focusing tropism of gene therapy vectors in the context of systemic vector delivery. As shown herein, a pseudotyped viral envelopes lacking receptor-binding regions maintains efficient transduction of splenic B cells despite loss of original receptor-binding activity, suggesting that transduction of splenic B cells involves a novel mechanism(s) of virus binding.

[0043] In addition to selectively focusing viral tropism on key antiviral response cells, BCR-mediated viral entry may also selectively perturb the development, differentiation, and effector activity of those cells by generating BCR signaling. The present data link BCR- selective entry to BCR signaling activity and the molecular and enumerative indicators of B cell proliferation and differentiation. It was found that viral tropism can be directed to highlydistinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes and specific stages of B cell maturation. Accordingly, the selective gene transduction mediated by the unexpectedly specific binding of BCRs to viral envelope proteins to facilitate viral entry into B lymphocytes is associated with BCR signal transduction, cell proliferation, and differentiation.

[0044] Given the vast diversity-generating mechanisms of BCR genomic rearrangement, the B cell entry mechanism described herein may have a wide-ranging scope and may allow for entry of virtually any enveloped virus into the B cell pool. The physiological, pathological, and immunological significance of this generic B cell entry pathway could potentially contribute to the wide range of diseases that emanate from B cell dysregulation, as well as the critical role played by BCR / antibody-mediated recognition in host defense against viral infection. Although this viral epitope / BCR- selective mechanism may be narrow in total B cell scope, it will be highly selective to the specific B cell clonal subsets capable of generating effective envelope-neutralizing antibodies. BCRs may also recognize exterior features of nonenveloped viruses, viral vectors, and other gene delivery vehicles, and thereby facilitate their entry into B cells.

[0045] In the context of gene therapy and vector-based vaccines, the present discovery suggests new opportunities to selectively target specific B cell subpopulations in an antigenspecific manner through strategic pseudotyping of vector particles. As demonstrated, epitopes might be engineered into vector particles to direct transduction based on B cell antigen specificity (e.g., conjugating pseudotyped viral envelopes with target antigens, etc.). Transgenes may also be engineered to selectively enhance or suppress antibody production, isotype switching, etc., resulting in directed immune response or tolerance as the therapeutic context may require.

[0046] Beyond the general opportunity for BCR- specific vector targeting by engineered envelope proteins, the present results also identify empirical opportunities for optimizing the biological impacts of the approach through strategic selection of envelope backbones. For example, one pseudotyped viral envelope may promote more efficient plasma cell differentiation and may thereby down-regulate cell surface BCR expression more efficiently and consequently protect antibody-producing cells from subsequent entry by a pathogenic virus (e.g., in the context of a viral vaccine) compared to other pseudotyped viral envelopes. A pseudotyped viral envelope may transduce splenic B cells more efficiently without being trapped by the liver and / or other cell types in the blood and spleen, which may increase the effective multiplicity of binding, entry, and transduction and thereby enhance BCRsignaling, transgene expression, and antibody production by plasma and memory B cells compared to other pseudotyped viral envelopes. Because the half-life of plasma cells and memory B cells can be as long as the life of the host and B cells can induce tolerance to their transgene products, in vivo B cell transduction with certain pseudotyped viral envelopes may have more durable biological effects than other pseudotypes.

[0047] The selective tropism of pseudotyped viral envelopes disclosed herein for splenic B cells represents a significant advantage for BCR- and antigen- specific immune modulation in vivo. Two previous studies have utilized ex vivo transduction of antiviral antibodies and BCRs (HIV-1 and HCV) using a single vector46 47, which allows for selective upregulation of antibody production by transduced B cells when encountering the target virus and / or viral antigens. These B cell-based gene therapies require isolating B cells from a body, in vitro transduction of isolated B cells, and infusion of the transduced cells back into the body. By contrast, the present pseudotyped viral envelopes couple the high transduction efficiency of viral vectors with engineerable pseudo typing for antigen- specific immune modulation in vivo (i.e.. without any need for ex vivo B cell manipulation).

[0048] In addition to mediating BCR-specific gene transduction and viral pathogenesis, also identified is a genetic strategy for isolating BCR / antibody sequences that encode neutralizing antibodies. Current protocols for mapping neutralizing antibodies by single-cell sequencing depend on immunization with viral antigens followed by staining of BCRs fluorescence-conjugated viral proteins48. The presently disclosed methods use native forms of viral envelope proteins loaded on the virion and transduces the cells expressing the BCR against the envelope protein to endogenously express a fluorescent protein(s); therefore, this approach can provide more efficient and sensitive identification of antibody sequences with high affinity to native envelope proteins.

[0049] In addition to the implications for vector and vaccine strategy, the presently disclosed novel BCR-mediated viral entry pathway also has implications in the context of viral entry mechanisms. For example, as described herein, anti-SARS-CoV-2 BCRs can induce fusion without ACE2. Several anti-SARS-CoV-2 antibodies are known to induce conformational changes of the envelope protein even if it is only partial53. Complete conformational changes of the envelope protein require exposure to a low pH environment in endosomes and / or cleavage by proteases54 55; thus, binding of antibodies alone might not be sufficient to induce complete conformational changes of the envelope protein, but their BCR form will induce endocytosis and / or bring S proteins proximal to the proteases. Zika virus (ZIKV), SARS-CoV-2, and HIV were surprisingly demonstrated to enter B cells via thispathway. This mechanism allows for viral infection of B cells that lack any expression of the cell surface molecules that would otherwise restrict viral tropism for other cell types. The present results therefore raise the potential for more complex forms of humoral immunity- involved viral entry mechanisms than have previously been analyzed for soluble anti-viral antibodies.

[0050] In some aspects, these results suggest viral envelope protein compositions can be manipulated to selectively target B cells in antigen- specific profiles. In some aspects, these results suggest new strategies for antigen-specific gene therapy by pseudotyped viral vectors and generation of neutralizing antibodies, including for safe and effective antiviral vaccines, as well as new concepts for understanding immunogenetic adaptation to viral pathogens.I. Examples of Definitions

[0051] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure have the meanings that are commonly understood by those of ordinary skill in the art.

[0052] As used herein, the term “about” and “approximately” and “substantially” when used to modify a numerically defined parameter (e.g., the dose of “X”) means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter. Therefore, in any disclosed aspect, the terms may be substituted with “within [a percentage] of’ what is specified. In one non-limiting aspect, the percentage includes 0.1, 0.5, 1, 5, and 10 percent.

[0053] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein.

[0054] As used herein, the use of the word “a” or “an” or “the” when used in conjunction with the terms “comprising,” “including,” “having,” or “containing,” or variations of these terms, may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0055] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0056] Where aspects of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members.

[0057] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0058] The compositions and methods disclosed herein may “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. As a result, the compositions and methods of the present disclosure that “comprise,” “have,” “include” or “contain” one or more elements possesses those one or more elements but are not limited to possessing only those one or more elements. Likewise, an element of a composition or method of the present disclosure that “comprises,” “has,” “includes” or “contains” one or more features possesses those one or more features but is not limited to possessing only those one or more features.

[0059] It is contemplated that aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.” Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of’ (and any form of consisting of, such as “consist of’ and “consists of’) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0060] Reference throughout this specification to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” “a further aspect,” “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” “a further embodiment,”or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect or embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect or embodiment. Furthermore, the particular features, structures, and / or characteristics may be combined in any suitable manner in one or more aspects or embodiments.

[0061] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0062] The terms “increase,” improve,” “decrease” or “reduce” refer to values that are relative to a baseline measurement, such as a measurement in a reference, standard, or control. As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and / or determined substantially simultaneously and / or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and / or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.

[0063] The term “identity” or “identical to” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules or RNA molecules) or between polypeptide molecules. “Identity” measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model of computer programs (e.g., algorithms), which are well known in the art. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. In some aspects, the degree of similarity or identity is given for a region that is at least, at most, exactly, between (inclusive or exclusive) any two of, or about 50%, 60%, 70%, 80%, 90%, or 100% of the entire length of the reference sequence. For example, if the reference sequence consists of 200 residues, the degree of identity is given for at least, at most, exactly, between (inclusive or exclusive) any two of, or about 100, 120, 140, 160, 180, or 200 residues,in some aspects, continuous residues. In some aspects, the degree of similarity or identity is given for the entire length of the reference sequence.

[0064] A “polypeptide” or “protein” (used interchangeably herein) refers to a chain of amino acids of any length. The chain may be linear or branched. The chain may comprise one or more of modified amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, and / or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing polypeptides can be excluded from the polypeptides of the disclosure. Polypeptides may be a single molecule or may be a multi- molecular complex such as a dimer, trimer or tetramer. A protein comprises one or more peptides or polypeptides, and may be folded into a 3-dimensional form, which may be required for the protein to exert its biological function.

[0065] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O -phospho serine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0066] A “polynucleotide” or “nucleic acid,” (used interchangeably herein) refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases or their analogs, or any substrate, which are synthetic, naturally occurring, and non-naturally occurring, and that can be metabolized in a manner similar to the reference nucleotides, e.g., have similar bindingproperties as the reference nucleic acid and / or are incorporated into a chain by DNA or RNA polymerase. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-0-methyl ribonucleotides, peptide-nucleic acids (PNAs). Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single- stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid may be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0067] As used herein, the term “gene” refers to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post- translational modification, and / or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or a substantially similar polypeptide.

[0068] As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some aspects, a gene product may be a transcript. In some aspects, a gene product may be a polypeptide. In some aspects, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein. In some aspects,1, 2, 3, or more of the foregoing steps can be excluded from expression of nucleic acid sequences of the disclosure.

[0069] “Pseudotype” refers to a virus particle, where the envelope or capsid includes heterologous viral proteins.

[0070] ‘ ‘Nucleic acid genome” refers to the genomic or nucleic acid component of a virus particle, which encodes the genome of the virus particle, including any proteins required for replication and / or integration of the genome, if required, and optionally a heterologous protein operably linked to a promoter, the promoter being either native to the protein or heterologous (viral or non-viral). The nucleic acid genome can be based on any virus, and have an RNA or DNA genome, either single stranded or double stranded. In some aspects, the nucleic acid genome is from the family Retroviridae. In some aspects, the nucleic acid genome is from the Lentiviral genus of the family Retroviridae.

[0071] Nucleic acids encoding polypeptide-encoding gene(s) (e.g., a mutated viral envelope protein and / or a targeting moiety) may be comprised in a vector. A “vector” refers to a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest (e.g., an antibody-encoding gene) in a host cell. Examples of vectors include, but are not limited to viral vectors, e.g., retroviral vectors and retroviruses comprising such vectors, and may include naked nucleic acids, or may include nucleic acids associated with delivery-aiding materials (e.g., cationic condensing agents, liposomes, etc.). Vectors may include DNA or RNA. An “expression vector” as used herein refers to a vector that includes at least one polypeptide-encoding gene (e.g., a mutated viral envelope protein and / or a targeting moiety), and optionally, at least one regulatory element (e.g., promoter sequence, poly(A) sequence) relating to the transcription or translation of the gene. The same nucleic acid may encode both the viral genome and the polypeptide-encoding gene(s). Alternatively, the viral genome and the polypeptide-encoding gene(s) may be encoded by different nucleic acids.

[0072] “Targeting moiety” refers to a heterologous protein that binds to a protein on the cell surface of a selected cell type. Representative targeting moieties include one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, antibodies, receptor ligands, or a combination thereof. An “antigen” or “antigenic molecule” refers broadly to target molecules that are specifically recognized by an antibody, including fragments or mimics thereof, or targeting moiety disclosed herein.

[0073] A viral “envelope” protein, or “Env” protein, as used herein, refers to any polypeptide sequence that resides on the surface lipid bilayer of a viral virion whose function is to mediate the adsorption to and the penetration of host cells susceptible to infection. A viralenvelope is formed by a cell-derived lipid bilayer into which proteins encoded by the env region of the viral genome are inserted. Envelope proteins are typically glycoproteins and usually comprise a transmembrane (TM) and a surface (SU) component linked together by disulfide bonds. Virus structure is described in detail in, for example, Coffin, et al., RETROVIRUSES, 1997, Cold Spring Harbor Laboratory Press.

[0074] A viral “capsid,” as used herein, refers to the principal structural protein of the virion core derived from the central region of the Gag polyprotein. The capsid protein in a mature viral particle forms a shell surrounding the ribonucleoprotein complex that contains the genomic nucleic acid. This shell, which includes additional proteins, is also referred to as a capsid. A capsid shell can exist as a component of a virion without surrounding a genomic nucleic acid.

[0075] A “virion” refers to a virus body, including the outer lipid bilayer which surrounds a capsid shell which in turn surrounds a genomic nucleic acid, when present. A virion of the disclosure, can, but need not, have a genomic nucleic acid.

[0076] A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.

[0077] The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid can be recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature e.g., a fusion protein).

[0078] An “isolated” virus refers to a virus that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., species, cell from which it is expressed, library, etc., (3) is expressed by a cell from a different species, or (4) does not occur in nature. For example, a virus naturally present in a living cell or animal is not “isolated,” but a synthetic virus, or a virus partially or completely separated from thecoexisting materials of its natural state, is “isolated.” Thus, a virus that is chemically synthesized, or expressed in a cellular system different from the system from which it naturally originates, will be “isolated” from its naturally associated components. A virus also may be rendered substantially free of naturally associated components by isolation, using harvesting and purification techniques well known in the art, from cells transduced with the virus. An “isolated virus” or “isolated pseudotyped virus” may exist in substantially purified form, or may exist in a non-native environment such as, for example, a cell into which the virus has been delivered.

[0079] ‘ ‘Individual, “subject,” and “patient” are used interchangeably and can refer to any animal, including mammals. Mammals according to aspects include canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, humans, and the like, and encompass mammals in utero. In an aspect, humans are suitable subjects. Human subjects may be of any gender and at any stage of development. In some aspects, a subject is a patient with an infection or cancer.

[0080] As used herein, “treat,” “treating,” or “treatment” or equivalent terminology refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change, condition, or disorder, such as the growth, development, or spread of an infection or cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of the disease or condition, stabilized (z.e., not worsening) state of the disease or condition, delay or slowing of progression of the disease or condition, amelioration or palliation of the disease or condition, and / or remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or condition as well as those prone to have the disease or condition or those in which the disease or condition is to be prevented. The results of treatment can be determined by methods known in the art, such as determination of reduction of infectious agent load, determination of reduction of tumor burden, determination of restoration of function, or other methods known in the art.

[0081] The terms “prevent” or “prevention” refer to one or more of delay of onset, reduction in frequency, and / or reduction in severity of at least one sign or symptom of a particular disease, disorder, or condition (e.g., an infection or cancer). In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder, or condition if a statistically significant decrease in thedevelopment, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete when onset of disease, disorder, or condition has been delayed for a predefined period of time.

[0082] As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Pharmaceutical compositions may be immunogenic compositions. In some aspects, active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some aspects, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous and / or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation.

[0083] The term “therapeutically effective amount” refers to the amount of active ingredient that elicits the biological or medicinal response in a tissue, system, animal, individual, and / or human that is being sought by a researcher, veterinarian, medical doctor, and / or other clinician, which may include one or more of the following: (1) preventing the disease or condition; for example, preventing a disease, condition, or disorder in an individual that may be predisposed to the disease, condition, or disorder but does not yet experience and / or display the pathology and / or symptomatology of the disease or condition; (2) inhibiting the disease or condition; for example, inhibiting a disease, condition, or disorder in an individual that is experiencing and / or displaying the pathology and / or symptomatology of the disease, condition, or disorder (z.e., arresting and / or slowing further development of the pathology and / or symptomatology); and / or (3) ameliorating the disease or condition in an individual that is experiencing and / or displaying the pathology and / or symptomatology of the disease, condition, or disorder (z.e., reversing the pathology and / or symptomatology).

[0084] The term “sample,” as used herein, refers to samples for use in the methods described herein and may be obtained using any method known to the art that can provide a sample suitable for the methods described herein. The methods of obtaining a sample from a subject provided herein may include, for example, obtaining tissue, cells, or biological material by scraping, swabbing, biopsying, or otherwise collecting the tissue, cells, or biological material. A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. General methods for obtaining biological samples arealso known in the art. For example, publications such as Ramzy, Ibrahim, Clinical Cytopathology and Aspiration Biopsy (2001), which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. In certain aspects, the sample is obtained from sources including, but not limited to, urine, blood, serum, plasma, sputum, mucus, saliva, exhaled breath condensate, bronchoalveolar lavage fluid, cerebrospinal fluid, sweat, tissue, hair follicle, buccal tissue, tears, menses, or feces. In specific aspects, the sample is obtained from serum. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional. In some aspects of the present methods, the biological sample is obtained from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained after the subject, a medical professional, or a third party acquires and sends the biological sample to a third party. In some cases, the third party may provide suitable containers, and excipients for storage and transport of the biological sample to the third party.II. Pseudotyped Viral Envelopes

[0085] The disclosure provides for transduction of B cells with a heterologous gene by contacting the B cells with a nucleic acid encoding a pseudotyped viral envelope. The nucleic acid comprising the pseudotyped viral envelope may be comprised in a viral vector. The nucleic acid comprising the pseudotyped viral envelope may be comprised in a viral genome. The pseudotyped viral envelope may be derived from any enveloped virus. In specific aspects, the pseudotyped viral envelope is a pseudotyped retroviral (e.g., oncoretro viral, gammaretroviral, lentiviral) envelope. The viral vector comprising a nucleic acid encoding pseudotyped retroviral (e.g., oncoretroviral, gammaretroviral, lentiviral) envelope may be a retroviral (e.g., oncoretroviral, gammaretroviral, lentiviral) vector. The viral genome comprising a nucleic acid encoding pseudotyped retroviral (e.g., oncoretroviral, gammaretroviral, lentiviral) envelope may be a retroviral (e.g., oncoretroviral, gammaretroviral, lentiviral) genome. The retroviral (e.g., oncoretroviral, gammaretroviral, lentiviral) vector or retroviral (e.g., oncoretroviral,gammaretroviral, lentiviral) genome may be comprised in a retrovirus (e.g., oncoretrovirus, gammaretrovirus, lentivirus).

[0086] Enveloped viruses express envelope glycoproteins that enable the infection of host cells by mediating fusion between the viral envelope and host cell membranes. For many enveloped viruses, the endoplasmic reticulum (ER) of the host cell is used to support viral entry, replication, and / or assembly. Exemplary envelope virus classes include measles viruses, herpes viruses, lentiviruses, alpha viruses, pox viruses, and vaccinia viruses. Exemplary enveloped viruses include, for example, Sindbis virus and vesicular stomatitis virus.

[0087] The enveloped viruses of the disclosure may comprise viruses that do not normally cause disease or other negative effects in the infected host or cell. Alternatively, the virus may be an attenuated virus, i.e., a virus which has mutated or which has been engineered or otherwise treated such that it does not support pathogenic infection of healthy or nontarget cells.

[0088] In some aspects, the enveloped virus can be derived from the Lentivirus genus. In some aspects, the enveloped virus can be derived from HIV, SINV, or FIV. In further aspects, the enveloped virus can be derived from the Sindbis virus (SINV) or vesicular stomatitis virus (VSV) in the Lentivirus genus.

[0089] Lentiviruses are a type of retrovirus that belong to the family of Lentiviridae. They are characterized by their ability to infect and replicate in non-dividing cells, such as neurons, macrophages, and dendritic cells. Lentiviruses have a single-stranded RNA genome that is reverse transcribed into DNA before integrating into the host cell genome. Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle thereof, as in the course of latent infection. As such, lentiviruses can be a useful tool for genetic engineering and gene therapy. Lentiviral vectors can be used to deliver genes into target cells for therapeutic purposes. These vectors can be derived from lentiviruses and have been modified to remove the viral genes necessary for replication while retaining the ability to integrate into the host cell genome.A. Sindbis Virus Envelopes

[0090] A typical lentivirus is the Sindbis virus (SINV). SINV is a positive-sense singlestranded RNA virus and is a member of the Alphavirus genus within the Togaviridae family. It was first isolated in 1952 from mosquitoes in Egypt and has since been found in many partsof the world, including Africa, Europe, and Asia. SINV is an arbovirus, meaning that it is transmitted to humans and animals through the bite of infected mosquitoes. Alphaviruses are a small group of RNA viruses that are enveloped by a host cell-derived lipid membrane. In contrast to the loose and unorganized envelopes of the orthomyxovirus and paramyxovirus families, the envelope of alphaviruses is a highly organized icosahedral structure of transmembranal virus glycoproteins and a lipid bilayer.

[0091] SINV is a small virus, with a genome of approximately 11.7 kilobases in length. The genome contains two open reading frames (ORFs), which encode the structural and non- structural proteins of the virus. The structural proteins include the capsid, envelope glycoproteins El, E2, and E3, and the membrane protein. These proteins are responsible for viral entry into host cells, assembly of new viral particles, and release of mature virions.

[0092] Sindbis virus is composed of a 49S positive-sense RNA genome and 240 copies each of three structural proteins: two transmembranal glycoproteins (El and E2) and an internal capsid (C) protein. The structural proteins assemble into two concentric icosahedral shells arranged about the genome in a T=4 icosahedral lattice. The inner lattice is the nucleocapsid, which has an outer radius of approximately 200 A and consists of 240 copies of capsid protein. The envelope extends from an inner radius of 200 A to an outer radius of 325 A and is composed of 80 trimers of E1 / E2 heterodimers which are embedded in a lipid bilayer. The (El / E2)3 heterotrimers protrude 50 A from the surface of the virus and flare out into three distinct lobes forming a trimer 230 A in diameter.

[0093] The structural proteins are translated from a 26S subgenomic RNA as a 130- kDa precursor protein that is subsequently processed into its individual components. The 30- kDa capsid protein, the first structural protein to be translated, is autocatalytically cleaved from the nascent polypeptide shortly after synthesis. After release from the ribosome translation complex, the capsid protein condenses onto newly synthesized genomic RNA to form nucleocapsids in the cytoplasm of the infected cell. The cleavage of capsid from the growing polypeptide chain exposes a signal sequence that delivers the polypeptide to the endoplasmic reticulum, where protein synthesis continues and glycosylation begins. Translation of protein from the mRNA into the rough endoplasmic reticulum produces PE2, the precursor of E2, and El spike proteins, which associate rapidly into PE2 / E1 heterodimers and then almost immediately into (PE2 / E1)3 hetero trimers. PE2 contains a 64-amino-acid-long E3 sequence preceding the 423-amino-acid-long E2 glycoprotein. The cleavage between E3 and E2 occurs in a post-Golgi compartment prior to the delivery of the spike trimers to the plasma membrane.This cleavage, mediated by a cellular protease, occurs at a position immediately after a four- amino-acid motif characterized as basic-X-basic -basic.

[0094] The El protein is a type I transmembrane protein that is composed of three domains: a large N-terminal ectodomain, a transmembrane domain, and a short C-terminal cytoplasmic tail. The ectodomain contains three disulfide bonds that stabilize the protein structure and two fusion loops that mediate fusion of the viral and cellular membranes. The El protein undergoes a conformational change during viral entry that exposes the fusion loops and enables the protein to interact with the host cell membrane. This triggers a series of events that lead to fusion of the viral and cellular membranes and release of the viral genome into the host cell. The E2 protein is a type I transmembrane protein that consists of three domains: an N- terminal domain, a central domain, and a C-terminal domain. The N-terminal domain contains a beta-barrel structure that is responsible for binding to host cell receptors. The central domain is a flexible hinge region that allows the protein to undergo conformational changes during viral entry. The C-terminal domain is a globular domain that is involved in the formation of the viral envelope. The E2 protein is responsible for binding the virus to host cells and initiating the process of viral entry. It also plays a role in determining the host range of the virus. The E3 protein is a small, 64-amino-acid protein that is useful for virus assembly and budding. It is located in the endoplasmic reticulum (ER) and plays a role in the folding and trafficking of the E2 glycoprotein. E3 also interacts with the host cell cytoskeleton, which is important for virus release. The E3 protein has been shown to be involved in a number of other functions, including: Inhibiting the host immune response: E3 can inhibit the production of interferons, which are proteins that help to protect the host from infection; promoting cell death: E3 can promote the death of infected cells, which can help to clear the virus from the body; and evasive mutagenesis to help the virus to evade the host immune response by mutating its genes.

[0095] The non- structural proteins of SINV are involved in viral RNA replication and other aspects of viral replication. These proteins include nsPl, nsP2, nsP3, and nsP4. NsP4 is the RNA-dependent RNA polymerase responsible for viral RNA synthesis.

[0096] SINV infects a wide range of vertebrate and invertebrate hosts, including humans, birds, rodents, and mosquitoes. In humans, SINV infection can cause a febrile illness known as Sindbis fever. Symptoms of Sindbis fever typically include fever, rash, joint pain, and muscle pain. In rare cases, more severe complications, such as encephalitis, may occur.

[0097] SINV has been used as a model system for understanding virus-host interactions, viral replication, and immune responses to viral infection. It has also been used as a vector for gene therapy and vaccine development. In some aspects, SINV can infect a widerange of cell types, produce high levels of transgene expression, and exhibit low pathogenicity in humans. In some aspects, SINV vectors are used to deliver therapeutic genes to treat cancer, genetic disorders, and other diseases.

[0098] The pseudotyped viral envelope used to transduce B cells of the disclosure may be derived from a lentiviral envelope (e.g., a SINV envelope). Structurally, at least one of the El, E2, and / or E3 proteins has one or more mutations in comparison to a wild type sequence of the El, E2, and / or E3 proteins to provide altered titer, specificity, specificity index, tropism, or host immune reaction. Combinations of mutated El, E2, and / or E3 are also contemplated. Functionally, pseudotyped viral envelopes of the present disclosure have a decreased ability to bind to endogenous cognate receptors, including glycosaminoglycans (e.g., heparin sulfate). In certain aspects, lentiviruses comprising a nucleic acid encoding the pseudotyped viral envelope, whether in a vector or the viral genome, are isolated.

[0099] In specific aspects, the pseudotyped viral envelope used to transduce B cells of the disclosure is derived from a SINV envelope comprising Sindbis El, E2, and / or E3 proteins. As used herein, the terms “Sindbis El protein,” “Sindbis E2 protein,” and “Sindbis E3 protein” or a nucleic acid encoding “Sindbis El protein,” “Sindbis E2 protein,” and “Sindbis E3 protein” refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have a nucleotide sequence that has at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater, nucleotide sequence identity, over a region of at least, at most, exactly, or between (inclusive or exclusive) any two of 25, 50, 100, 200, 500, 1000, or more nucleotides, and up to the full length sequence, of the nucleotide sequence of Sindbis El protein, Sindbis E2 protein, and / or Sindbis E3 protein; (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against an immunogen comprising Sindbis El protein, Sindbis E2 protein, and / or Sindbis E3 protein, and modified variants having at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater, sequence identity thereto; (3) specifically hybridize under stringent hybridization conditions to an anti- sense strand corresponding to a nucleic acid sequence of Sindbis El protein, Sindbis E2 protein, and / or Sindbis E3 protein and modified variants having at least, at most, exactly, or between (inclusive or exclusive) any two of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater, sequence identity thereto; and / or (4) encode a protein having an amino acid sequence that has at least, at most, exactly, or between (inclusive or exclusive) any two of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or greater, aminoacid sequence identity, over a region of at least, at most, exactly, or between (inclusive or exclusive) any two of 25, 50, 100, 200, 500, 1000, or more amino acids, and up to the full length sequence, of the amino acid sequence of Sindbis El protein, Sindbis E2 protein, and / or Sindbis E3 protein. The nucleic acids and proteins of the disclosure include both naturally occurring or recombinant molecules, as well as point mutations, including randomly generated point mutations and those generated by site-directed mutagenesis.

[0100] Sindbis El protein, Sindbis E2 protein, and Sindbis E3 protein are encoded by a polyprotein, the amino acid sequence of which is provided, e.g., by the following sequences, where the residues corresponding to Sindbis El protein are bolded, residues corresponding to Sindbis E2 protein are italicized, and residues corresponding to Sindbis E3 protein are underlined:

[0101] MNRGFFNMLGRRPFPAPTAMWRPRRRRQAAPMPARNGLASQIQ QLTTAVSALVIGQATRPQPPRPRPPPRQKKQAPKQPPKPKKPKTQEKKKKQPA KPKPGKRQRMALKLEADRLFDVKNEDGDVIGHALAMEGKVMKPLHVKGTIDH PVLSKLKFTKSSAYDMEFAQLPVNMRSEAFTYTSEHPEGFYNWHHGAVQYSGG RFTIPRGVGGRGDSGRPIMDNSGRVVAIVLGGADEGTRTALSVVTWNSKGKTIK TTPEGTEEWSAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEA YDTLLNATLRCGSSGRSKRSVIDDFTLTSPYLGTCSYCHHTVPCFSPVKIEQVWDEADD NTIRIQTSAQFGYDQSGAASANKYRYMSLKQDHTVKEGTMDDIKISTSGPCRRLSYKGYFL LAKCPPGDSVTVSIVSSNSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLKETTA GYITMHRPRPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQ CVAYKSDQTKWVFNSPDLIRHDDHTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISL QLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAP GDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPN AWPTSLALLCCVR&4AAETFTETMSYEWSNS0PFFWVQECIPEAAFIVEMRCCSCCEPF EVVAGAYEAKVDAYEHATTVPNVPQIPYKAEVERAGYAPENEEITVMSSEVEPSTN OEYITCKFTTVVPSPKIKCCGSLECOPAAHADYTCKVFGGVYPFMWGGAQCFCDSE NSOMSEAYVEESADCASDHAQAIKVHTAAMKVGERIVYGNTTSFEDVYVNGVTPG TSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIOATSLTSKD LIASTDIRLLKPSAKNVHVPYTOASSGFEMWKNNSGRPLOETAPFGCKIAVNPLRAV DCSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLOYVSDREGOC PVHSHSSTATLOESTVHVLEKGAVTVHFSTASPOANFIVSLCGKKTTCNAECKPPAD HIVSTPHKNDOEFQAAISKTSWSWEFAEFGGASSEEIIGEMIFACSMMETSTRR (SEQ ID NO:1) (UniProt Accession No. P03316).

[0102] MNRGFFNMLGRRPFPAPTAMWRPRRRRQAAPMPARNGLASQIQ QLTTAVSALVIGQATRPQPPRPRPPPRQKKQAPKQPPKPKKPKTQEKKKKQPA KPKPGKRQRMALKLEADRLFDVKNEDGDVIGHALAMEGKVMKPLHVKGTIDH PVLSKLKFTKSSAYDMEFAQLPVNMRSEAFTYTSEHPEGFYNWHHGAVQYSGG RFTIPRGVGGRGDSGRPIMDNSGRVVAIVLGGADEGTRTALSVVTWNSKGKTIK TTPEGTEEWSAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEA YDTLLNATLRCGSSGRSKRSVIDDFTLTSPYLGTCSYCHHTVPCFSPVKIEQVWDEADD NTIRIQTSAQFGYDQSGAASANKYRYMSLKQDHTVKEGTMDDIKISTSGPCRRLSYKGYFL LAKCPPGDSVTVSIVSSNSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLKETTA GYITMHRPRPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQ CVAYKSDQTKWVFNSPDLIRHDDHTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISL QLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAP GDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPN AWPrSZALLCCVRSAAAETFTETMSYLWSNSOPFFWVQLCIPLAAFIVLMRCCSCCLPF LVVAGAYLAKVDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTN OEYITCKFTTVVPSPKIKCCGSLECOPAAHADYTCKVFGGVYPFMWGGAQCFCDSE NSOMSEAYVELSADCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPG TSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIOATSLTSKD LIASTDIRLLKPSAKNVHVPYTOASSGFEMWKNNSGRPLOETAPFGCKIAVNPLRAV DCSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLOYVSDREGOC PVHSHSSTATLOESTVHVLEKGAVTVHFSTASPOANFIVSLCGKKTTCNAECKPPAD HIVSTPHKNDOEFQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR (SEQ ID NO:2) (UniProt Archive Identifier No. UPI0000131EC1; Protein Information Resource Entry No. VHWVB).

[0103] MNRGFFNMLGRRPFPAPTAMWRPRRRRQAAPMPARNGLASQIQ QLTTAVSALVIGQATRPQPPRPRPPPRQKKQAPKQPPKPKKPKTQEKKKKQPA KPKPGKRQRMALKLEADRLFDVKNEDGDVIGHALAMEGKVMKPLHVKGTIDH PVLSKLKFTKSSAYDMEFAQLPVNMRSEAFTYTSEHPEGFYNWHHGAVQYSGG RFTIPRGVGGRGDSGRPIMDNSGRVVAIVLGGADEGTRTALSVVTWNSKGKTIK TTPEGTEEWSAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEA YDTLLNATLRCGSSGRSKRSVIDGFTLTSPYLGTCSYCHHTEPCFSPVKIEQVWDEADD NTIRIQTSAQFGYDQSGAASANKYRYMSLKQDHTVKEGTMDDIKISTSGPCRRLSYKGYFL LAKCPPGDSVTVSIVSSNSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLKETTA GYITMHRPRPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCVAYKSDQTKWVFNSPDLIRHDDHTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISL QLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAP GDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPN AVZPTSZALLCCVRSAVAETFTETMSYLWSNSQPFFWVQLCIPLAAFIVLMRCCSCCLPF LVVAGAYLAKVDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTN QEYITCKFTTVVPSPKIKCCGSLECQPAAHADYTCKVFGGVYPFMWGGAQCFCDSE NSQMSEAYVELSAVCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPG TSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIOATSLTSKD LIASTDIRLLKPSAKNVHVPYTOASSGFEMWKNNSGRPLOETAPFGCKIAVNPLRAV DCSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLQYVSDREGOC PVHSHSSTATLOESTVHVLEKGAVTVHFSTASPOANFIVSLCGKKTTCNAECKPPAD HIVSTPHKNDQEFQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR (SEQ ID NOG) (UniProt Archive Identifier No. UPI00001710C3; Protein Information Resource Entry No. VHWVB2).

[0104] The nucleic acid sequence encoding Sindbis El protein, Sindbis E2 protein, and Sindbis E3 protein is provided, e.g., in Rice & Strauss, Proc. Natl. Acad. Sci. USA 78(4):2062- 2066 (April 1981), and Strauss, Rice, & Strauss, Virology 133:92-110 (1984), both of which are incorporated by reference herein in their entirety.

[0105] In other aspects, other Togaviridae family envelopes, e.g., from the Alphavirus genus, e.g., Semliki Forest Virus, Ross River Virus and equine encephalitis virus, can also be used to pseudotype the viral envelopes of the disclosure. The envelope protein sequences for such Alphaviruses are known in the art and incorporated by reference herein.

[0106] In some aspects, the pseudotyped viral envelope, e.g., a pseudotyped Sindbis envelope, comprises a protein binding domain. In some aspects, the protein binding domain is a ZZ domain, which is the IgG Fc-binding peptide derived from Staphylococcus aureus protein A, as an adaptor molecule inserted into or fused with the virus envelope protein, e.g., Sindbis E2 protein, to permit virus binding and entrance to cells bearing specific cell surface antigens in the presence of the appropriate monoclonal antibody. As an example, an amino acid and a nucleotide sequence encoding a pseudotyped Sindbis viral envelope including a ZZ domain (in bold; SEQ ID NOs:25 and 26) (i.e., a Sindbis ZZ envelope), is provided, e.g., by:

[0107] MASAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEAY DTLLNAILRCGSSGRSKRSVIDDFTLTSPYLGTCSYCHHTVPCFSPVKIEQVWDEADD NTIRIQTSAQFGYDQSGAASANKYRYMSLKQVTDNKFNKEQQNAFYEILHLPNLN EEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDANSSSVPGDPVTTV KEGTMDDIKISTSGPCRRLSYKGYFLLAKCPPGDSVTVSIVSSNSATSCTLARKIKPKF VGREKYDLPPVHGKKIPCTVYDRLKETTAGYITMHRPRPHAYTSYLEESSGKVYAKP PSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCVAYKSDQTKWVFNSPDLIRHDD HTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISLQLDTDHLTLLTTRRLGANPEP TTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAPGDPHGWPHEIVQHYYH RHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPNAVIPTSLALLCCV RSANAETFTETMSYLWSNSQPFFWVQLCIPLAAFIVLMRCCSCCLPFLVVAGAYLAK VDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTNQEYITCKFTTVV PSPKIKCCGSLECQPAAHADYTCKVFGGVYPFMWGGAQCFCDSENSQMSEAYVELS ADCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPGTSKDLKVIAGPIS ASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIQATSLTSKDLIASTDIRLLKPS AKNVHVPYTQASSGFEMWKNNSGRPLQETAPFGCKIAVNPLRAVDCSYGNIPISIDIP NAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLQYVSDREGQCPVHSHSSTATLQ ESTVHVLEKGAVTVHFSTASPQANFIVSLCGKKTTCNAECKPPADHIVSTPHKNDQE FQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR (SEQ ID N0:4).

[0108] ATGGCGTCCGCAGCACCACTGGTCACGGCAATGTGTTTGCTCGGAA ATGTGAGCTTCCCATGCGACCGCCCGCCCACATGCTATACCCGCGAACCTTCCAG AGCCCTCGACATCCTTGAAGAGAACGTGAACCATGAGGCCTACGATACCCTGCT CAATGCCATATTGCGGTGCGGATCGTCTGGCAGAAGCAAAAGAAGCGTCATTGA CGACTTTACCCTGACCAGCCCCTACTTGGGCACATGCTCGTACTGCCACCATACT GTACCGTGCTTCAGCCCTGTTAAGATCGAGCAGGTCTGGGACGAAGCGGACGAT AACACCATACGCATACAGACTTCCGCCCAGTTTGGATACGACCAAAGCGGAGCA GCAAGCGCAAACAAGTACCGCTACATGTCGCTTAAGCAGGTAACCGACAACAAA TTCAACAAAGAACAACAAAACGCGTTCTATGAGATCTTACATTTACCTAACT TAAACGAAGAACAACGAAACGCCTTCATCCAAAGTTTAAAAGATGACCCAAG CCAAAGCGCTAACCTTTTAGCAGAAGCTAAAAAGCTAAATGATGCTCAGGC GCCGAAAGTAGACAACAAATTCAACAAAGAACAACAAAACGCGTTCTATGA GATCTTACATTTACCTAACTTAAACGAAGAACAACGAAACGCCTTCATCCAA AGTTTAAAAGATGACCCAAGCCAAAGCGCTAACCTTTTAGCAGAAGCTAAAA AGCTAAATGATGCTCAGGCGCCGAAAGTAGACGCGAATTCGAGCTCGGTAC CCGGGGATCCGGTAACCACCGTTAAAGAAGGCACCATGGATGACATCAAGATT AGCACCTCAGGACCGTGTAGAAGGCTTAGCTACAAAGGATACTTTCTCCTCGCAA AATGCCCTCCAGGGGACAGCGTAACGGTTAGCATAGTGAGTAGCAACTCAGCAACGTCATGTACACTGGCCCGCAAGATAAAACCAAAATTCGTGGGACGGGAAAAATATGATCTACCTCCCGTTCACGGTAAAAAAATTCCTTGCACAGTGTACGACCGTCTGAAAGAAACAACTGCAGGCTACATCACTATGCACAGGCCGAGACCGCACGCTTATACATCCTACCTGGAAGAATCATCAGGGAAAGTTTACGCAAAGCCGCCATCTGGGAAGAACATTACGTATGAGTGCAAGTGCGGCGACTACAAGACCGGAACCGTTTCGACCCGCACCGAAATCACTGGTTGCACCGCCATCAAGCAGTGCGTCGCCTATAAGAGCGACCAAACGAAGTGGGTCTTCAACTCACCGGACTTGATCAGACATGACGACCACACGGCCCAAGGGAAATTGCATTTGCCTTTCAAGTTGATCCCGAGTACCTGCATGGTCCCTGTTGCCCACGCGCCGAATGTAATACATGGCTTTAAACACATCAGCCTCCAATTAGATACAGACCACTTGACATTGCTCACCACCAGGAGACTAGGGGCAAACCCGGAACCAACCACTGAATGGATCGTCGGAAAGACGGTCAGAAACTTCACCGTCGACCGAGATGGCCTGGAATACATATGGGGAAATCATGAGCCAGTGAGGGTCTATGCCCAAGAGTCAGCACCAGGAGACCCTCACGGATGGCCACACGAAATAGTACAGCATTACTACCATCGCCATCCTGTGTACACCATCTTAGCCGTCGCATCAGCTACCGTGGCGATGATGATTGGCGTAACTGTTGCAGTGTTATGTGCCTGTAAAGCGCGCCGTGAGTGCCTGACGCCATACGCCCTGGCCCCAAACGCCGTAATCCCAACTTCGCTGGCACTCTTGTGCTGCGTTAGGTCGGCCAATGCTGAAACGTTCACCGAGACCATGAGTTACTTGTGGTCGAACAGTCAGCCGTTCTTCTGGGTCCAGTTGTGCATACCTTTGGCCGCTTTCATCGTTCTAATGCGCTGCTGCTCCTGCTGCCTGCCTTTTTTAGTGGTTGCCGGCGCCTACCTGGCGAAGGTAGACGCCTACGAACATGCGACCACTGTTCCAAATGTGCCACAGATACCGTATAAGGCACTTGTTGAAAGGGCAGGGTATGCCCCGCTCAATTTGGAGATCACTGTCATGTCCTCGGAGGTTTTGCCTTCCACCAACCAAGAGTACATTACCTGCAAATTCACCACTGTGGTCCCCTCCCCAAAAATCAAATGCTGCGGCTCCTTGGAATGTCAGCCGGCCGCTCATGCAGACTATACCTGCAAGGTCTTCGGAGGGGTCTACCCCTTTATGTGGGGAGGAGCGCAATGTTTTTGCGACAGTGAGAACAGCCAGATGAGTGAGGCGTACGTCGAATTGTCAGCAGATTGCGCGTCTGACCACGCGCAGGCGATTAAGGTGCACACTGCCGCGATGAAAGTAGGACTGCGTATTGTGTACGGGAACACTACCAGTTTCCTAGATGTGTACGTGAACGGAGTCACACCAGGAACGTCTAAAGACTTGAAAGTCATAGCTGGACCAATTTCAGCATCGTTTACGCCATTCGATCATAAGGTCGTTATCCATCGCGGCCTGGTGTACAACTATGACTTCCCGGAATATGGAGCGATGAAACCAGGAGCGTTTGGAGACATTCAAGCTACCTCCTTGACTAGCAAGGATCTCATCGCCAGCACAGACATTAGGCTACTCAAGCCTTCCGCCAAGAACGTGCATGTCCCGTACACGCAGGCCTCATCAGGATTTGAGATGTGGAAAAACAACTCAGGCCGCCCACTGCAGGAAACCGCACCTTTCGGGTGTAAGATTGCAGTAAATCCGCTCCGAGCGGTGGACTGTTCATACGGGAACATTCCCATTTCTAT TGACATCCCGAACGCTGCCTTTATCAGGACATCAGATGCACCACTGGTCTCAACA GTCAAATGTGAAGTCAGTGAGTGCACTTATTCAGCAGACTTCGGCGGGATGGCC ACCCTGCAGTATGTATCCGACCGCGAAGGTCAATGCCCCGTACATTCGCATTCGA GCACAGCAACTCTCCAAGAGTCGACAGTACATGTCCTGGAGAAAGGAGCGGTGA CAGTACACTTTAGCACCGCGAGTCCACAGGCGAACTTTATCGTATCGCTGTGTGG GAAGAAGACAACATGCAATGCAGAATGTAAACCACCAGCTGACCATATCGTGAG CACCCCGCACAAAAATGACCAAGAATTTCAAGCCGCCATCTCAAAAACATCATG GAGTTGGCTGTTTGCCCTTTTCGGCGGCGCCTCGTCGCTATTAATTATAGGACTTA TGATTTTTGCTTGCAGCATGATGCTGACTAGCACACGAAGATGA (SEQ ID N0:5)

[0109] In some aspects, the ZZ domain has at least, at most, exactly, between (inclusive or exclusive) any two of, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, with SEQ ID NOs:25 or 26. In some aspects, the ZZ domain comprises any one of SEQ ID NOs:25 or 26. In some aspects, the ZZ domain consists of SEQ ID NOs:25 or 26.

[0110] In some aspects, protein binding domain comprises a biotin binding sequence. The biotin binding sequence may be avidin, streptavidin, monomeric rhizavidin, or a monomeric streptavidin / rhizavidin hybrid. As examples, amino acid and nucleotide sequences encoding a pseudotyped Sindbis viral envelope including a biotin binding sequence (in bold, SEQ ID NOs:27-30), is provided, e.g., by:

[0111] Sindbis eMA envelope:

[0112] MASAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEAY DTLLNAILRCGSSGASVIDDFTLTSPYLGTCSYCHHTEPCFSPVKIEQVWDEADDNTIR IOTSAOFGYDOSGAASANKYRYMAAAAVTGGGGSLGVTSSGSGSGGGGGSGGGGSG GGGSASFDASNFKDFSSIASASSSWQNQHGSTMIIQVDSFGNVSGQYVNRAEGTG CQNSPYPLTGRVNGTFIDFSVKWNNSTENCNSNTQWTGYAQVNGNNTEIVTRW NLKYEGGSGPAIWOGODTFQYVPTTEIDAGGGGSGSGSGGGGGSSGSGSGGGGSG SGSGGGGGSLVTGGGGSVTTVKEGTMDDIKISTSGPCRRLSYKGYFLLAKCPPGDSV TVSIVSSNSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLA4TTAGYITMH RPGPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCV AYKSDQTKWVFNSPDLIRHDDHTVQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHIS LQLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYA QESAPGDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPNAVIPTSLALLCCVRSANAETFTETMSYLWSNSQPFFWVQLCIPLAAFI VLMRCCSCCLPFLVVAGAYLAKVDAYEHATTVPNVPQIPYKALVERAGYAPLNLEI TVMSSEVLPSTNQEYITCKFTTVVPSPKIKCCGSLECQPAAHAGYTCKVFGGVYPFMWGGAQCFCDSENSQMSEAYVELSADCASDHAQAIKVHTAAMKVGLRIVYGNTTSF LDVYVNGVTPGTSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIQATSLTSKDLIASTDIRLLKPSSGNVHVPYTQASSGFEMWKNNSGRPLQETAPFG CKIAVNPLRAVDCSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMAT LQYVSDREGQCPVHSHSSTATLQESTVHVLEKGAVTVHFSTASPQANFIVSLCGKKT TCNAECKPPADHIVSTPHKNDQEFQAAISKTSWSWLFALFGGASSLLIIGLMIFACSM MLTSTRR (SEQ ID NO:6).

[0113] ATGGCCTCTGCCGCCCCTCTGGTGACAGCCATGTGCCTGCTGGGCA ACGTGTCCTTCCCCTGCGACAGACCCCCTACCTGCTACACCAGAGAGCCCAGCAG AGCCCTGGACATCCTGGAAGAGAACGTGAACCACGAGGCCTACGACACCCTGCT GAACGCCATCCTGAGATGCGGCAGCTCTGGCAAGCGTGATCGACGACTTCACCCT GACCAGCCCCTACCTGGGCACCTGTAGCTACTGCCACCACACCGAGCCCTGCTTC AGCCCCGTGAAGATCGAACAGGTGTGGGACGAGGCCGACGATAACACCATCCGG ATCCAGACCAGCGCCCAGTTCGGCTACGATCAGAGCGGAGCCGCCAGCGCCAAC AAGTACCGGTACATGGCCGCTGCTGCCGTGACAGGCGGCGGAGGCAGCCTAGGC GTGACCAGCAGCGGCTCTGGATCTGGCGGAGGCGGAGGAAGTGGAGGGGGAGG AAGCGGCGGAGGGGGCTCTGCTAGCTTCGACGCCTCCAATTTCAAGGACTTCA GCAGCATCGCCTCTGCCTCTAGCTCCTGGCAGAACCAGCACGGCAGCACCA TGATCATCCAGGTGGACTCTTTTGGCAATGTGAGCGGACAGTACGTGAACAGGGCCGAGGGCACAGGATGCCAGAATTCCCCCTATCCTCTGACCGGCAGAGTGAACGGCACCTTCATCGACTTCAGCGTGAAGTGGAACAATTCTACCGAGAACTGTAATAGCAACACCCAGTGGACAGGCTACGCCCAAGTGAATGGCAACA ATACCGAGATCGTGACACGGTGGAACCTGAAGTACGAGGGAGGATCTGGAC CAGCAATCTGGCAGGGCCAGGACACATTCCAGTATGTGCCCACCACAGAAATCGATGCTGGCGGGGGAGGCTCTGGAAGTGGATCTGGGGGTGGCGGCGGAAGCTCTGGATCTGGAAGCGGAGGGGGAGGCAGCGGCAGTGGATCAGGGGGGGGAGGG GGATCTCTGGTAACCGGGGGAGGCGGAAGCGTGACCACCGTGAAAGAGGGCAC CATGGACGACATCAAGATCAGCACCAGCGGCCCCTGCAGAAGGCTTAGCTACAAAGGATATTTTCTGCTGGCCAAGTGTCCCCCTGGAGACAGCGTGACCGTGTCCATC GTGAGCTCCAACAGCGCCACATCCTGCACTCTGGCTCGGAAGATCAAGCCAAAG TTCGTGGGAAGAGAGAAGTACGACCTGCCACCCGTGCACGGCAAGAAAATCCCCTGTACCGTGTACGATAGGCTGGCCGCTACTACCGCCGGATATATTACAATGCACAGGCCAGGACCTCATGCTTACACCAGCTATCTGGAGGAATCTAGTGGCAAAGTGTACGCCAAGCCTCCATCAGGGAAAAACATCACCTACGAGTGCAAATGTGGGGATTATAAGACCGGAACAGTGAGCACTAGGACCGAAATCACCGGCTGCACAGCCATTAAGCAGTGTGTGGCTTATAAATCAGACCAGACAAAGTGGGTGTTCAATAGCCCTGATCTGATCCGCCACGACGATCATACTGTGCAGGGAAAACTGCACCTGCCCTTCAAGCTGATTCCCAGCACATGCATGGTGCCTGTGGCCCATGCTCCAAACGTGATCCACGGCTTCAAACATATTTCCCTGCAGCTGGACACTGATCACCTGACCCTGCTGACAACTAGGCGCCTGGGCGCTAATCCAGAGCCAACCACAGAATGGATCGTGGGGAAGACAGTGAGGAACTTTACTGTGGACCGCGATGGCCTGGAGTACATTTGGGGGAATCACGAACCAGTGCGGGTGTATGCTCAGGAGTCCGCTCCAGGAGACCCTCACGGATGGCCACATGAAATCGTGCAGCATTACTATCACAGACATCCCGTGTACACAATCCTGGCTGTGGCCAGCGCCACTGTGGCTATGATGATTGGCGTGACCGTGGCTGTGCTGTGCGCTTGTAAGGCGCGCAGAGAGTGCCTGACCCCCTACGCTCTGGCCCCTAACGCCGTGATCCCAACATCCCTGGCTCTGCTGTGCTGTGTGCGGTCTGCTAACGCCGAGACATTCACTGAAACCATGTCCTATCTGTGGAGTAATTCACAGCCCTTCTTTTGGGTGCAGCTGTGTATCCCTCTGGCCGCTTTCATTGTGCTGATGAGGTGCTGTTCTTGCTGTCTGCCTTTTCTGGTGGTGGCTGGCGCCTACCTGGCTAAAGTGGACGCCTATGAGCACGCTACTACCGTGCCAAATGTGCCCCAGATCCCTTACAAGGCCCTGGTGGAAAGGGCTGGATATGCTCCTCTGAACCTGGAGATTACCGTGATGTCAAGCGAAGTGCTGCCATCCACCAATCAGGAGTACATCACATGCAAGTTCACCACTGTGGTGCCTTCTCCAAAAATTAAGTGCTGTGGGAGTCTGGAATGCCAGCCCGCCGCTCATGCCGGATACACCTGTAAGGTGTTCGGGGGAGTGTATCCTTTTATGTGGGGCGGGGCCCAGTGCTTCTGTGATTCTGAGAATAGTCAGATGTCAGAAGCGTACGTCGAGCTGTCCGCCGATTGTGCCTCTGACCACGCCCAGGCCATCAAAGTGCACACCGCCGCCATGAAAGTGGGCCTGCGGATCGTGTACGGCAACACAACCAGCTTTCTGGACGTGTACGTGAACGGCGTGACCCCCGGCACCAGCAAGGACCTGAAAGTGATCGCCGGACCCATCAGCGCCAGCTTCACCCCCTTCGACCACAAGGTGGTGATCCACCGGGGCCTGGTGTACAACTACGACTTCCCCGAGTACGGCGCCATGAAGCCTGGCGCCTTCGGCGATATCCAGGCCACCTCCCTGACCTCCAAGGATCTGATCGCCTCCACCGACATCAGACTGCTGAAGCCCAGCAGCGGCAATGTGCACGTGCCTTACACCCAGGCCAGCTCCGGCTTCGAGATGTGGAAGAACAACAGCGGCAGACCCCTGCAGGAAACCGCCCCCTTCGGCTGCAAGATCGCCGTGAACCCCCTGAGAGCCGTGGATTGCAGCTACGGCAACATCCCCATCAGCATCGACATCCCCAACGCCGCCTTTATCAGAACCAGCGACGCCCCACTGGTGTCCACCGTGAAGTGCGAGGTGTCCGAGTGCACCTACAGCGCCGA CTTTGGCGGCATGGCCACCCTGCAGTACGTGTCCGACCGGGAGGGCCAGTGTCCT GTGCACAGCCACAGCTCCACCGCCACACTGCAGGAATCCACAGTCCACGTCCTG GAAAAGGGCGCCGTGACCGTGCACTTTAGCACCGCCAGCCCCCAGGCCAACTTC ATCGTGTCCCTGTGCGGCAAAAAGACCACCTGTAACGCCGAGTGCAAGCCCCCT GCCGACCACATCGTGTCCACCCCCCACAAGAACGACCAGGAATTCCAGGCCGCC ATCAGCAAGACCTCTTGGTCCTGGCTGTTCGCCCTGTTTGGCGGAGCCAGCAGCC TGCTGATTATCGGCCTGATGATCTTCGCCTGCAGCATGATGCTGACCAGCACCCG GCGCTGA (SEQ ID N0:7).

[0114] Sindbis mSAH envelope:

[0115] MASAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEAY DTLLNAILRCGSSGASVIDDFTLTSPYLGTCSYCHHTEPCFSPVKIEQVWDEADDNTIR IOTSAOFGYDOSGAASANKYRYMAAAAVTGGGGSLGVTSSGSGSGGGGGSGGGGSG GGGSASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTL TGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGG SGPATEQGODTFTKVKIDAGGGGSGSGSGGGGGSSGSGSGGGGSGSGSGGGGGSL VTGGGGSVTTVKEGTMDDIKISTSGPCRRLSYKGYFLLAKCPPGDSVTVSIVSSNSAT SCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLA4TTAGYITMHRPGPHAYTSY LEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCVAYKSDQTKW VFNSPDLIRHDDHTVQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISLQLDTDHLTL LTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAPGDPHG WPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPNA VIPTSLALLCCVRSANAETFTETMSYLWSNSQPFFWVQLCIPLAAFIVLMRCCSCCLP FLVVAGAYLAKVDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTN QEYITCKFTTVVPSPKIKCCGSLECQPAAHAGYTCKVFGGVYPFMWGGAQCFCDSE NSQMSEAYVELSADCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPG TSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIQATSLTSKD LIASTDIRLLKPSSGNVHVPYTQASSGFEMWKNNSGRPLQETAPFGCKIAVNPLRAVD CSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLQYVSDREGQCP VHSHSSTATLQESTVHVLEKGAVTVHFSTASPQANFIVSLCGKKTTCNAECKPPADHI VSTPHKNDQEFQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR (SEQ ID NO:8).

[0116] ATGGCCTCTGCCGCCCCTCTGGTGACAGCCATGTGCCTGCTGGGCAACGTGTCCTTCCCCTGCGACAGACCCCCTACCTGCTACACCAGAGAGCCCAGCAGAGCCCTGGACATCCTGGAAGAGAACGTGAACCACGAGGCCTACGACACCCTGCTGAACGCCATCCTGAGATGCGGCAGCTCTGGCAAGCGTGATCGACGACTTCACCCTGACCAGCCCCTACCTGGGCACCTGTAGCTACTGCCACCACACCGAGCCCTGCTTCAGCCCCGTGAAGATCGAACAGGTGTGGGACGAGGCCGACGATAACACCATCCGGATCCAGACCAGCGCCCAGTTCGGCTACGATCAGAGCGGAGCCGCCAGCGCCAACAAGTACCGGTACATGGCCGCTGCTGCCGTGACAGGCGGCGGAGGCAGCCTAGGCGTGACCAGCAGCGGCTCTGGATCTGGCGGAGGCGGAGGAAGTGGAGGGGGAGGAAGCGGCGGAGGGGGCTCTGCTAGCGCCGAGGCCGGCATCACCGGCACATGGTACAACCAGCACGGCTCTACCTTCACAGTGACCGCAGGAGCAGACGGAAACCTGACAGGACAGTATGAGAATAGGGCACAGGGAACCGGATGCCAGAACAGCCCCTACACACTGACCGGCAGGTATAATGGCACAAAGCTGGAGTGGCGCGTGGAGTGGAACAATAGCACAGAGAATTGTCACTCCCGGACCGAGTGGAGAGGACAGTACCAGGGAGGAGCCGAGGCCAGGATCAACACACAGTGGAATCTGACCTATGAGGGAGGAAGCGGACCTGCAACCGAGCAGGGCCAGGATACCTTCACCAAGGTGAAAATCGATGCTGGCGGGGGAGGCTCTGGAAGTGGATCTGGGGGTGGCGGCGGAAGCTCTGGATCTGGAAGCGGAGGGGGAGGCAGCGGCAGTGGATCAGGGGGGGGAGGGGGATCTCTGGTAACCGGGGGAGGCGGAAGCGTGACCACCGTGAAAGAGGGCACCATGGACGACATCAAGATCAGCACCAGCGGCCCCTGCAGAAGGCTTAGCTACAAAGGATATTTTCTGCTGGCCAAGTGTCCCCCTGGAGACAGCGTGACCGTGTCCATCGTGAGCTCCAACAGCGCCACATCCTGCACTCTGGCTCGGAAGATCAAGCCAAAGTTCGTGGGAAGAGAGAAGTACGACCTGCCACCCGTGCACGGCAAGAAAATCCCCTGTACCGTGTACGATAGGCTGGCCGCTACTACCGCCGGATATATTACAATGCACAGGCCAGGACCTCATGCTTACACCAGCTATCTGGAGGAATCTAGTGGCAAAGTGTACGCCAAGCCTCCATCAGGGAAAAACATCACCTACGAGTGCAAATGTGGGGATTATAAGACCGGAACAGTGAGCACTAGGACCGAAATCACCGGCTGCACAGCCATTAAGCAGTGTGTGGCTTATAAATCAGACCAGACAAAGTGGGTGTTCAATAGCCCTGATCTGATCCGCCACGACGATCATACTGTGCAGGGAAAACTGCACCTGCCCTTCAAGCTGATTCCCAGCACATGCATGGTGCCTGTGGCCCATGCTCCAAACGTGATCCACGGCTTCAAACATATTTCCCTGCAGCTGGACACTGATCACCTGACCCTGCTGACAACTAGGCGCCTGGGCGCTAATCCAGAGCCAACCACAGAATGGATCGTGGGGAAGACAGTGAGGAACTTTACTGTGGACCGCGATGGCCTGGAGTACATTTGGGGGAATCACGAACCAGTGCGGGTGTATGCTCAGGAGTCCGCTCCAGGAGACCCTCACGGATGGCCACATGAAATCGTGCAGCATTACTATCACAGACATCCCGTGTACACAATCCTGGCTGTGGCCAGCGCCACTGTGGCTATGATGATTGGCGTGACCGTGGCTGTGCTGTGCGCTTGTAAGGCGCGCAGAGAGTGCCTGACCCCCTACGCTCT GGCCCCTAACGCCGTGATCCCAACATCCCTGGCTCTGCTGTGCTGTGTGCGGTCT GCTAACGCCGAGACATTCACTGAAACCATGTCCTATCTGTGGAGTAATTCACAGC CCTTCTTTTGGGTGCAGCTGTGTATCCCTCTGGCCGCTTTCATTGTGCTGATGAGGTGCTGTTCTTGCTGTCTGCCTTTTCTGGTGGTGGCTGGCGCCTACCTGGCTAAAGT GGACGCCTATGAGCACGCTACTACCGTGCCAAATGTGCCCCAGATCCCTTACAAG GCCCTGGTGGAAAGGGCTGGATATGCTCCTCTGAACCTGGAGATTACCGTGATGT CAAGCGAAGTGCTGCCATCCACCAATCAGGAGTACATCACATGCAAGTTCACCACTGTGGTGCCTTCTCCAAAAATTAAGTGCTGTGGGAGTCTGGAATGCCAGCCCGC CGCTCATGCCGGATACACCTGTAAGGTGTTCGGGGGAGTGTATCCTTTTATGTGG GGCGGGGCCCAGTGCTTCTGTGATTCTGAGAATAGTCAGATGTCAGAAGCGTAC GTCGAGCTGTCCGCCGATTGTGCCTCTGACCACGCCCAGGCCATCAAAGTGCACACCGCCGCCATGAAAGTGGGCCTGCGGATCGTGTACGGCAACACAACCAGCTTTC TGGACGTGTACGTGAACGGCGTGACCCCCGGCACCAGCAAGGACCTGAAAGTGA TCGCCGGACCCATCAGCGCCAGCTTCACCCCCTTCGACCACAAGGTGGTGATCCA CCGGGGCCTGGTGTACAACTACGACTTCCCCGAGTACGGCGCCATGAAGCCTGGCGCCTTCGGCGATATCCAGGCCACCTCCCTGACCTCCAAGGATCTGATCGCCTCC ACCGACATCAGACTGCTGAAGCCCAGCAGCGGCAATGTGCACGTGCCTTACACC CAGGCCAGCTCCGGCTTCGAGATGTGGAAGAACAACAGCGGCAGACCCCTGCAG GAAACCGCCCCCTTCGGCTGCAAGATCGCCGTGAACCCCCTGAGAGCCGTGGATTGCAGCTACGGCAACATCCCCATCAGCATCGACATCCCCAACGCCGCCTTTATCA GAACCAGCGACGCCCCACTGGTGTCCACCGTGAAGTGCGAGGTGTCCGAGTGCA CCTACAGCGCCGACTTTGGCGGCATGGCCACCCTGCAGTACGTGTCCGACCGGG AGGGCCAGTGTCCTGTGCACAGCCACAGCTCCACCGCCACACTGCAGGAATCCACAGTCCACGTCCTGGAAAAGGGCGCCGTGACCGTGCACTTTAGCACCGCCAGCC CCCAGGCCAACTTCATCGTGTCCCTGTGCGGCAAAAAGACCACCTGTAACGCCG AGTGCAAGCCCCCTGCCGACCACATCGTGTCCACCCCCCACAAGAACGACCAGG AATTCCAGGCCGCCATCAGCAAGACCTCTTGGTCCTGGCTGTTCGCCCTGTTTGGCGGAGCCAGCAGCCTGCTGATTATCGGCCTGATGATCTTCGCCTGCAGCATGATG CTGACCAGCACCCGGCGCTGA (SEQ ID N0:9).

[0117] In some aspects, the biotin binding sequence has at least, at most, exactly, between (inclusive or exclusive) any two of, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or valuederivable therein, with SEQ ID NOs:27-30. In some aspects, the biotin binding sequence comprises any one of SEQ ID NOs:27-30. In some aspects, the biotin binding sequence consists of SEQ ID NOs:27-30.

[0118] In some aspects, the pseudotyped viral envelope, e.g., a pseudotyped Sindbis envelope, comprises a detectable label. In some aspects, the detectable label is one that confers a phenotype that allows for the discrimination of transduced cells based on affinity pull down, such as a FLAG tag. A FLAG tag is a short, eight-amino acid peptide (DYKDDDDK; SEQ ID NO: 12) that is commonly used as a protein tag for recombinant protein expression and purification. The FLAG tag is non-immunogenic and does not affect the function of the protein to which it is attached. It can be added to the N-terminus or C-terminus of a protein using recombinant DNA technology.

[0119] A linker sequence may be included in the construction of the pseudotyped viral envelope. For example, a linker having at least, at most, or exactly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (or any derivable range therein) may separate nucleic acids of the disclosure, such as nucleic acid encoding viral envelope protein subunits, protein binding domains, and / or detectable labels. In some aspects, the linker comprises GGGGSLGVTSSGSGSGGGGGSGGGGSGGGGSAS (SEQ ID NO: 13), encoded byGGCGGCGGAGGCAGCCTAGGCGTGACCAGCAGCGGCTCTGGATCTGGCGGAGGC GGAGGAAGTGGAGGGGGAGGAAGCGGCGGAGGGGGCTCTGCTAGC (SEQ ID NO: 14). In some aspects, the linker comprises IDAGGGGSGSGSGGGGGSSGSGSGGGGSGSGSGGGGGSLVTGGGGS (SEQ ID NO: 15), encoded byATCGATGCTGGCGGGGGAGGCTCTGGAAGTGGATCTGGGGGTGGCGGCGGAAGC TCTGGATCTGGAAGCGGAGGGGGAGGCAGCGGCAGTGGATCAGGGGGGGGAGG GGGATCTCTGGTAACCGGGGGAGGCGGAAGCG (SEQ ID NO: 16). In some aspects, the linker comprises GGGGSLG (SEQ ID NO: 17), encoded by GTAACAGGCGGAGGAGGCAGCCTAGGG (SEQ ID NO: 18). In some aspects, the linker comprises VTGGGGS (SEQ ID NO: 19), encoded by GTAACCGGAGGCGGCGGATCC (SEQ ID NO:20). In some aspects, the linker comprises two linkers, and the two linkers separate nucleic acids of the disclosure, such as nucleic acid encoding viral envelope proteinsubunits, protein binding domains, and / or detectable labels. In some aspects, the protein binding domain and / or detectable label is provided between the two linkers. As an example, a nucleic acid sequence encoding a first linker can be inserted into the nucleic acid sequence encoding the Sindbis envelope immediately following the nucleic acid sequence encoding amino acid residue 70 of the Sindbis E2 protein. The first linker can be followed by a nucleic acid sequence encoding a protein binding domain and / or detectable label, and the protein binding domain and / or detectable label can be followed by a nucleic acid sequence encoding a second linker. The second linker can be followed by the nucleic acid sequence encoding the remainder of the Sindbis E2 protein (z.e., the remaining portion of the Sindbis E2 protein following amino acid residue 70). Exemplary linker sequences in Sindbis E2 proteins are underlined in SEQ ID NOs:4-9, above.

[0120] Exemplary amino acid and nucleotide sequences encoding a pseudotyped Sindbis viral envelope including two linkers and a FLAG tag (in bold) are provided, e.g., by:

[0121] MASAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEAY DTLLNAILRCGSSGASVIDDFTLTSPYLGTCSYCHHTEPCFSPVKIEQVWDEADDNTIR IQTSAOFGYDOSGAASANKYRYMAAAAVTGGGGSLGDYKDDDDKVTGGGGSVTTV KEGTMDDIKISTSGPCRRLSYKGYFLLAKCPPGDSVTVSIVSSNSATSCTLARKIKPKF VGREKYDLPPVHGKKIPCTVYDRLA4TTAGYITMHRPGPHAYTSYLEESSGKVYAKP PSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCVAYKSDQTKWVFNSPDLIRHDD HTVQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISLQLDTDHLTLLTTRRLGANPEP TTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAPGDPHGWPHEIVQHYYH RHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPNAVIPTSLALLCCV RSANAETFTETMSYLWSNSQPFFWVQLCIPLAAFIVLMRCCSCCLPFLVVAGAYLAK VDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTNQEYITCKFTTVV PSPKIKCCGSLECQPAAHAGYTCKVFGGVYPFMWGGAQCFCDSENSQMSEAYVELS ADCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPGTSKDLKVIAGPIS ASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIQATSLTSKDLIASTDIRLLKPS SGNVHVPYTQASSGFEMWKNNSGRPLQETAPFGCKIAVNPLRAVDCSYGNIPISIDIP NAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLQYVSDREGQCPVHSHSSTATLQ ESTVHVLEKGAVTVHFSTASPQANFIVSLCGKKTTCNAECKPPADHIVSTPHKNDQE FQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR (SEQ ID NO: 10).

[0122] ATGGCGTCCGCAGCACCACTGGTCACGGCAATGTGTTTGCTCGGAA ATGTGAGCTTCCCATGCGACCGCCCGCCCACATGCTATACCCGCGAACCTTCCAG AGCCCTCGACATCCTTGAAGAGAACGTGAACCATGAGGCCTACGATACCCTGCTCAATGCCATATTGCGGTGCGGATCGTCTGGCAAGCGTCATTGACGACTTTACCCTGACCAGCCCCTACTTGGGCACATGCTCGTACTGCCACCATACTGAACCGTGCTTCAGCCCTGTTAAGATCGAGCAGGTCTGGGACGAAGCGGACGATAACACCATACGCATACAGACTTCCGCCCAGTTTGGATACGACCAAAGCGGAGCAGCAAGCGCAAACAAGTACCGCTACATGGCGGCTGCGGCGGTAACAGGCGGAGGAGGCAGCCTAGGGGACTACAAAGACGATGACGACAAGGTAACCGGAGGCGGCGGATCCGTAACCACCGTTAAAGAAGGCACCATGGATGACATCAAGATTAGCACCTCAGGACCGTGTAGAAGGCTTAGCTACAAAGGATACTTTCTCCTCGCAAAATGCCCTCCAGGGGACAGCGTAACGGTTAGCATAGTGAGTAGCAACTCAGCAACGTCATGTACACTGGCCCGCAAGATAAAACCAAAATTCGTGGGACGGGAAAAATATGATCTACCTCCCGTTCACGGTAAAAAAATTCCTTGCACAGTGTACGACCGTCTGGCAGCAACAACTGCAGGCTACATCACTATGCACAGGCCGGGACCGCACGCTTATACATCCTACCTGGAAGAATCATCAGGGAAAGTTTACGCAAAGCCGCCATCTGGGAAGAACATTACGTATGAGTGCAAGTGCGGCGACTACAAGACCGGAACCGTTTCGACCCGCACCGAAATCACTGGTTGCACCGCCATCAAGCAGTGCGTCGCCTATAAGAGCGACCAAACGAAGTGGGTCTTCAACTCACCGGACTTGATCAGACATGACGACCACACGGTCCAAGGGAAATTGCATTTGCCTTTCAAGTTGATCCCGAGTACCTGCATGGTCCCTGTTGCCCACGCGCCGAATGTAATACATGGCTTTAAACACATCAGCCTCCAATTAGATACAGACCACTTGACATTGCTCACCACCAGGAGACTAGGGGCAAACCCGGAACCAACCACTGAATGGATCGTCGGAAAGACGGTCAGAAACTTCACCGTCGACCGAGATGGCCTGGAATACATATGGGGAAATCATGAGCCAGTGAGGGTCTATGCCCAAGAGTCAGCACCAGGAGACCCTCACGGATGGCCACACGAAATAGTACAGCATTACTACCATCGCCATCCTGTGTACACCATCTTAGCCGTCGCATCAGCTACCGTGGCGATGATGATTGGCGTAACTGTTGCAGTGTTATGTGCCTGTAAAGCGCGCCGTGAGTGCCTGACGCCATACGCCCTGGCCCCAAACGCCGTAATCCCAACTTCGCTGGCACTCTTGTGCTGCGTTAGGTCGGCCAATGCTGAAACGTTCACCGAGACCATGAGTTACTTGTGGTCGAACAGTCAGCCGTTCTTCTGGGTCCAGTTGTGCATACCTTTGGCCGCTTTCATCGTTCTAATGCGCTGCTGCTCCTGCTGCCTGCCTTTTTTAGTGGTTGCCGGCGCCTACCTGGCGAAGGTAGACGCCTACGAACATGCGACCACTGTTCCAAATGTGCCACAGATACCGTATAAGGCACTTGTTGAAAGGGCAGGGTATGCCCCGCTCAATTTGGAGATCACTGTCATGTCCTCGGAGGTTTTGCCTTCCACCAACCAAGAGTACATTACCTGCAAATTCACCACTGTGGTCCCCTCCCCAAAAATCAAATGCTGCGGCTCCTTGGAATGTCAGCCGGCCGCTCATGCAGGCTATACCTGCAAGGTCTTCGGAGGGGTCTACCCCTTTATGTGGGGAGGAGCGCAATGTTTTTGCGACAGTGAGAACAGCCAGATGAGTGAGGCGTACGTCGAATTGTCAGCAGATTGCGCGTCTGACCACGCGCAGGCGATT AAGGTGCACACTGCCGCGATGAAAGTAGGACTGCGTATTGTGTACGGGAACACT ACCAGTTTCCTAGATGTGTACGTGAACGGAGTCACACCAGGAACGTCTAAAGAC TTGAAAGTCATAGCTGGACCAATTTCAGCATCGTTTACGCCATTCGATCATAAGG TCGTTATCCATCGCGGCCTGGTGTACAACTATGACTTCCCGGAATATGGAGCGAT GAAACCAGGAGCGTTTGGAGACATTCAAGCTACCTCCTTGACTAGCAAGGATCT CATCGCCAGCACAGACATTAGGCTACTCAAGCCTTCCTCAGGAAACGTGCATGTC CCGTACACGCAGGCCTCATCAGGATTTGAGATGTGGAAAAACAACTCAGGCCGC CCACTGCAGGAAACCGCACCTTTCGGGTGTAAGATTGCAGTAAATCCGCTCCGA GCGGTGGACTGTTCATACGGGAACATTCCCATTTCTATTGACATCCCGAACGCTG CCTTTATCAGGACATCAGATGCACCACTGGTCTCAACAGTCAAATGTGAAGTCAG TGAGTGCACTTATTCAGCAGACTTCGGCGGGATGGCCACCCTGCAGTATGTATCC GACCGCGAAGGTCAATGCCCCGTACATTCGCATTCGAGCACAGCAACTCTCCAA GAGTCGACAGTACATGTCCTGGAGAAAGGAGCGGTGACAGTACACTTTAGCACC GCGAGTCCACAGGCGAACTTTATCGTATCGCTGTGTGGGAAGAAGACAACATGC AATGCAGAATGTAAACCACCAGCTGACCATATCGTGAGCACCCCGCACAAAAAT GACCAAGAATTTCAAGCCGCCATCTCAAAAACATCATGGAGTTGGCTGTTTGCCC TTTTCGGCGGCGCCTCGTCGCTATTAATTATAGGACTTATGATTTTTGCTTGCAGC ATGATGCTGACTAGCACACGAAGATGA (SEQ ID NO: 11).

[0123] ‘ ‘Mutated Sindbis envelope” refers to a point mutation, insertion, or deletion in the amino acid sequence of a wild type Sindbis El, E2, or E3 protein. The El, E2, or E3 protein can have one or more mutations. In addition, combinations of mutations in El , E2, and E3 are encompassed by the disclosure, e.g., mutations in El and E2, or in E2 and E3, or E3 and El, or El, E2, and E3.

[0124] In certain aspects, the pseudotyped viral envelope comprises one or more of the following mutations in a wild type Sindbis envelope (e.g., SEQ ID NOs:l-3), or mutations corresponding to the same wild type amino acid residues in a Sindbis ZZ envelope sequence (e.g., SEQ ID NO:4), a Sindbis eMA envelope sequence (e.g., SEQ ID NO:6), a Sindbis mSAH envelope sequence (e.g., SEQ ID NO:8), or a Sindbis 1LFLAG1L envelope (e.g., SEQ ID NO: 10). Deletions from a wild type Sindbis envelope (e.g., SEQ ID NOs: l-3) are illustrated by a A in the amino acid and nucleic acid sequences of SEQ ID NOs:4-10. Substitutitions to a wild type Sindbis envelope (e.g., SEQ ID NOs:l-3) are illustrated by the italicized amino acid and nucleotide residues in SEQ ID NOs:4-10. Exemplary mutations including the following.

[0125] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA.

[0126] Substitution or deletion at amino acid residue 1 of Sindbis E2 protein. For example, in specific aspects, the amino acid R at residue 1 of the Sindbis E2 protein may be substituted with the amino acid D.

[0127] Substitution or deletion at amino acid residues 68-71 of Sindbis E2 protein. For example, the sequence SEKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted or deleted. In certain aspects, the sequence SEKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted. In certain aspects, the sequence SEKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA.

[0128] Substitution or deletion at amino acid residue 114 of Sindbis E2 protein. For example, in specific aspects, the amino acid S at residue 114 of the Sindbis E2 protein may be substituted with the amino acid P.

[0129] Substitution or deletion at amino acid residues 159 and 160 of Sindbis E2 protein. For example, the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted or deleted. In certain aspects, the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA.

[0130] Substitution or deletion at amino acid residues 216 and 218 of Sindbis E2 protein. For example, the amino acid E at residue 216 and / or the amino acid T at residue 218 of Sindbis E2 protein can be substituted or deleted. In certain aspects, the amino acid E at residue 216 of Sindbis E2 protein is substituted with the amino acid A. In certain aspects, the amino acid T at residue 218 of Sindbis E2 protein is substituted with the amino acid A. In certain aspects, the amino acid E at residue 216 the amino acid T at residue 218 of Sindbis E2 protein are substituted with the amino acid A.

[0131] Substitution or deletion of amino acid residues 226 and 227 of Sindbis El protein. For example, the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted or deleted. In certain aspects, the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted with the sequence SG. In some aspects, substitution or deletion of amino acid residues 226 and 227 of Sindbis El protein eliminates cholesterol dependence for cell entry. In some aspects, substitution or deletion of amino acid residues 226 and 227 of Sindbis El protein increases titer by 2-10 fold.

[0132] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein and substitution or deletion at amino acid residue 1 of Sindbis E2 protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted, and the sequence R at amino acid residue 1 of the Sindbis E2 protein can be substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the amino acid R at residue 1 of the Sindbis E2 protein may be substituted with the amino acid D. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the amino acid R at residue 1 of the Sindbis E2 protein may be substituted with the amino acid D.

[0133] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein and substitution or deletion at amino acid residues 159 and 160 of Sindbis E2 protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA.

[0134] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein and substitution or deletion at amino acid residues 216 and 218 of Sindbis E2 protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted, and the amino acid E at residue 216 and / or the amino acid T at residue 218 of Sindbis E2 protein can be substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the amino acid E at residue 216 of Sindbis E2 protein is substituted with the amino acid A. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the amino acid T at residue 218 of Sindbis E2 protein is substituted with the amino acid A. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the amino acid E at residue 216 the amino acid T at residue 218 of Sindbis E2 protein are substituted with the amino acid A. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the amino acid E at residue 216 of Sindbis E2 protein is substituted with the amino acid A. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein issubstituted with the sequence AAAAA, and the amino acid T at residue 218 of Sindbis E2 protein is substituted with the amino acid A. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the amino acid E at residue 216 the amino acid T at residue 218 of Sindbis E2 protein are substituted with the amino acid A.

[0135] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein and substitution or deletion at amino acid residues 68-71 of Sindbis E2 protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted.

[0136] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein, and substitution or deletion at amino acid residues 68-71 of Sindbis E2 protein, and substitution or deletion at amino acid residues 159 and 160 of Sindbis E2 protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted or deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and thesequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA.

[0137] Substitution or deletion at amino acid residues 60-64 of Sindbis E3 protein, and substitution or deletion at amino acid residues 68-71 of Sindbis E2 protein, and substitution or deletion at amino acid residues 159 and 160 of Sindbis E2 protein, and substitution or deletion of amino acid residues 226 and 227 of Sindbis El protein. For example, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein can be substituted or deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted or deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted or deleted, and the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted or deleted. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA, and the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted with the sequence SG. In certain aspects, the sequence RSKR at amino acid residues 61-64 of the Sindbis E3 protein is deleted, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA, and the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted with the sequence SG. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is substituted with the sequence AAAA, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substituted with the sequence AA, and the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted with the sequence SG. In certain aspects, the sequence RSKRS at amino acid residues 60-64 of the Sindbis E3 protein is substituted with the sequence AAAAA, and the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein is deleted, and the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein is substitutedwith the sequence AA, and the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein is substituted with the sequence SG.

[0138] In one aspect, the pseudotyped viral envelope comprises the following mutations in a wild type Sindbis envelope (e.g., SEQ ID NOs:l-3), or mutations corresponding to the same wild type amino acid residues in a Sindbis ZZ envelope sequence (e.g., SEQ ID NO:6), a Sindbis AV envelope sequence (e.g., SEQ ID NO:8), a Sindbis STAV envelope sequence (e.g., SEQ ID NO: 10), a Sindbis eMA envelope sequence (e.g., SEQ ID NO: 12), or a a Sindbis mSAH envelope sequence (e.g., SEQ ID NO: 14): deletion of amino acid residues 61-64 of Sindbis E3 protein, and substitution of the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein with the sequence AAAA, and substitution of the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein with the sequence AA.

[0139] In one aspect, the pseudotyped viral envelope comprises the following mutations in a wild type Sindbis envelope (e.g., SEQ ID NOs:l-3), or mutations corresponding to the same wild type amino acid residues in a Sindbis ZZ envelope sequence (e.g., SEQ ID NO:6), a Sindbis AV envelope sequence (e.g., SEQ ID NO:8), a Sindbis STAV envelope sequence (e.g., SEQ ID NO: 10), a Sindbis eMA envelope sequence (e.g., SEQ ID NO: 12), or a a Sindbis mSAH envelope sequence (e.g., SEQ ID NO: 14): deletion of amino acid residues 61-64 of Sindbis E3 protein, and substitution of the sequence SLKQ at amino acid residues 68 to 71 of the Sindbis E2 protein with the sequence AAAA, substitution of the sequence KE at amino acid residues 159 and 160 of the Sindbis E2 protein with the sequence A A, and substitution of the sequence AK at amino acid residues 226 and 227 of the Sindbis El protein with the sequence SG.

[0140] In some aspects, the mutated Sindbis envelope has at least, at most, exactly, between (inclusive or exclusive) any two of, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, or any range or value derivable therein, with SEQ ID NOs:4-l l. In some aspects, the mutated Sindbis envelope comprises any one of SEQ ID NOs:4-l l. In some aspects, the mutated Sindbis envelope consists of SEQ ID NOs:4-l l.

[0141] Mutations into one or more of the Sindbis viral envelope protein sequences can be introduced using any known methods in the art. Mutations can be targeted or random. For example, targeted mutations can be introduced using site-directed mutagenesis, for instance employing overlapping PCR or overlap extension PCR (see, e.g., Aiyar, et al., Methods Mol Biol (1996) 57:177-91; and Pogulis, et al., Methods Mol Biol (1996) 57:167- 76). Alternatively,mutations can be introduced by taking advantage of the error prone replication process of Sindbis viruses, which lack proof-reading and mismatch repair activities, and recombination between quasispecies in a virus population, for instance, by using a replication competent virus (see Domingo and Holland, Annu Rev Microbiol (1997) 51:151-178). Mutant Sindbis virus envelope proteins of particular interest have a diminished ability to bind to endogenous receptors and therefore demonstrate decreased background infectivity in comparison to wild type sequences. Preferably, the mutated Sinbis virus envelope proteins of the present disclosure have a decreased ability to bind to glycosaminoglycans (GAGs), including heparin sulfate (HS), in comparison to wild type sequences.III. Packaging Cells & Systems

[0142] In the methods and compositions provided herein, the recombinant retroviral genomes, in non-limiting illustrative examples, lentiviral genomes, have a limitation to the number of polynucleotides that can be packaged into the viral particle. In some aspects provided herein, the polypeptides encoded by the polynucleotide encoding region can be truncations or other deletions that retain a functional activity such that the polynucleotide encoding region is encoded by less nucleotides than the polynucleotide encoding region for the wild type polypeptide. In some aspects, the polypeptides encoded by the polynucleotide encoding region can be fusion polypeptides that can be expressed from one promoter. In some aspects, the fusion polypeptide can have a cleavage signal to generate two or more functional polypeptides from one fusion polypeptide and one promoter. Furthermore, some functions that are not required after initial ex vivo transduction are not included in the retroviral genome, but rather are present on the surface of the vims or retrovirus via the packaging cell membrane. These various strategies are used herein to maximize the functional elements that are packaged within the virus.

[0143] In some aspects, the recombinant retroviral genome to be packaged can be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides on the low end of the range and 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides on the high end of the range. Functions discussed elsewhere herein that can be packaged include required retroviral sequences for retroviral assembly and packaging, such as a retroviral env, gag, and pol coding regions, as well as a 5' LTR and a 3' LTR, or an active truncated fragment thereof.

[0144] In one aspect, provided herein are packaging cells or systems comprising the pseudotyped viral envelope, the targeting moiety, and / or viral support elements as described herein. Packaging cells and systems, packaging techniques and vectors for packaging the nucleic acid genome into the pseudotyped viral particle are also known to those of skill in the art and can be made according to methods known to those of skill in the art (see, e.g., Polo, el al., Proc Natl Acad Sci USA, (1999) 96:4598-4603). Methods of packaging include using packaging cells that permanently express the envelope components, or by transiently transfecting cells with plasmids encoding the components of the vector, or by using an adenoviral system that encodes the components of the vector. Virus packaging cells and kits are commercially available, for example, from BD Sciences / Clontech in Mountain View, CA).

[0145] The packaging cells may be any cell suitable for virus production. In some aspects, the packaging cells is a mammalian cell that is used to make virus. Any of a wide variety of cells can be selected for in vitro production of a virus. Eukaryotic cells arc typically used, particularly mammalian cells including human, simian, canine, feline, equine and rodent cells. In illustrative examples, the cells are human cells. In further illustrative aspects, the cells reproduce indefinitely, and are therefore immortal. Examples of cells that can be advantageously used in the present invention include NIH 3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells and any cells derived from such cells. Highly transfectable cells, such as human embryonic kidney 293T cells, can be used. By “highly transfectable” it is meant that at least about 50%, more preferably at least about 70% and most preferably at least about 80% of the cells can express the genes of the introduced DNA.

[0146] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCLIO), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0147] In any of the aspects disclosed herein, the methods of making virus can include growing a mammalian packaging cells to 50%, 60%, 70%, 80%, 90% or 95% confluence or confluence to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density and thensplitting or diluting the cells. In some aspects, a stirred tank reactor can be used to grow the cells. In some aspects, the cells can be split at least about 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, or 1:20 using methods a skilled artisan will understand. In some aspects, the cells can be diluted to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% peak cell density.

[0148] The present disclsoure also provides methods of purifying the virus from cells. In one embodiment, the virus is purified from cells by the following method: Virus is filtered through 0.22-microM-pore-size filter before concentration. Virus (30mL) was loaded onto sucrose cushion (7mL) and spinned using SW32 (Beckman) roter. 20% Sucrose (wt / wt) in IX PBS with ImM EDTA was used for cushion. The spinning condition is 40000 X g for 90min at 4 degree. The supernatant is discarded and pellet is resuspended in 300 microL of Hanks Balanced Salt Solution. The concentrated virus is filtered again using same size filter before administration into animal.

[0088] The present invention also provides methods of transducing cells with the virus, as follows: some primarily hematopoietic cells are resistant to gene transduction (primary T cells and stem cell) in vitro. However changing the pH of the medium (7.4 to 5.5) during gene transduction makes gene transduction efficiency higher. (3-20 fold). This method is useful for ex vivo and in vitro transduction of some cell types.IV. Targeting Moieties

[0149] In some aspects, the pseudotyped viral envelopes of the disclosure are associated with a targeting moiety. The targeting moiety can be covalently or non-covalently conjugated or linked to the viral envelope protein(s) (e.g., the Sinbis El , E2, or E3 protein). Additionally, or alternatively, the targeting moiety can be a fusion protein with the viral envelope protein(s) (e.g., the Sinbis El , E2, or E3 protein), z.e., the nucleic acid encoding the pseudotyped viral envelope can also encode the targeting moiety. The targeting moiety may be encoded by a nucleic acid encoding the pseudotyped viral envelope (e.g., as a fusion protein with the viral envelope protein(s)) and expressed on the surface of the pseudotyped virus particles disclosed herein. The targeting moiety can be encoded by a nucleic acid that is different from the nucleic acid encoding the pseudotyped viral envelope. The nucleic acid(s) encoding the targeting moiety and the pseudotyped viral envelope may be included in the same or different vectors. A targeting moiety (e.g., a nucleic acid or polypeptide encoding the targeting moiety) may be also administered to a subject or contacted with a cell separately from administration of or contact with a cell by the pseudotyped viral envelope. For example, the targeting moiety (e.g., a nucleic acid or polypeptide encoding the targeting moiety), or acomposition thereof, can be administered to a subject or contacted with a cell before, after, or simultaneously with administration of or contact with a cell by the pseudotyped viral envelope or a composition thereof.

[0150] In some aspects, the targeting moiety is covalently or non-covalently conjugated or linked to the viral envelope protein (e.g., the Sindbis El, E2, or E3 protein), or is linked to another portion of the envelope. In one aspect, the targeting moiety is linked to the viral envelope protein (e.g., the Sindbis El, E2, or E3 protein) via non-covalent interactions with a protein binding domain, where the protein binding domain is fused with the viral envelope protein (e.g., the Sindbis El , E2, or E3 protein). In one aspect, the protein binding domain is fused with the Sindbis E2 or E3 protein. Exemplary protein binding domains include, e.g., the ZZ domain of protein A, streptavidin, avidin, a leucine zipper, a STAT protein N terminal domain, an FK506 binding protein, the 4C-RGD integrin binding sequence (CDCRGDCFC (SEQ ID NO:21), encoded by TGCGACTGTAGAGGCGACTGTTTCTGC (SEQ ID NO:22)), and the B6 transferrin receptor targeting sequence (GHKAKGPRK (SEQ ID NO:23), encoded by GGACATAAAGCTAAGGGTCCTAGAAAG (SEQ ID NO:24)) (see, e.g., O’Shea, Science 254: 539 (1991), Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211 : 121- 128 (1996); Klemm et al., Annu. Rev. Immunol. 16:569-592 (1998); Klemm et al., Annu. Rev. Immunol. 16:569-592 (1998); Ho etal., Nature 382:822-826 (1996); Pomeranz etal., Biochem. 37:965 (1998); and Xia, et al., J Virol (2000) 74:11359-66).

[0151] In another aspect, the targeting moiety is a fusion protein with the viral envelope protein(s) (e.g., Sinbis El, E2, or E3 protein). The nucleic acid encoding the pseudotyped viral envelope can also encode the targeting moiety. Such a nucleic acid may be comprised in a viral vector (e.g., a retroviral vector) or in a viral genome (e.g., a retroviral genome). In one aspect, the targeting moiety is fused with the Sindbis E2 or the Sindbis E3 protein.

[0152] The targeting moiety can be, e.g., an antibody, such as a monoclonal or single chain antibody that specifically binds to an antigen or a cell surface molecule, or any antigen, ligand, or binding partner of a cell surface molecule (e.g., a viral envelope protein) (e.g., Sindbis El, E2, or E3 protein).

[0153] The targeting moiety can target normal or diseased tissue in the context of any disease, condition, or cell type. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and / or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas, as well as solid tumors (e.g., breast, lung, ovarian, prostate, colon, and melanoma). Also included are immunedisorders, such as graft versus host disease, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, inflammatory bowel disease, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves’ disease, Hashimoto’s thyroiditis, myasthenia gravis, and vasculitis. Also included are infections caused by pathogenic or infectious disease microorganisms. In some aspects, the targeting moiety is directed to antigens or cell surface molecules, or ligands, or binding partners of cell surface molecules, that are selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor, immune, or pathogenic cells, as compared to normal or nontargeted cells or tissues. In other aspects, the targeting moiety is directed to antigens or cell surface molecules, or ligands, or binding partners of cell surface molecules, that are expressed on normal cells and / or is expressed on the engineered cells.

[0154] The targeting moiety also can also comprise or be directed to marker proteins indicative of diseases including cancers; autoimmune disease (e.g., multiple sclerosis, systemic lupus erythymatosis, rheumatoid arthritis, and diabetes mellitus); infectious disease, including infection by HIV, HCV, HBV, CMV, and HPV; and genetic diseases including sickle cell anemia, cystic fibrosis, Tay-Sachs, P -thalassemia, neurofibromatosis, polycystic kidney disease, hemophilia, etc. In certain embodiments, the targeting moiety targets a cell surface antigen specific to a particular cell or tissue type, e.g., lymphocytes, myocytes, keratinocytes, neurons, hepatocytes, lung, kidney, muscle, vascular, thyroid, ocular, breast, ovarian, testis, prostate tissue.

[0155] Exemplary antigens that the targeting moiety may comprise or to which the targeting moiety may be directed include, but are not limited to, antigenic molecules from infectious agents, auto- / self-antigens, tumor- / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In particular aspects, the antigens include EBNA, CD123, HER1, HER2, CA-125, CA 19-9, CA 72-4, CA 15-3\CA 27.29VBCAA, CA-195, CA- 242, CA-50, CA LX, MN-CA IX, TRAIL / DR4, CD2, CD5, CD7, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD47, CD56, CD68 / P1, CD70, CD97, CD99, CD123, CD171, CD179, CD200, CD319 (CS1), HLA-G, carcinoembryonic antigen, alphafetoprotein, b-human chorionic gonadotropin, AKT, Her3, epithelial tumor antigen, ROR1, folate binding protein, folate receptor, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, HERV-K, 11-6, IL-l lRa, IL-13Ra, kappa chain, lambda chain, CSPG4, CLL-1, U5snRNP200, BAFF-R, BCMA, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1,glioma-associated antigen, melanoma-associated antigen, BAGE, DAM-6, DAM- 10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, pi 5, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, MUC16, MUC18, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, P16, RU1, RU2, SART-1, SART-3, Wilms’ tumor antigen (WT1), AFP, |3-catenin, Caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF- 2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notchl-4), NY ESO 1, pl85erbB2, pl80erbB-3, c-Met, nm-23Hl, beta-HCG, BCA225, BTAA, CAM 17.1, CAM43, LI CAM, NCAM, NuMa, 43-9F, 791Tgp72, CO-029, FGF-5, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1 , RCASI, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, EBMA-1, BARF-1, CS-1, ADRB3, thyroglobulin, EVT6-AML, TGS5, plysialic acid, neutrophil elastase, intestinal carboxyl esterase, prostase, prostein, lewisY, LY6K, PAP, OR51 E2, PANX3, SSEA-4, TARP, CXORF61, Flt3, TEM1, TEM7R, TSHR, UPK2, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, k-ras, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, EphnnB2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA 17, PAX3, ALK, androgen receptor, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, cyclin Bl, poly sialic acid, M-CSF, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, GPRC5D, GPR20, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, HAVCR1, AKAP-4, SSX2, XAGE 1, B7H3, B7H6, Kit, legumain, TN Ag, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2,CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, TSP- 180, and LRRN1. Examples of sequences for antigens are known in the art, for example, in the GenBank® database: CD19 (Accession No. NG_007275.1), EBNA (Accession No. NGJ302392.2), WT1 (Accession No. NG_009272.1), CD123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage- A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-AlO (Accession No. NCJ300023.i l), TRAIL / DR4 (Accession No. NCJ300003.12), and / or CEA (Accession No. NCJ3OOO19.1O).

[0156] Tumor-associated antigens the targeting moiety may comprise or to which the targeting moiety may be directed can be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancers, as examples. Exemplary tumor- associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO 99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. See, e.g., U.S. Patent No. 6,544,518. Prostate cancer tumor- associated antigens include, for example, prostate specific membrane antigen (PSMA), pro state- specific antigen (PSA), prostate-carcinoma tumor antigen-1 (PCTA-1), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP). Other tumor associated antigens include Plu-1, HASH-1, HasH-2, Cripto and Criptin. Additionally, a tumor antigens may be a self -peptide hormone, such as whole length gonadotrophin hormone releasing hormone (GnRH), a short 10 amino acid long peptide, useful in the treatment of many cancers.

[0157] Antigens may include epitopic regions or epitopic peptides derived from genes expressed by or mutated in normal or tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, telomerase reverse transcriptase, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Herl, Her2 / neu, mutated or wild type p53, cytochrome P450 1B1, and abnormally expressed intron sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens that include epitopic regions or epitopic peptides derived from oncoviral processes, such as humanpapilloma virus proteins E6 and E7; Epstein bar virus proteins LMP1 and LMP2; nonmutated oncofetal proteins with a tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein .

[0158] In other aspects, instead of a human cellular antigen, such as a cancer antigen (tumor antigen), antigens the targeting moiety may comprise or to which the targeting moiety may be directed are obtained or derived from a pathogenic microorganism or from an opportunistic pathogenic microorganism (also called herein an infectious disease microorganism), such as a virus, fungus, parasite, and bacterium. In certain embodiments, antigens derived from such a microorganism to which the targeting moiety can be directed include full-length proteins.

[0159] Illustrative pathogenic organisms having antigens the targeting moiety may comprise or to which the targeting moiety may be directed include, but are not limited to, severe acute respiratory syndrome (SARS) (e.g., SARS-CoV-2), human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Influenza A, B, and C, vesicular stomatitis virus (VSV), vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, Staphylococcus species including Methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species including Streptococcus pneumoniae. As would be understood by the skilled person, proteins derived from these and other pathogenic microorganisms for use as antigen as described herein and nucleotide sequences encoding the proteins may be identified in publications and in public databases such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0160] Antigens derived from severe acute respiratory syndrome (SARS) (e.g., SARS- CoV-2) include the spike protein (SI and / or S2 subunit), the nucleocapsid protein, the membrane protein, and / or the envelope protein, and / or antigenic portions or fragments thereof. The spike protein is a large, complex protein that is essential for the entry of SARS-CoV-2 into host cells. It has two main domains: the N-terminal domain (NTD) and the C-terminal domain (CTD). The NTD binds to the host cell receptor, ACE2, while the CTD mediates fusion of the viral envelope with the host cell membrane. The nucleocapsid protein is a small, globular protein that forms a helical capsid that surrounds the viral RNA genome. It is essential for the stability and replication of the viral RNA genome. The membrane protein is a small, hydrophobic protein that is located between the spike protein and the nucleocapsid protein. It helps to anchor the spike protein to the surface of the virus and to protect the viral RNA genome from the environment. The envelope protein is a small, glycosylated protein that is located onthe surface of the virus. It helps to protect the virus from the environment and to mediate the entry of the virus into host cells.

[0161] Antigens derived from human immunodeficiency virus (HIV) include any of the HIV virion structural proteins (e.g., gpl20, gp41, pl7, and p24), protease, reverse transcriptase, or HIV proteins encoded by tat, rev, nef, vif, vpr and vpu.

[0162] Antigens derived from herpes simplex virus (e.g., HSV 1 and HSV2) include, but are not limited to, proteins expressed from HSV late genes. The late group of genes predominantly encodes proteins that form the virion particle. Such proteins include the five proteins from (UL) which form the viral capsid: UL6, UL18, UL35, UL38 and the major capsid protein UL19, UL45, and UL27, each of which may be used as an antigen as described herein. Other illustrative HSV proteins contemplated for use as antigens herein include the ICP27 (Hl, H2), glycoprotein B (gB) and glycoprotein D (gD) proteins. The HSV genome comprises at least 74 genes, each encoding a protein that could potentially be used as an antigen.

[0163] Antigens derived from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed during the immediate early and early phases of virus replication, glycoproteins I and III, capsid protein, coat protein, lower matrix protein pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the cluster of genes from UL128-UL150 (Rykman, et al., 2006), envelope glycoprotein B (gB), gH, gN, and ppi 50. As would be understood by the skilled person, CMV proteins for use as antigens described herein may be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[0164] Antigens derived from Epstein-Ban virus (EBV) that are contemplated for use in certain embodiments include EBV lytic proteins gp350 and gpl lO, EBV proteins produced during latent cycle infection including Epstein-Ban nuclear antigen (EBNA)-l, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP) and latent membrane proteins (LMP)-l, LMP-2A and LMP-2B (see, e.g., Lockey et al., 2008).

[0165] Antigens derived from respiratory syncytial virus (RSV) that are contemplated for use herein include any of the eleven proteins encoded by the RSV genome, or antigenic fragments thereof: NS 1, NS2, N (nucleocapsid protein), M (Matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription regulation), RNA polymerase, and phosphoprotein P.

[0166] Antigens derived from vesicular stomatitis virus (VSV) that are contemplated for use include any one of the five major proteins encoded by the VSV genome, and antigenicfragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

[0167] Antigens derived from an influenza virus that are contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins Ml and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0168] Exemplary viral antigens the targeting moiety may comprise or to which the targeting moiety may be directed also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., a calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (a hepatitis B core or surface antigen, a hepatitis C virus El or E2 glycoproteins, core, or non-structural proteins), herpesvirus polypeptides (including a herpes simplex virus or varicella zoster virus glycoprotein), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papilloma virus polypeptides, parainfluenza virus polypeptides (e.g., the hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picorna virus polypeptides (e.g., a poliovirus capsid polypeptide), pox virus polypeptides (e.g., a vaccinia virus polypeptide), rabies virus polypeptides (e.g., a rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0169] In certain aspects, the antigens the targeting moiety may comprise or to which the targeting moiety may be directed may be bacterial antigens. In certain aspects, bacterial of interest antigens may be a secreted polypeptide. In other certain aspects, bacterial antigens include antigens that have a portion or portions of the polypeptide exposed on the outer cell surface of the bacteria.

[0170] Antigens derived from Staphylococcus species including Methicillin-resistant Staphylococcus aureus (MRS A) that are contemplated for use include virulence regulators, such as the Agr system, Sar and Sae, the Ari system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP. Other Staphylococcus proteins that may serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes for two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As would beunderstood by the skilled person, Staphylococcus proteins for use as antigens may also be identified in other public databases such as GenBank®, Swiss-Prot®, and TrEMBL®.

[0171] Antigens derived from Streptococcus pneumoniae that are contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline -binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as an antigen in some embodiments (see, e.g., Zysk et al., 2000). The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced and, as would be understood by the skilled person, S. pneumoniae proteins for use herein may also be identified in other public databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for antigens according to the present disclosure include virulence factors and proteins predicted to be exposed at the surface of the pneumococci (see, e.g., Frolet et al., 2010).

[0172] Examples of bacterial antigens the targeting moiety may comprise or to which the targeting moiety may be directed include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae type b outer membrane protein), Helicobacter polypeptides, Klebsiella polypeptides, L-form bacteria polypeptides, Eeptospira polypeptides, Eisteria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcus polypeptides (e.g., S. pyogenes M proteins), group B streptococcus (.S'. agalactiae') polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis Fl and V antigens).

[0173] Examples of fungal antigens the targeting moiety may comprise or to which the targeting moiety may be directed include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Altemaria polypeptides, Aspergillus polypeptides, Basidioboluspolypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

[0174] Examples of protozoan parasite antigens the targeting moiety may comprise or to which the targeting moiety may be directed include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Eeishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides. Examples of helminth parasite antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Eagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, N anophy etus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides.(e.g., P. falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSAl c-term), and exported protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0175] Examples of ectoparasite antigens the targeting moiety may comprise or to which the targeting moiety may be directed include, but are not limited to, polypeptides (including antigens as well as allergens) from fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs.V. Antibodies

[0176] Aspects of the disclosure relate to targeting moieties useful, alone or in combination with pseudotyped viral envelopes, for activation, proliferation, and / or differentation of B cells. The targeting moieties can be, e.g., an antibody that specifically binds to an antigen or a cell surface molecule, or any antigen, ligand, or binding partner of a cell surface molecule (e.g., a viral envelope protein) (e.g., Sindbis El, E2, or E3 protein).

[0177] In some aspects, the targeting moiety is an antibody, an antibody-like molecule, or an antigen-binding fragment thereof. In some aspects, the targeting moiety is an antibody, a nanobody, a minibody, an scFv fragment, or a Fab fragment. In some aspects, the targeting moiety is a human antibody, humanized antibody, recombinant antibody, chimeric antibody, an antibody derivative, a veneered antibody, a diabody, a monoclonal antibody, or a polyclonal antibody. In some aspects, the targeting moiety is a monoclonal antibody. In some aspects, the targeting moiety is a murine antibody. In some aspects, the targeting moiety is a chimeric antibody. In some aspects, the targeting moiety is a humanized antibody. In some aspects, the targeting moiety is a chimeric antibody. In some aspects, the targeting moiety is a human antibody.

[0178] As used herein, the term “antibody” refers to an intact immunoglobulin of any class or isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen. An isotype refers to the genetic variations or differences in the constant regions of the heavy and light chains of an antibody. In humans, there are five heavy chain isotypes: IgA, IgD, IgG, IgE, and IgM and two light chain isotypes: kappa and lambda. The IgG class is divided into four isotypes: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl,IgG2a, IgG2b and IgG3 in mice. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region.

[0179] The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a human antibody, a veneered antibody, a diabody, a humanized antibody, an antibody derivative, a recombinant antibody, a recombinant humanized antibody, an engineered antibody, a multi- specific antibody, a DARPin, or a derivative or fragment of each thereof. Also contemplated are antibodies having specificity for more than one antigen or target, including bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and other multispecific antibodies.

[0180] As used herein, an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgM, IgD, IgG, IgA, IgE, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity. Examples of such include but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region or any portion thereof or at least one portion of a binding protein. In certain aspects, the antibody or antigen binding fragment specifically binds the target antigen to which the targeting moiety is directed.

[0181] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that can interact with different antibodies.

[0182] The term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. Epitope determinants may include a plurality of chemical atoms or groups on an antigen surface, such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In some aspects, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some aspects, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some aspects, at least some such chemical atoms or groups are physically separated from one another when the antigen adoptsan alternative conformation e.g., is linearized). Generally, antibodies specific for a particular target antigen would recognize an epitope on the target antigen within a complex mixture.

[0183] The epitope regions of a given polypeptide can be identified using many different epitope mapping techniques well known in the art, including: x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, and hydrogen-deuterium exchange see, e.g., Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986), each of which is incorporated by reference herein in their entirety. Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0184] The term “immunogenic sequence” means a molecule that includes an amino acid sequence of at least one epitope such that the molecule can stimulate the production of antibodies in an appropriate host. The term “immunogenic composition” means a composition that comprises at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0185] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains, such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies as disclosed herein may be derived solely from a single source or may be “chimeric,” that is, different portions of the antibody may be derived from two different antibodies. For example, for chimeric antibodies, the variable regions may be derived from a rat or murine source, while the constant region is derived from a different animal source, such as a human. The antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term “antibody” includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013).

[0186] The term “variable region” refers to a portion of the antibody that gives the antibody its specificity for binding antigen. The variable region is typically located at the ends of the heavy and light chains. Variable loops of P-strands, three each on the light (VL) and heavy (Vn) chains are responsible for binding to the antigen. These loops are referred to as the “complementarity determining regions” (CDRs). In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0187] The term “constant region” refers to a portion of the antibody that is identical in all antibodies of the same isotype. The constant region differs in antibodies of different isotypes.

[0188] The term “light chain” may describe a full-length light chain or fragments thereof. A full-length light chain has a molecular weight of around 25,000 Daltons and includes a variable region domain (abbreviated herein as VL), and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (K) and lambda (X). The term “VL fragment” means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region, including CDRs. A VL fragment can further include light chain constant region sequences. The variable region domain of the light chain is at the amino-terminus of the polypeptide.

[0189] The term “heavy chain” may describe a full-length heavy chain or fragments thereof. For example, a full-length heavy chain for human IgGl has a molecular weight of around 50,000 Daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CHI, CH2, and CH3). The term “VH fragment” means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including CDRs. A VH fragment can further include heavy chain constant region sequences. The number of heavy chain constant region domains will depend on the isotype. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE and is defined by the heavy chains present of which there are five classifications: mu (p), delta (d), gamma (y), alpha (a), or epsilon (a) chains, respectively. Human IgG has several subtypes, including, IgGl, IgG2, IgG3, and IgG4.A. Types of Antibodies

[0190] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens. They may also be fragments (e.g., F(ab’)2, Fab’, Fab, Fv, and the like), including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins.

[0191] The term “monomer” means an antibody containing only one immunoglobulin unit. Monomers are the basic functional units of antibodies. The term “dimer” means an antibody containing two immunoglobulin units attached to one another via constant domainsof the antibody heavy chains (the Fc, or fragment crystallizable, region). The complex may be stabilized by a joining (J) chain protein. The term “multimer” means an antibody containing more than two immunoglobulin units attached to one another via constant domains of the antibody heavy chains (the Fc region). The complex may be stabilized by a joining (J) chain protein.

[0192] The term “bivalent antibody” means an antibody that comprises two antigenbinding sites. The two binding sites may have the same antigen specificities, or they may be bi-specific, meaning the two antigen-binding sites have different antigen specificities.

[0193] Bispecific antibodies are a class of antibodies that have paratopes (z.e., antigenbinding sites) for two or more distinct epitopes. Bispecific antibodies can be biparatopic, wherein a bispecific antibody may specifically recognize a different epitope from the same antigen. Bispecific antibodies can be constructed from a pair of different single domain antibodies termed “nanobodies.” Single domain antibodies may be sourced and modified from cartilaginous fish and camelids. Nanobodies can be joined together by a linker using techniques typical to a person skilled in the art; such methods for selection and joining of nanobodies are described in PCT Publication No. WO2015044386A1, No. W02010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which are specifically incorporated herein by reference in their entirety.

[0194] Bispecific antibodies can be constructed as: a whole IgG, Fab ’2, Fab ’PEG, a diabody, or alternatively as a single chain variable fragment (scFv). Diabodies and scFvs can be constructed without an Fc region, using only variable domains. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315- 321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which are specifically incorporated by reference in their entirety.

[0195] In certain aspects, the antigen-binding domain may be multispecific or hetero specific by multimerizing with VH and VL region pairs that bind a different antigen. For example, the antibody may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Accordingly, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and to other targets, such as Fc receptors on effector cells.

[0196] Multispecific antibodies can be used and directly linked via a short flexible polypeptide chain, using routine methods known in the art. One such example is diabodies thatare bivalent, bispecific antibodies with two antigen-binding sites in which the VH and VL domains are expressed on a single polypeptide chain and utilize a linker that is too short to allow for pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain creating two antigen binding sites. The linker functionality is applicable for aspects of triabodies, tetrabodies, and higher order antibody multimers, (see, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001), each of which is incorporated herein by reference in their entirety).

[0197] The part of the Fv fragment of an antibody molecule that binds with high specificity to the epitope of the antigen is referred to herein as the “paratope.” The paratope consists of the amino acid residues that contact the epitope of an antigen to facilitate antigen recognition. Each of the two Fv fragments of an antibody is composed of the two variable domains, VH and VL, in dimerized configuration. The primary structure of each of the variable domains includes three hypervariable loops separated by, and flanked by, framework regions (FRs). The hypervariable loops are the regions of highest primary sequences variability among the antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term “complementarity determining region” (CDR). The length of the hypervariable loops (or CDRs) varies between antibody molecules. The framework regions of all antibody molecules from a given mammal have high primary sequence similarity / consensus. The consensus of framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) which are interspersed among the framework regions. The hypervariable loops are given identifying names which distinguish their position within the polypeptide, and on which domain they occur. CDRs in the VL domain are identified as LI, L2, and L3, with LI occurring at the most distal end and L3 occurring closest to the CL domain. The CDRs may also be given the names CDR-L1, CDR-L2, and CDR-L3. The L3 (CDR-L3) is generally the region of highest variability among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain arranged linearly in the primary structure and separated from each other by ERs. The amino terminal (N-terminal) end of the VL chain is named ERL The region identified as ER2 occurs between LI and L2 hypervariable loops. ER3 occurs between L2 and L3 hypervariable loops, and the ER4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which includes three CDRs identified as Hl, H2, and H3, or CDR-H1, CDR-H2 and CDR-H3. The majority of amino acid residues in the variable domains, or Ev fragments (VH and VL), are part of the ERs (approximately 85%).

[0198] Several methods have been developed and can be used by one skilled in the art to identify the exact amino acids that constitute each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms, which identify the conserved amino acid residues that make up the framework regions, therefore identifying the CDRs that may vary in length but are located between framework regions. Three commonly used methods have been developed for identification of the CDRs of antibodies: Kabat (as described in T. T. Wu and E. A. Kabat, J Exp Med, 132(2): 211-50 (1970)); Chothia (as described in C. Chothia et al., Nature, 342(6252): 877-83 (1989)); and IMGT (as described in M.-P. Lefranc et al., Developmental & Comparative Immunology , 27(1): 55-77 (2003)). These methods each include unique numbering systems for the identification of the amino acid residues that constitute the variable regions. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen occur in the CDRs, although in some cases, residues within the framework regions contribute to antigen binding. Depending on the type and size of the antigen, different CDR residues may contact the antigen. See Almagro JC. J Mol Recognit. 17(2): 132-43 (2004), incorporated herein by reference.

[0199] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include:

[0200] 1) Computational predictions of the tertiary structure of the antibody / epitope binding interactions based on the chemical nature of the amino acid sequence of the antibody variable region and composition of the epitope.

[0201] 2) Hydrogen-deuterium exchange and mass spectroscopy.

[0202] 3) Polypeptide fragmentation and peptide mapping approaches in which one generates multiple overlapping peptide fragments from the full length of the polypeptide and evaluates the binding affinity of these peptides for the epitope.

[0203] 4) Antibody Phage Display Library analysis in which the antibody Fab fragment encoding genes of the mammal are expressed by bacteriophage in such a way as to be incorporated into the coat of the phage. This population of Fab expressing phage are then allowed to interact with the antigen which has been immobilized or may be expressed in by a different exogenous expression system. Non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be readily isolated and the genes which encode them determined. This approach can also be used for smaller regions of the Fab fragment including Fv fragments or specific VH and VL domains as appropriate.

[0204] In certain aspects, affinity matured antibodies are enhanced with one or more modifications in one or more CDRs thereof (and / or one or more FRs thereof) that result in an improvement in the affinity of the antibody for a target antigen as compared to a parent antibody that does not possess those alteration(s). Certain affinity matured antibodies will have nanomolar or picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art, e.g., Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and / or framework residues employed in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009) in conjugation with computation methods as demonstrated in Tiller et al., Front. Immunol. 8:986 (2017), each of which references are incorporated herein by reference in their entirety.

[0205] Chimeric immunoglobulins are the products of fused genes derived from different species (the various domains of the antibodies’ heavy and light chains are coded for by DNA from more than one species; see, e.g., U.S. Pat. No. 4,816,567); “humanized” antibodies generally have the FRs from human immunoglobulins and one or more CDRs are from a non-human source e.g., murine).

[0206] As used herein, the term “humanized antibody” or “humanized immunoglobulin” refers to a human / non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a variable region of the recipient are replaced by residues from a variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and capacity. Humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. The humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, a non-human antibody containing one or more amino acids in a framework region, a constant region or a CDR, that have been substituted with a correspondingly positioned amino acid from a human antibody. In general, humanized antibodies are expected to produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody. The humanized antibodies may have conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions. Conservative substitutions groupings include: glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, serinethreonine and asparagine-glutamine. Humanization or engineering of antibodies can beperformed using any known method such as, but not limited to, those described in U.S. Pat. Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567; each incorporated by reference herein in its entirety.

[0207] Minimizing the antibody polypeptide sequence from the non-human species can optimize chimeric antibody function and reduces immunogenicity. Specific amino acid residues of the non-human antibody are modified to be homologous to corresponding residues in a human antibody. One example is the “CDR-grafted” antibody, in which an antibody comprises one or more CDRs from a particular species or belonging to a specific antibody class or subclass, while the remainder of the antibody chain(s) is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. In some instances, corresponding non-human (e.g., murine) residues replace framework region residues of the human immunoglobulin. Replacement of human framework region residues with non-human framework region residues may serve to improve and / or restore antigen binding. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody to further refine performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988); Almagro et al., Front Immunol 8; 1751 (2018); and Payes et al., “Genetic Engineering of Antibody Molecules,” In: Reviews in Cell Biology and Molecular Medicine. John Wiley & Sons, Inc., Hoboken, New Jersey, USA. 1(3): 1-52 (2015), each of which is incorporated by reference herein in its entirety.

[0208] Intrabodies are intracellularly localized immunoglobulins that bind to intracellular antigens as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0209] Antibodies also include “linear antibodies.” The procedure for making linear antibodies is known in the art and described in Zapata et al., 1995. Briefly, these antibodies comprise a pair of tandem Ed segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0210] The terms “polyclonal antibody” or “polyclonal antibody composition” as used herein refer to a preparation of antibodies that are derived from different B-cell lines. They area mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. Thus, polyclonal antibody preparations typically include different antibodies against different determinants (epitopes). In order to produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with the antigen or antigen fragment, generally with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are subsequently collected from the sera of the host. The polyclonal antibody can be affinity purified against the antigen rendering it monospecific.

[0211] A monoclonal antibody or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies from an exclusive parental cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant (epitope). Monoclonal antibodies are highly specific, as each monoclonal antibody is directed against a single determinant on the antigen.B. Functional Antibody Fragments and Antigen-Binding Fragments1. Antigen-Binding Fragments

[0212] Certain aspects relate to antibody fragments, such as antibody fragments that bind to antigen, and targeting moieties may comprise antibody fragments. The term functional antibody fragment includes antigen-binding fragments of an antibody that retain the ability to specifically bind to an antigen. These fragments are constituted of various arrangements of the variable region heavy chain (VH) and / or light chain (VL) and can include constant region heavy chain 1 (CHI) and light chain (CL). In some aspects, they lack the Fc region constituted of heavy chain 2 (CH2) and 3 (CH3) domains. Aspects of antigen binding fragments and the modifications thereof may include: (i) the Fab fragment type constituted with the VL, VH, CL, and CHI domains; (ii) the Fd fragment type constituted with the VH and CHI domains; (iii) the Fv fragment type constituted with the VH and VL domains; (iv) the single domain fragment type, dAb, (Holt et al. Trends Biotechnol. 21(11):484-90 (2003)) constituted with a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Fane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston etal., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015), each of which are incorporated by reference in their entirety.

[0213] Antigen-binding fragments also include fragments of an antibody that retain exactly, at least, or at most 1, 2, or 3 CDRs from a light chain variable region. Fusions of CDR- containing sequences to an Fc region (or a CH2 or CH3 region thereof) are included within the scope of this definition including, for example, scFv fused, directly or indirectly, to an Fc region are included herein.

[0214] The term Fab fragment means a monovalent antigen-binding fragment of an antibody containing the variable (VL and VH) and the constant (CL and CHI) domains. The term Fab’ fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab’ fragment includes the VL, VH, CL and CHI domains and all or part of the hinge region. The term F(ab’)2 fragment means a bivalent antigen-binding fragment of a monoclonal antibody comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. An F(ab’)2 fragment includes, for example, all or part of the two VH and VL domains and can further include all or part of the two CL and CHI domains.

[0215] The term Fd fragment means a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the VH, including the CDRs. An Fd fragment can further include CHI region sequences.

[0216] The term Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of the VL and VH, and absent of the CL and CHI domains. The VL and VH include, for example, the CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding fragment. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are herein incorporated by reference. The term (scFv)2 means bivalent or bispecific sFv polypeptide chains that include oligomerization domains at their C-termini, separated from the sFv by a hinge region. The oligomerization domain comprises self-associating a-helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as “miniantibodies” or “minibodies.”

[0217] A single domain antibody is an antigen-binding fragment containing only a VH or the VL domain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.2. Fragment Crystallizable (Fc) Region

[0218] An Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing a hinge region that promotes dimerization are included.C. Polypeptides with Antibody CDRs & Scaffolding Domains that Display the CDRs

[0219] Antigen-binding peptide scaffolds, such as CDRs, are used to generate proteinbinding molecules in accordance with the aspects. Generally, a person skilled in the art can determine the type of protein scaffold on which to graft at least one of the CDRs. It is known that scaffolds, optimally, must meet a number of criteria such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and / or be easy to produce, express, and purify. Skerra, J Mol Recognit, 13:167- 87 (2000).

[0220] The protein scaffolds can be sourced from but are not limited to fibronectin type III FN3 domain (known as “monobodies”), fibronectin type III domain 10, lipocalin, anticalin, Z- domain of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the “ankyrin repeat”, the “armadillo repeat”, the “leucine-rich repeat” and the “tetratricopeptide repeat”. Such proteins are described in US Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. W02006 / 056464, each of which are specifically incorporated herein by reference in their entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibitors of neuronal nitric oxide synthase (PIN) may also be used.D. Antibody Binding

[0221] The term “selective-binding agent”, “antigen-binding agent”, or “antigenbinding protein” refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab’, F(ab’)2, single chain antibodies, peptides, peptide fragments and proteins.

[0222] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. “Immunologically reactive” means that the selective binding agent or antibody of interest will bind with antigens present in a biological sample. The term “immune complex” refers the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.1. Affinity / Avidity

[0223] The term “affinity” refers the strength with which an antibody or selective binding agent binds an epitope. In antibody binding reactions, this is expressed as the affinity constant (Kaor kasometimes referred to as the association constant) for any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of the antibody to its antigen relative to the binding strength of the antibody to an unrelated amino acid sequence. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and / or selective binding agent.

[0224] There are several experimental methods that can be used by one skilled in the art to evaluate the binding affinity of any given antibody or selective binding agent for its antigen. This is generally done by measuring the equilibrium dissociation constant (KD or Ka), using the equation KD = koff / kon= [A] [B] / [AB] . The term koff is the rate of dissociation between the antibody and antigen per unit time and is related to the concentration of antibody and antigen present in solution in the unbound form at equilibrium. The term konis the rate of antibody and antigen association per unit time and is related to the concentration of the bound antigen-antibody complex at equilibrium. The units used for measuring the KD are mol / L (molarity, or M), or concentration. The Kaof an antibody is the inverse of the KD and is determined by the equation Ka= 1 / KD. Examples of some experimental methods that can be used to determine the KD value are enzyme-linked immunosorbent assays (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE).

[0225] Antibodies deemed useful in certain aspects may have an equilibrium dissociation constant of at least, at most, exactly, or between (inclusive or exclusive) any two of about 10'6, IO’7, 10'8, 10'9, IO0M, 101M, 1042M, or any range derivable therein. These values are reported for antibodies discussed herein and the same assay may be used to evaluate the binding properties of such antibodies. An antibody of the disclosure is said to “specificallybind” its target antigen when the dissociation constant (KD) is about 10'8M. The antibody specifically binds antigen with “high affinity” when the KD is about 5xl0'9M, and with “very high affinity” when the KD is about 5xl0'12M.2. Epitope Specificity

[0226] The epitope of an antigen is the specific region of the antigen for which an antibody has binding affinity. In the case of protein or polypeptide antigens, the epitope is the specific residues (or specified amino acids or protein segment) that the antibody binds with high affinity. An antibody does not necessarily contact every residue within the protein. Nor does every single amino acid substitution or deletion within a protein necessarily affect binding affinity. For purposes of this specification and the accompanying claims, the terms “epitope” and “antigenic determinant” are used interchangeably to refer to the site on an antigen to which B and / or T cell receptors respond or recognize. Polypeptide epitopes can be formed from both contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide. In some aspects, an epitope includes at least 3, for example 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, in a unique spatial conformation.

[0227] Epitope specificity of an antibody can be determined in a variety of ways. One approach, for example, involves testing a collection of overlapping peptides of about 15 amino acids spanning the full sequence of the protein and differing in increments of a small number of amino acids (e.g., 3 to 30 amino acids). The peptides are immobilized in separate wells of a microtiter dish. Immobilization can be accomplished, for example, by biotinylating one terminus of the peptides. This process may affect the antibody affinity for the epitope, therefore different samples of the same peptide can be biotinylated at the N and C terminus and immobilized in separate wells for the purposes of comparison. This is useful for identifying end-specific antibodies. Optionally, additional peptides can be included terminating at a particular amino acid of interest. This approach is useful for identifying end- specific antibodies to internal fragments. An antibody or antigen-binding fragment is screened for binding to each of the various peptides. The epitope is defined as a segment of amino acids that is common to all peptides to which the antibody shows high affinity binding.

[0228] It also is possible to determine without undue experimentation, whether an antibody has the same specificity as the antibody of this disclosure by determining whether the antibody being tested prevents an antibody of this disclosure from binding the protein or polypeptide with which the antibody is normally reactive. If the antibody being tested competeswith the antibody of the disclosure as shown by a decrease in binding by the monoclonal antibody of this disclosure, then it is likely that the two antibodies bind to the same or a closely related epitope. Alternatively, one can pre-incubate the antibody of this disclosure with a protein with which it is normally reactive, and determine if the antibody being tested is inhibited in its ability to bind the antigen. If the antibody being tested is inhibited then, likely, it has the same, or a closely related, epitopic specificity as the antibody of this disclosure.3. Modification of Antigen-Binding Domains

[0229] It is understood that the antibodies of the present disclosure may be modified to yield variant antibodies. Variant antibodies are substantially identical to the antibody polypeptide sequences, or fragments thereof, and still bind the epitopes of the present disclosure. Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP, or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.

[0230] As discussed herein, minor variations in the amino acid sequences of antibodies or antigen-binding regions thereof are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and most preferably at least 99% sequence identity. In some aspects, conservative amino acid replacements are contemplated.

[0231] The variable region of the antibodies of the present disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0232] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0233] In addition, the antibodies of the disclosure may be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Such modifications include, but are not limited to, alterations of the number of cysteine residues in the hinge region to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody (U.S. Pat. No. 5,677,425) and amino acid mutations in the Fc hinge region to decrease the biological half-life of the antibody (U.S. Pat. No. 6,165,745).

[0234] Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Certain preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Standard methods to identify protein sequences that fold into a known three-dimensional structure are available to those skilled in the art; Dill and McCallum., Science 338:1042-1046 (2012). Several algorithms for predicting protein structures and the gene sequences that encode these have been developed, and many of these algorithms can be found at the National Center for Biotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov / guide / proteins / ) and at the Bioinformatics Resource Portal (on the World Wide Web at expasy.org / proteomics). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure.

[0235] It is also contemplated that the antigen-binding domain may be multi- specific or multivalent by multimerizing the antigen-binding domain with VH and VL region pairs that bind either the same antigen (multi- valent) or a different antigen (multi- specific).E. Enzymatic or Chemical Modification of Antibodies

[0236] Additionally, the antibodies of the disclosure may be enzymatically or chemically modified to produce further derivatives of the antibodies and antigen binding fragments that are described herein. The term “antibody derivative” can include post- translational modification to the linear polypeptide sequence of the antibody or fragment. Thederivatized antibody or fragment thereof may comprise any molecule or substance that imparts a desired property to the antibody or fragment.

[0237] The derivatized antibody can comprise, for example, a chemical post- translational modification, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, or a detectable bead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a particular use (e.g. , administration to a subject, such as a human subject, or other in vivo or in vitro uses). An antibody or fragment thereof can be covalently attached to a molecule or substance, such as a labeling moiety or a therapeutic moiety; covalent attachment does not prevent the antibody from generating an anti-idiotypic response. An antibody or fragment thereof can be non-covalently attached to a molecule or substance, such as a labeling moiety or a therapeutic moiety.

[0238] Optionally, an antibody or an antigen-binding fragment can be chemically conjugated to, or expressed as, a fusion protein with other proteins. In some aspects, polypeptides may be chemically modified by conjugating or fusing the polypeptide to serum protein, such as human serum albumin, to increase half-life of the resulting molecule. See, e.g., EP 0322094 and EP 0486525. In some aspects, the polypeptides may be conjugated to a diagnostic agent and used diagnostically, for example, to monitor the development or progression of a disease and determine the efficacy of a given treatment regimen. In some aspects, the polypeptides may also be conjugated to a therapeutic agent to provide a therapy in combination with the therapeutic effect of the polypeptide.

[0239] In some aspects, disclosed are antibodies and antibody-like molecules that are linked to at least one agent to form an antibody conjugate or payload. To increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides and the like. By contrast, a reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands.1. Post- Translational Modifications

[0240] The antigen-binding protein can have or lack one or more post-translational modifications such as myristoylation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glypiation, acylation, acetylation, formylation, alkylation, methylation, amide bond formation, amidation at C-terminus, arginylation, polyglutamylation, polyglycylation, butyrylation, glycosylation, glycation, polysialylation, malonylation, hydroxylation, iodination, phosphorylation, adenylylation, propionylation, S- glutathionylation, S-nitrosylation, S-sulfenylation (aka S-sulphenylation), succinylation, sulfation, biotinylation, pegylation, SUMOylation, ubiquitination, neddylation, pupylation, disulfide bridges, or racemization. The antigen-binding protein can have reduced or increased amounts of one or more post-translational modifications as compared to the same antigenbinding protein expressed in the cell that is native to the encoded gene. The reduction or increase may be by at least or at most 25, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500% or more (or any range derivable therein).

[0241] In some aspects, contemplated are glycosylation variants of antibodies, wherein the number and / or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861, incorporated herein by reference). Antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. In other aspects, one or more new N-linked glycosylation sites are created.

[0242] Additional antibody variants include cysteine variants, wherein one or more cysteine residues in the parent or native amino acid sequence are deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia, when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewercysteine residues than the native antibody and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0243] In some aspects, the polypeptides can be pegylated to increase biological halflife by reacting the polypeptide with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the polypeptide. Polypeptide pegylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). Methods for pegylating proteins are known in the art and can be applied to the polypeptides of the disclosure to obtain PEGylated derivatives of antibodies. See, e.g., EP 0154316 and EP 0401384, incorporated herein by reference. In some aspects, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly (n- vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols, and polyvinyl alcohols. As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins.2. Conjugates

[0244] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. “Detectable labels” are compounds and / or elements that can be detected due to their specific functional properties, and / or chemical characteristics, the use of which allows the antibody to be detected, and / or further quantified if desired. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. The label may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and / or other property. Examples of detectable labels include, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. Examples of labels are, but not limited to, horseradish peroxidase (HRP), fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, nicotinamide adenine dinucleotide phosphate (NADPH), and a- or B-galactosidase.

[0245] In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases. Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).

[0246] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker. Suitable functional groups, including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0247] Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker. Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens / antibodies, e.g., rhodamine / anti-rhodamine, biotin / avidin and biotin / streptavidin.

[0248] Antibodies may also be coupled to low molecular weight haptens to increase the sensitivity of the antibody in an assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, whichreacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific antihapten antibodies. See, Harlow and Lane (1988) supra.

[0249] Antibody conjugates also include those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and / or to an enzyme to generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The uses of such labels are well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241; each incorporated herein by reference. Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light.

[0250] Additional suitable conjugated molecules include ribonuclease (RNase), DNase I, an antisense oligonucleotide, an inhibitory RNA molecule such as a siRNA molecule, an immuno stimulatory nucleic acid, aptamers, ribozymes, triplex forming molecules, and external guide sequences (e.g., guide RNAs). Aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets, and can bind small molecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Ribozymes are nucleic acid molecules that can catalyze a chemical reaction, either intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. Triplex forming function nucleic acid molecules can interact with double- stranded or single- stranded nucleic acid by forming a triplex, in which three strands of DNA form a complex dependent on both Watson-Crick and Hoogsteen basepairing. Triplex molecules can bind target regions with high affinity and specificity. The functional nucleic acid molecules may act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules may possess a de novo activity independent of any other molecules.

[0251] The antibodies of the disclosure or antigen-binding regions thereof can also be linked to another functional molecule such as another antibody or ligand for a receptor to generate a bi-specific or multi- specific molecule that binds to at least two or more different binding sites or target molecules. Linking of the antibody to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, can bedone, for example, by chemical coupling, genetic fusion, or noncovalent association. Multispecific molecules can further include a third binding specificity, in addition to the first and second target epitope.

[0252] The antibodies or fragments thereof of the present disclosure may be linked to a moiety that is toxic to a cell to which the antibody is bound to form “depleting” antibodies.

[0253] The antibodies of the disclosure may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0254] The antibodies also can be bound to many different carriers. Thus, this disclosure also provides compositions containing the antibodies and another substance, active or inert. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the disclosure. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.

[0255] In some aspects, contemplated are immunoconjugates comprising an antibody or antigen-binding fragment thereof conjugated (e.g., covalently attached) to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (z.e. , a radioconjugate). In this way, the agent of interest can be targeted directly to cells bearing the targeted cell surface antigen. The antibody and agent may be associated through non-covalent interactions such as through electrostatic forces, or by covalent bonds. Various linkers, known in the art, can be employed to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a genetic fusion protein. In one aspect, an antibody may be conjugated to various therapeutic substances to target the cell surface antigen. Examples of conjugated agents include, but are not limited to, metal chelate complexes, drugs, toxins, and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboranes, and radioactive halogens.

[0256] In antibody drug conjugates (ADC), an antibody is conjugated to one or more drug moieties through a linker. The ADC may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, toform antibody-L, via a covalent bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. ADC may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

[0257] Examples of ADC known to a person skilled in the art are pro-drugs useful for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu- Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278). In contrast, systematic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the target tumor cells (Baldwin et al. , Lancet 1:603-5 (1986); Thorpe, “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” In: Monoclonal Antibodies ‘84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506) (1985). Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21:183-87 (1986)).

[0258] In certain aspects, ADCs include covalent or aggregative conjugates of antibodies, or antigen-binding fragments thereof, with other proteins or peptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag (e.g., V5-His). Antibody-containing fusion proteins may comprise peptides added to facilitate purification or identification of the antibody (e.g., poly- His). An antibody polypeptide also can be linked to the FLAG® (Sigma- Aldrich, St. Louis, Mo.) peptide as described in Hopp et al., Bio / Technology 6:1204 (1988), and U.S. Pat. No. 5,011,912.

[0259] The conjugated agents can be linked to the antibody directly or indirectly, using any of a large number of available methods. Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety (Amon etal., 1985; Hellstrom et al., 1987; Thorpe, 1985; Baldwin et al., 1985; Thorpe et al., 1982).

[0260] Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6-diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).

[0261] Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.

[0262] Conjugates may also be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).

[0263] In some aspects, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site, are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).

[0264] Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature (O’Shannessy et al., J. Immunol. Methods 99(2): 153-61 (1987)).

[0265] Bi-specific and multi- specific molecules can be prepared using methods known in the art. For example, each binding unit of the hi-specific molecule can be generated separately and then conjugated to one another. When the binding molecules are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl- thioacetate (SATA), 5,5'-dithiobis(2-nitroberizoic acid) (DTNB), o-phenylenedimaleimide (oRDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfo succinimidyl 4-(N- maleimidomethyl)cyclohaxane-I-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). When the binding molecules are antibodies, they can be conjugated by sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains.VI. Antibody Production

[0266] Disclosed herein, in some aspects, are methods for generating antibodies. The methods can include identifying an antibody directed against a targeting moiety by contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudotyped viral envelope; identifying a B cell receptor that recognizes the targeting moiety; and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody. The nucleic acid molecules may be used to express large quantities of recombinant antibodies or to produce chimeric antibodies, single chain antibodies, antigen-binding fragments, immunoadhesins, diabodies, bi-specific antibodies, mutated antibodies, and other antibody derivatives. If the nucleic acid molecules are derived from a non-human, non-transgenic animal, the nucleic acid molecules may be used for antibody humanization. For example, the antibody, or an antigenbinding fragment thereof, can be generated by culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell. Such a process is described elsewhere herein.

[0267] In one aspect, the antibodies produced are neutralizing antibodies or antigenbinding fragment thereof. The term “neutralizing” refers to an antibody that may do one or more of: interfere with the receptor / protein interaction; reduce the concentration of receptor / protein interacted species in a subject or a cell; prevent the receptor / protein interaction in a subject or a cell; and / or reduce the biological function of a protein, which may include, but is not limited to, one or more of: promoting tumorigenesis or tumor progression; promoting cell growth and metastases; promoting inflammation, cell differentiation, adhesion, tumorigenesis, migration, invasion, and angiogenesis; promoting disease; and / or promoting infection.

[0268] Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, for purification, and for use as therapeutics are well known in the art as disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745, each incorporated herein by reference (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab’)2 fragments, Fab fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question. In certain aspects, polypeptides, peptides, and proteinsand immunogenic fragments thereof can also be synthesized in solution or on a solid support in accordance with conventional techniques.

[0269] Unless specified otherwise, the antibodies can be isolated from any suitable biological source, e.g., murine, rat, rabbit, goat, camelid, sheep, or canine.

[0270] In an example, a polyclonal antibody is prepared by immunizing an animal with an antigen or a portion thereof and collecting antisera from that immunized animal. The antigen may be altered compared to an antigen sequence found in nature. A variant or altered antigenic peptide or polypeptide can be employed to generate antibodies. Inocula are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent to form an aqueous composition. Antisera is subsequently collected by methods known in the arts, and the serum may be used as-is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

[0271] Methods of making monoclonal antibodies are also well known in the art e.g., U.S. Patent 4,196,265, herein incorporated by reference in its entirety for all purposes). Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition, e.g., a purified or partially purified protein, polypeptide, peptide, or domain. Resulting antibody-producing B-cells from the immunized animal, or all dissociated splenocytes, are then induced to fuse with cells from an immortalized cell line to form hybridomas. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing and have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Typically, the fusion partner includes a property that allows selection of the resulting hybridomas using specific media. For example, fusion partners can be hypoxanthine / aminopterin / thymidine (HAT)-sensitive.

[0272] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Next, selection of hybridomas can be performed by culturing the cells by singleclone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. Fusion procedures for making hybridomas, immunization protocols, and techniques for isolation of immunized splenocytes for fusion are known in the art.

[0273] For example, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3, Sp2 MAI, Sp2 SSI, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2 / O) or the like, or heteromyelomas, fusion products thereof, or any cell or fusion cell derived there from, or any other suitable cell line as known in the art, with antibody producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing-heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present disclosure. The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods.

[0274] Other techniques for producing monoclonal antibodies include the viral or oncogenic transformation of B -lymphocytes, a molecular cloning approach may be used to generate a nucleic acid or polypeptide, the selected lymphocyte antibody method (SLAM) (see, e.g., Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996), the preparation of combinatorial immunoglobulin phagemid libraries from RNA isolated from the spleen of the immunized animal and selection of phagemids expressing appropriate antibodies, or producing a cell expressing an antibody from a genomic sequence of the cell comprising a modified immunoglobulin locus using Cre-mediated site-specific recombination (see, e.g., U.S. 6,091,001).

[0275] Monoclonal antibodies may be further purified using filtration, centrifugation, and various chromatographic methods such as high-performance liquid chromatography (HPLC). Monoclonal antibodies may be further screened or optimized for properties relating to specificity, avidity, half-life, immunogenicity, binding association, binding disassociation,or overall functional properties relative to being a treatment for infection. Thus, monoclonal antibodies may have alterations in the amino acid sequence of CDRs, including insertions, deletions, or substitutions with a conserved or non-conserved amino acid.

[0276] Chimeric, humanized, or primatized antibodies of the present disclosure can be prepared based on the sequence of a reference monoclonal antibody prepared using standard molecular biology techniques. DNA encoding the heavy and light chain immunoglobulins can be obtained from the hybridoma of interest and engineered to contain non-reference (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (U.S. Pat. No. 4,816,567). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art (U.S. Pat. No. 5,225,539 and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370). Similarly, to create a primatized antibody, the murine CDR regions can be inserted into a primate framework using methods known in the art (WO 93 / 02108 and WO 99 / 55369).

[0277] Techniques for making partially to fully human antibodies are known in the art and any such techniques can be used. According to one aspect, fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody. (See for example, Russel et al., 2000; Gallo et al., 2000; Green, 1999; Yang et al., 1999A; Yang, 1999B; Jakobovits, 1998; Green and Jakobovits, 1998; Jakobovits, 1998; Tsuda et al., 1997; Sherman-Gold, 1997; Mendez et al., 1997; Jakobovits, 1996; Jakobovits, 1995; Mendez et al., 1995; Jakobovits, 1994; Arbones et al., 1994; Jakobovits, 1993; Jakobovits et al., 1993; U.S. Pat. No. 6,075,181).

[0278] Alternatively, the antibodies of this disclosure can also be modified to create veneered antibodies. Veneered antibodies are those in which the exterior amino acid residues of the antibody of one species are judiciously replaced or “veneered” with those of a second species so that the antibodies of the first species will not be immunogenic in the second species thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein is primarily dependent on the nature of its surface, the immunogenicity of an antibody could be reduced by replacing the exposed residues which differ from those usually found in another mammalian species antibodies. This judicious replacement of exterior residues should havelittle, or no, effect on the interior domains, or on the interdomain contacts. Thus, ligand binding properties should be unaffected because of alterations which are limited to the variable region framework residues. The process is referred to as “veneering” since only the outer surface or skin of the antibody is altered, the supporting residues remain undisturbed.

[0279] The procedure for “veneering” makes use of the available sequence data for human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Nonlimiting examples of the methods used to generate veneered antibodies include EP 519596; U.S. Pat. No. 6,797,492; and described in Padlan et al., 1991.

[0280] Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, cDNA, or the like, display library; e.g., as available from various commercial vendors such as MorphoSys (Martinsreid / Planegg, Del.), BioInvent (Lund, Sweden), Affitech (Oslo, Norway) using methods known in the art. Art known methods are described in the patent literature some of which include U.S. Pat. Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternative methods rely upon immunization of transgenic animals (e.g., SCID mice) (Nguyen et al., 1977; Sandhu et al., 1996); Eren et al., 1998), that can produce a repertoire of human antibodies, as known in the art and / or as described herein. Such techniques, include, but are not limited to, ribosome display (Wanes et al., 1997; Hanes et al., 1998); single cell antibody producing technologies (e,g., selected lymphocyte antibody method (“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al., 1987; Babcook et al., 1996); gel microdroplet and flow cytometry (Powell et al., 1990; Gray et al., 1995; Kenny et al., 1995); B-cell selection (Steenbakkers et al., 1994).

[0281] The antibodies of this disclosure can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification.

[0282] The immunogenicity of a particular immunogen composition can be enhanced using non-specific stimulators of the immune response, known as adjuvants. Adjuvants thatmay be used include, but are not limited to, interleukin- 1 (IL-1), IL-2, IL-4, IL-7, IL- 12, y- interferon (INF-y), granulocyte-macrophage colony- stimulating factor (GMCSF), Bacillus Calmette- Guerin (BCG), aluminum hydroxide, muramyl dipeptide (MDP) compounds, muramyl tripeptide phosphatidyl ethanolamine (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Exemplary adjuvants may include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and / or aluminum hydroxide adjuvant. In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM), such as but not limited to, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines such as INF-B, IL-2, or IL- 12, or genes encoding proteins involved in immune helper functions, such as B7-1 (CD80) or B7-2 (CD86). A phagedisplay system can be used to expand antibody molecule populations in vitro. Saiki, et al., Nature 324:163 (1986); Scharf et al., Science 233:1076 (1986); U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol. 254:392 (1995); Barbas, III et al., Methods: Comp. Meth Enzymol. (1995) 8:94; Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991).

[0283] Antibody fragments that retain the ability to recognize the antigen of interest will also find use herein. Several antibody fragments are known in the art that comprise antigenbinding sites capable of exhibiting immunological binding properties of an intact antibody molecule and can be subsequently modified by methods known in the arts. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. See, e.g., Inbar et al., Proc. Nat. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem. 19:4091-4096 (1980).

[0284] Single-chain variable fragments (scFvs) may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). SCFVS can form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., Prot. Eng. 10:423 (1997); Kort et al., Biomol. Eng. 18:95-108 (2001)). By combining different VL- and Vn-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., Biomol. Eng. 18:31-40 (2001)). Antigenbinding fragments are typically produced by recombinant DNA methods known to those skilled in the art. Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined using recombinant methods by a synthetic linker that enables themto be made as a single chain polypeptide (known as single chain Fv (sFv or scFv); see e.g., Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879- 5883 (1988). Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues and may include glutamic acid and lysine residues inserted to enhance solubility. Antigen-binding fragments are screened for utility in the same manner as intact antibodies. Such fragments include those obtained by N-terminal and / or C-terminal deletions, where the remaining amino acid sequence is substantially identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full- length cDNA sequence.

[0285] Also contemplated herein are non-peptide compounds having properties analogous to those of a template peptide. These types of non-peptide compounds are termed “peptide mimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987).

[0286] Also contemplated are “antibody like binding peptidomimetics” (ABiPs), which are peptide-like molecules that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods. These analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchere, Adv. Drug Res. 15:29 (1986); Veber and Freidiner, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference in their entirety for any purpose. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Such compounds are often developed with the aid of computerized molecular modeling. Generally, peptidomimetics of the disclosure are proteins that are structurally similar to an antibody displaying a desired biological activity, such as the ability to bind a protein, but have one or more peptide linkages optionally replaced by a linkage selected from: — CH2NH — , — CH2S — , — CH2 — CH2 — , — CH-CH — (cis and trans), — COCH2 — , — CH(OH)CH2 — , and — CH2SO — by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type e.g., D-lysine in place of L- lysine) may be used to generate more stable proteins. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387 (1992), incorporatedherein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

[0287] Once generated, a phage display library can be used to improve the immunological binding affinity of Fab molecules using known techniques. See, e.g., Figini el al., J. Mol. Biol. 239:68 (1994). The coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used.VII. Proteins & Nucleic Acids

[0288] In certain aspects, the current disclosure concerns proteins, polypeptides, and peptides, and polynucleotides encoding the proteins, polypeptides, and peptides. Proteins of the disclosure include pseudotyped viral envelope proteins. In some aspects, proteins of the disclosure include targeting moieties.

[0289] As used herein, the terms “wild type” or “WT” or “native” refer to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild type versions of a protein or polypeptide are employed, however, in other aspects of the disclosure, a modified protein or polypeptide is employed. The terms described above may be used interchangeably.

[0290] The polypeptides and / or polynucleotides of the disclosure may be modified or variant polypeptides and / or polynucleotides. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide having a chemical structure, particularly an amino acid sequence, that is altered with respect to the wild type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild type activity or function in other respects, such as immunogenicity.

[0291] The polypeptides and / or polynucleotides may be fragments of polypeptides and / or polynucleotides. The term “fragment,” with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of atruncated open reading frame that lacks the 3 '-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90%, or at least 99% of the amino acid residues from an amino acid sequence. In the present disclosure, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having sequence identity of at least, at most, exactly, or between (inclusive or exclusive) any two of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with a polypeptide, DNA nucleic acid or RNA nucleic acid sequence, from which it is derived.

[0292] In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 70% with a polypeptide or polynucleotide sequence, from which it is derived. In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 80% with a polypeptide or polynucleotide sequence, from which it is derived. In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 85% with a polypeptide or polynucleotide sequence, from which it is derived. In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 90% with a polypeptide or polynucleotide sequence, from which it is derived. In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 95% with a polypeptide or polynucleotide sequence, from which it is derived. In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 97% with a polypeptide or polynucleotide sequence, from which it is derived. In one aspect, a fragment of a polypeptide or polynucleotide sequence refers to a sequence having sequence identity of at least 99% with a polypeptide or polynucleotide sequence, from which it is derived.

[0293] As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some aspects, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciatedby those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some aspects, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).

[0294] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one aspect, one or more particular amino acid residues are changed using, for example, a site- directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed usin...

Claims

CLAIMS1. A method of transducing a B cell with a heterologous gene, comprising contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope.

2. A method of treating or preventing an infection, comprising transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope.

3. The method of claim 2, wherein the infection is a viral, bacterial, fungal, or parasitic infection.

4. A method of treating or preventing cancer, comprising transducing a B cell with a heterologous gene by contacting the B cell with a nucleic acid encoding a pseudotyped retroviral envelope.

5. The method of claim 4, wherein the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

6. A method of directing retroviral tropism to a B cell population, comprising contacting B cells of the B cell population with a nucleic acid encoding a pseudotyped retroviral envelope.

7. The method of claim 6, wherein the B cell population comprises B cells that express B cell receptors specific for the pseudotyped retroviral envelope and / or a targeting moiety.

8. The method of claim 6 or 7, wherein the B cell population comprises mature or differentiated B cells.

9. The method of claim 8, wherein the mature or differentiated B cells comprise plasma cells, germinal center-activated cells, and / or memory B cells.

10. A method of inducing B cell proliferation, comprising contacting B cells with a nucleic acid encoding a pseudo typed retroviral envelope.

11. A method of inducing B cell differentiation, comprising contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope.

12. A method of inducing B cell proliferation and differentiation, comprising contacting B cells with a nucleic acid encoding a pseudotyped retroviral envelope.

13. The method of claim 12, wherein B cell proliferation and B cell differentiation are induced simultaneously.

14. The method of any one of claims 1-13, wherein the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins.

15. The method of any one of claims 1-13, wherein the pseudotyped retroviral envelope comprises lentiviral envelope proteins.

16. The method of any one of claims 1-15, wherein the nucleic acid is comprised in a viral vector.

17. The method of claim 16, wherein the viral vector is a gammaretroviral vector or oncoretroviral vector.

18. The method of claim 16, wherein the viral vector is a lentiviral vector.

19. The method of any one of claims 1-15, wherein the nucleic acid is comprised in a viral genome.

20. The method of claim 19, wherein the viral genome is a gammaretroviral genome or oncoretroviral genome.

21. The method of claim 19, wherein the viral genome is a lentiviral genome.

22. The method of any one of claims 1-21, wherein the nucleic acid encoding the pseudotyped retroviral envelope or the viral vector or viral genome comprising the nucleic acid is comprised in a retrovirus.

23. The method of claim 22, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus.

24. The method of claim 22, wherein the retrovirus is a lentivirus.

25. The method of any one of claims 1-24, wherein the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3,wherein the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3, and wherein the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope.

26. The method of claim 25, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise: substitutions or deletions at amino acid residues 226 and / or 227 of El; substitutions or deletions at amino acid residues 68-71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60-64 of E3.

27. The method of claim 25 or 26, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted.

28. The method of claim 27, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG.

29. The method of any one of claims 25-28, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted.

30. The method of claim 29, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA.

31. The method of any one of claims 25-30, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted.

32. The method of claim 31, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA.

33. The method of any one of claims 25-32, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted.

34. The method of claim 33, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted.

35. The method of any one of claims 25-32, wherein the sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA.

36. The method of any one of claims 25-35, wherein the mutated Sindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68-71AAAA in E2, and the mutation KE159-160AA in E2.

37. The method of claim 36, wherein the mutated Sindbis envelope further comprises the mutation AK226-227SG in El.

38. The method of any one of claims 25-37, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viral genome comprising the nucleic acid.

39. The method of any one of claims 1-38, wherein the pseudotyped retroviral envelope further comprises one or more flexible linkers.

40. The method of any one of claims 1-39, wherein the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain.

41. The method of claim 40, wherein the detectable label comprises a FLAG peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12).

42. The method of claim 40 or 41, wherein the protein binding domain comprises an integrin-binding domain.

43. The method of claim 42, wherein the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21).

44. The method of claim 40 or claim 41, wherein the protein binding domain comprises an Fc-binding sequence.

45. The method of claim 44, wherein the Fc-binding sequence comprises a protein A ZZ amino acid sequence.

46. The method of claim 40 or claim 41, wherein the protein binding domain comprises a biotin-binding sequence.

47. The method of claim 46, wherein the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence.

48. The method of any one of claims 39-47, wherein the one or more flexible linkers comprise two flexible linkers.

49. The method of claim 46, wherein a first of the one or more flexible linkers is inserted at amino acid 70 of E2.

50. The method of claim 48 or 49, wherein the detectable label and / or the protein binding domain is comprised between the two flexible linkers.

51. The method of any one of claims 1-50, wherein the pseudotyped retroviral envelope further comprises a targeting moiety.

52. The method of any one of claims 1-51, wherein the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety.

53. The method of claim 52, wherein the nucleic acid is comprised in a viral vector.

54. The method of claim 52, wherein the nucleic acid is comprised in viral genome.

55. The method of any one of claims 1-55, wherein B cells express B cell receptors specific for the pseudotyped retroviral envelope, a targeting moiety, or a combination thereof.

56. The method of any one of claims 1-55, wherein the nucleic acid encoding the pseudo typed retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope and the targeting moiety is comprised in a retrovirus.

57. The method of claim 56, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus.

58. The method of claim 56, wherein the retrovirus is a lentivirus.

59. The method of any one of claims 51-58, wherein the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof.

60. The method of claim 59, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof.

61. The method of claim 59 or claim 60, wherein the pathogenic antigen comprises one or more viral envelope proteins.

62. The method of any one of claims 56-61 , wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

63. The method of claim 51, wherein the targeting moiety is conjugated to the pseudotyped retroviral envelope.

64. The method of claim 63, wherein the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope.

65. The method of claim 63 or 64, wherein the targeting moiety is covalently linked to the pseudo typed retroviral envelope.

66. The method of claim 63 or 64, wherein the targeting moiety is non-covalently linked to the pseudotyped retroviral envelope.

67. The method of any one of claims 63-66, wherein the targeting moiety comprises an antibody.

68. The method of claim 67, wherein the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof.

69. The method of claim 68, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof.

70. The method of claim 68 or claim 69, wherein the pathogenic antigen comprises one or more viral envelope proteins.

71. The method of any one of claims 68-70, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone,stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

72. The method of any one of claims 1-71, wherein transduction is mediated by molecular mechanisms independent of interaction of the pseudotyped retroviral envelope with cognate retrovirus receptors.

73. The method of any one of claims 1-72, wherein transduction is mediated by molecular mechanisms independent of interaction of the pseudotyped retroviral envelope with PtdSer receptors.

74. The method of any one of claims 1-73, wherein the nucleic acid encoding the pseudotyped retroviral envelope and, optionally, the targeting moiety, also encodes the heterologous gene.

75. The method of claim 74, wherein the heterologous gene is operably linked to a promoter.

76. The method of claim 75, wherein the promoter is a tissue-specific promoter.

77. The method of any one of claims 1-76, wherein the B cell is transduced in vivo.

78. The method of any one of claims 1-77, wherein the B cell is contacted in vivo.

79. The method of any one of claims 1-78, wherein the nucleic acid encoding a pseudo typed retroviral envelope is administered to a subject.

80. The method of claim 79, wherein the nucleic acid encoding the pseudotyped retroviral envelope is administered intravenously.

81. The method of any one of claims 1-76, wherein the B cell is transduced ex vivo.

82. The method of claim 81, wherein the B cell is a primary B cell.

83. The method of claim 81, wherein the B cell is a B cell from a B cell line.

84. The method of any one of claims 1-76, wherein the B cell is transduced in vitro.

85. The method of claim 84, wherein the B cell is a primary B cell.

86. The method of claim 84, wherein the B cell is a B cell from a B cell line.

87. A method of inducing an immune response against an infection, disease, or condition associated with a targeting moiety in a subject in need thereof, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudo typed retroviral envelope.

88. The method of claim 87, wherein the B cell expresses a receptor to which the targeting moiety is directed.

89. The method of claim 87 or 88, wherein the B cell expresses receptors specific for the pseudotyped retroviral envelope, the targeting moiety, or a combination thereof.

90. The method of any one of claims 1-88, wherein contacting the B cell with the targeting moiety and the nucleic acid encoding a pseudotyped retroviral envelope induces proliferation and / or differentiation of the B cell.

91. The method of any one of claims 87-90, wherein the immune response comprises endogenous antibody production by the B cell.

92. A method of inducing proliferation and / or differentiation of a B cell expressing a receptor to which a targeting moiety is directed, comprising contacting the B cell with the targeting moiety and a nucleic acid encoding a pseudotyped retroviral envelope.

93. The method of claim 92, wherein the B cell is contacted with the targeting moiety prior to contacting the B cell with the nucleic acid encoding the pseudotyped retroviral envelope.

94. The method of claim 92, wherein the B cell is simultaneously contacted with the targeting moiety and the nucleic acid encoding the pseudo typed retroviral envelope.

95. The method of claim 92, wherein the B cell is contacted with the targeting moiety after contacting the B cell with the nucleic acid encoding the pseudo typed retroviral envelope.

96. The method of claim 92, wherein the B cell is contacted with the targeting moiety prior to and simultaneously with contacting the B cell with the nucleic acid encoding the pseudo typed retroviral envelope.

97. The method of claim 92, wherein the B cell is contacted with the targeting moiety simultaneously with and after contacting the B cell with the nucleic acid encoding the pseudo typed retroviral envelope.

98. The method of claim 92, wherein the B cell is contacted with the targeting moiety prior to, simultaneously with, and after contacting the B cell with the nucleic acid encoding the pseudo typed retroviral envelope.

99. A method of preventing, treating, and / or ameliorating an infection, disease, or condition associated with a targeting moiety in a subject in need thereof, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudo typed retroviral envelope.

100. A method of vaccinating a subject against an infection, disease, or condition associated with a targeting moiety, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudo typed retroviral envelope.

101. A method of generating neutralizing antibodies against an infection, disease, or condition associated with a targeting moiety, comprising contacting a B cell with the targeting moiety and a nucleic acid encoding pseudotyped retroviral envelope.

102. The method of any one of claims 87-101, wherein the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins.

103. The method of any one of claims 87-101, wherein the pseudotyped retroviral envelope comprises lentiviral envelope proteins.

104. The method of any one of claims 87-103, wherein the nucleic acid is comprised in a viral vector.

105. The method of claim 104, wherein the viral vector is a gammaretroviral vector or oncoretroviral vector.

106. The method of claim 104, wherein the viral vector is a lentiviral vector.

107. The method of any one of claims 87-103, wherein the nucleic acid is comprised in a viral genome.

108. The method of claim 107, wherein the viral genome is a gammaretroviral genome or oncoretro viral genome.

109. The method of claim 107, wherein the viral genome is a lentiviral genome.

110. The method of any one of claims 87-109, wherein the nucleic acid encoding the pseudotyped retroviral envelope or the viral vector or viral genome comprising the nucleic acid is comprised in a retrovirus.

111. The method of claim 110, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus.

112. The method of claim 110, wherein the retrovirus is a lentivirus.

113. The method of any one of claims 87-112, wherein the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3, wherein the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3, and wherein the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope.

114. The method of claim 113, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise: substitutions or deletions at amino acid residues 226 and / or 227 of El; substitutions or deletions at amino acid residues 68-71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60-64 of E3.

115. The method of claim 113 or 114, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted.

116. The method of claim 115, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG.

117. The method of any one of claims 113-116, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted.

118. The method of claim 117, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA.

119. The method of any one of claims 113-118, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted.

120. The method of claim 119, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA.

121. The method of any one of claims 113-120, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted.

122. The method of claim 121, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted.

123. The method of any one of claims 113-120, wherein the sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA.

124. The method of any one of claims 113-123, wherein the mutated Sindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68-71AAAA in E2, and the mutation KE159-160AA in E2.

125. The method of claim 124, wherein the mutated Sindbis envelope further comprises the mutation AK226-227SG in El.

126. The method of any one of claims 113-125, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viral genome comprising the nucleic acid.

127. The method of any one of claims 87-126, wherein the pseudotyped retroviral envelope further comprises one or more flexible linkers.

128. The method of any one of claims 87-127, wherein the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain.

129. The method of claim 128, wherein the detectable label comprises a FLAG peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12).

130. The method of claim 128 or 129, wherein the protein binding domain comprises an integrin-binding domain.

131. The method of claim 130, wherein the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21).

132. The method of claim 128 or claim 129, wherein the protein binding domain comprises an Fc-binding sequence.

133. The method of claim 132, wherein the Fc-binding sequence comprises a protein A ZZ amino acid sequence.

134. The method of claim 128 or claim 129, wherein the protein binding domain comprises a biotin-binding sequence.

135. The method of claim 134, wherein the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence.

136. The method of any one of claims 127-135, wherein the one or more flexible linkers comprise two flexible linkers.

137. The method of claim 134, wherein a first of the one or more flexible linkers is inserted at amino acid 70 of E2.

138. The method of claim 136 or 137, wherein the detectable label and / or the protein binding domain is comprised between the two flexible linkers.

139. The method of any one of claims 87-138, wherein the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof.

140. The method of claim 139, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof.

141. The method of claim 139 or claim 140, wherein the one or more pathogenic antigens comprise one or more viral envelope proteins.

142. The method of any one of claims 139-141, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

143. The method of any one of claims 87-142, wherein the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety.

144. The method of claim 143, wherein the nucleic acid is comprised in a viral vector.

145. The method of claim 143, wherein the nucleic acid is comprised in viral genome.

146. The method of any one of claims 143-145, wherein the nucleic acid encoding the pseudo typed retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope the targeting moiety is comprised in a retrovirus.

147. The method of claim 146, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus.

148. The method of claim 146, wherein the retrovirus is a lentivirus.

149. The method of any one of claims 87-142, wherein the targeting moiety is conjugated to the pseudotyped retroviral envelope.

150. The method of claim 149, wherein the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope.

151. The method of claim 149 or 150, wherein the targeting moiety is covalently linked to the pseudotyped retroviral envelope.

152. The method of claim 149 or 150, wherein the targeting moiety is non-covalently linked to the pseudo typed retroviral envelope.

153. The method of any one of claims 149-152, wherein the targeting moiety comprises an antibody.

154. The method of claim 153, wherein the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof.

155. The method of claim 154, wherein the infectious agent is a viral, bacterial, fungal, or parasitic agent, or a combination thereof.

156. The method of claim 154 or claim 155, wherein the one or more antigenic molecules comprise one or more viral envelope proteins.

157. The method of any one of claims 154-156, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

158. The method of any one of claims 87-157, wherein B cells of the subject are contacted with the targeting moiety and the nucleic acid encoding the pseudotyped retroviral envelope a single time.

159. The method of any one of claims 87-158, wherein B cells of the subject are contacted with the targeting moiety and / or the nucleic acid encoding the pseudotyped retroviral envelope a multiple times.

160. The method of any one of claims 87-159, where B cells of the subject are contacted with the nucleic acid encoding the pseudotyped retroviral envelope multiple times.

161. The method of any one of claims 87-160, wherein B cells of the subject are not contacted with the targeting moiety more than a single time.

162. The method of any one of claims 87-161, wherein the targeting moiety and the nucleic acid encoding a pseudotyped retroviral envelope are comprised in a pharmaceutical composition.

163. The method of any one of claims 87-158, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

164. The method of any one of claims 87-163, wherein the subject is or is about less than 1 year of age, 1 year of age or older, 5 years of age or older, 10 years of age or older, 20 years ofage or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or older.

165. The method of any one of claims 87-164, wherein the B cell is contacted in vivo.

166. The method of any one of claims 87-165, wherein the targeting moiety and the nucleic acid encoding a pseudotyped retroviral envelope is administered to a subject.

167. The method of claim 87-166 wherein the targeting moiety and the nucleic acid encoding the pseudotyped retroviral envelope is administered intravenously.

168. The method of any one of claims 87-164, wherein the B cell is transduced ex vivo.

169. The method of claim 168, wherein the B cell is a primary B cell.

170. The method of claim 168, wherein the B cell is a B cell from a B cell line.

171. The method of any one of claims 87-164, wherein the B cell is transduced in vitro.

172. The method of claim 171, wherein the B cell is a primary B cell.

173. The method of claim 171, wherein the B cell is a B cell from a B cell line.

174. A method of identifying an antibody against a virus, comprising: contacting a B cell with a nucleic acid encoding a pseudo typed retroviral envelope; identifying a B cell receptor that recognizes the pseudotyped retroviral envelope; and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody.

175. A method of identifying an antibody directed against a targeting moiety, comprising: contacting a B cell with the targeting moiety and a nucleic acid encoding a pseudo typed retroviral envelope; identifying a B cell receptor that recognizes the targeting moiety; and sequencing the identified B cell receptor to provide a nucleic acid sequence encoding the antibody.

176. The method of claim 174 or 175, wherein the B cell expresses B cell receptors specific for the pseudotyped retroviral envelope, a targeting moiety, or a combination thereof.

177. The method of any one of claims 174-176, wherein the pseudotyped retroviral envelope comprises gammaretroviral envelope proteins or oncoretroviral envelope proteins.

178. The method of claim any one of claims 174-176, wherein the pseudotyped retroviral envelope comprises lentiviral envelope proteins.

179. The method of claim 178, wherein the nucleic acid is comprised in a viral vector.

180. The method of claim 179, wherein the viral vector is a gammaretroviral vector or oncoretroviral vector.

181. The method of claim 179, wherein the viral vector is a lentiviral vector.

182. The method of claim 178, wherein the nucleic acid is comprised in a viral genome.

183. The method of claim 182, wherein the viral genome is a gammaretroviral genome or oncoretroviral genome.

184. The method of claim 182, wherein the viral genome is a lentiviral genome.

185. The method of any one of claims 174-184, wherein the nucleic acid encoding the pseudotyped retroviral envelope or the viral vector or viral genome comprising the nucleic acid is comprised in a retrovirus.

186. The method of claim 185, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus.

187. The method of claim 185, wherein the retrovirus is a lentivirus.

188. The method of any one of claims 175-187, wherein the pseudotyped retroviral envelope is a mutated Sindbis envelope comprising Sindbis envelope proteins El, E2, and E3, wherein the mutated Sindbis envelope comprises substitutions or deletions at amino acid residues in El, E2, and / or E3, and wherein the substitutions or deletions at amino acid residues in El, E2, and / or E3 abrogate cognate receptor binding by the envelope.

189. The method of claim 188, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 comprise: substitutions or deletions at amino acid residues 226 and / or 227 of El;substitutions or deletions at amino acid residues 68-71, 159, 160, and / or X to X of E2; and / or substitutions or deletions at amino acid residues 60-64 of E3.

190. The method of claim 188 or 189, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted or deleted.

191. The method of claim 190, wherein the sequence AK at amino acid residues 226 and 227 of the El protein is substituted with the sequence SG.

192. The method of any one of claims 188-191, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted or deleted.

193. The method of claim 192, wherein the sequence SLKQ at amino acid residues 68 to 71 of the E2 protein is substituted with the sequence AAAA.

194. The method of any one of claims 188-193, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted or deleted.

195. The method of claim 194, wherein the sequence KE at amino acid residues 159 and 160 of the E2 protein is substituted with the sequence AA.

196. The method of any one of claims 188-195, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is substituted or deleted.

197. The method of claim 196, wherein the sequence RSKR at amino acid residues 61-64 of the E3 protein is deleted.

198. The method of any one of claims 188-195, wherein the sequence RSKRS at amino acid residues 60-64 of the E3 protein is substituted with the sequence AAAAA.

199. The method of any one of claims 188-198, wherein the mutated Sindbis envelop comprises a deletion of residues 61 to 64 in E3, the mutations SLKQ68-71AAAA in E2, and the mutation KE159-160AA in E2.

200. The method of claim 199, wherein the mutated Sindbis envelope further comprises the mutation AK226-227SG in El.

201. The method of any one of claims 188-200, wherein the substitutions or deletions at amino acid residues in Sindbis envelope proteins El, E2, and E3 decrease non-specific tropism, increase B cell tropism, and / or increase titer of a retrovirus comprising the nucleic acid encoding the mutated Sindbis envelope or the viral vector or viral genome comprising the nucleic acid.

202. The method of any one of claims 174-201, wherein the pseudotyped retroviral envelope further comprises one or more flexible linkers.

203. The method of any one of claims 174-202, wherein the pseudotyped retroviral envelope further comprises a detectable label and / or a protein binding domain.

204. The method of claim 203, wherein the detectable label comprises a FLAG peptide comprising the amino acid sequence DYKDDDDK (SEQ ID NO: 12).

205. The method of claim 203 or 204, wherein the protein binding domain comprises an integrin-binding domain.

206. The method of claim 205, wherein the integrin binding domain comprises the amino acid sequence CDCRGDCFC (SEQ ID NO:21).

207. The method of claim 203 or claim 204, wherein the protein binding domain comprises an Fc-binding sequence.

208. The method of claim 207, wherein the Fc-binding sequence comprises a protein A ZZ amino acid sequence.

209. The method of claim 203 or claim 204, wherein the protein binding domain comprises a biotin-binding sequence.

210. The method of claim 209, wherein the protein binding domain comprises an avidin, streptavidin, rhizavidin, or streptavidin / rhizavidin hybrid amino acid sequence.

211. The method of any one of claims 202-210, wherein the one or more flexible linkers comprise two flexible linkers.

212. The method of claim 209, wherein a first of the one or more flexible linkers is inserted at amino acid 70 of E2.

213. The method of claim 211 or 212, wherein the detectable label and / or the protein binding domain is comprised between the two flexible linkers.

214. The method of any one of claims 175-213, wherein the targeting moiety comprises one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof.

215. The method of claim 214, wherein the pathogen source of the one or more pathogenic antigens is a virus, bacteria, fungus, or parasite, or a combination thereof.

216. The method of claim 214 or claim 215, wherein the one or more pathogenic antigens comprise one or more viral envelope proteins.

217. The method of any one of claims 214-216, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

218. The method of any one of claims 175-217, wherein the nucleic acid encoding the pseudotyped retroviral envelope also encodes the targeting moiety.

219. The method of claim 218, wherein the nucleic acid is comprised in a viral vector.

220. The method of claim 218, wherein the nucleic acid is comprised in viral genome.

221. The method of any one of claims 218-220, wherein the nucleic acid encoding the pseudo typed retroviral envelope and the targeting moiety or the viral vector or viral genome comprising the nucleic acid encoding the pseudotyped retroviral envelope the targeting moiety is comprised in a retrovirus.

222. The method of claim 221, wherein the retrovirus is a gammaretrovirus or an oncoretrovirus.

223. The method of claim 221, wherein the retrovirus is a lentivirus.

224. The method of any one of claims 175-217, wherein the targeting moiety is conjugated to the pseudo typed retroviral envelope.

225. The method of claim 224, wherein the targeting moiety is conjugated to the protein binding domain of the pseudotyped retroviral envelope.

226. The method of claim 224 or 225, wherein the targeting moiety is covalently linked to the pseudotyped retroviral envelope.

227. The method of claim 224 or 225, wherein the targeting moiety is non-covalently linked to the pseudo typed retroviral envelope.

228. The method of any one of claims 224-227, wherein the targeting moiety comprises an antibody.

229. The method of claim 228, wherein the antibody is directed against one or more pathogenic antigens, one or more tumor- or cancer-associated antigens, or one or more auto- or self-antigens, or a combination thereof.

230. The method of claim 229, wherein the infectious agent is a viral, bacterial, fungal, or parasitic agent, or a combination thereof.

231. The method of claim 229 or claim 230, wherein the one or more antigenic molecules comprise one or more viral envelope proteins.

232. The method of any one of claims 229-231, wherein the tumor is derived from or the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer, or a combination thereof.

233. The method of any one of claims 174-232, wherein the B cell is contacted in vivo.

234. The method of any one of claims 174-233, wherein the targeting moiety, the nucleic acid encoding a pseudotyped retroviral envelope, or a comination thereof is administered to a subject.

235. The method of claim 174-234 wherein the targeting moiety, the nucleic acid encoding a pseudotyped retroviral envelope, or a comination thereof is administered intravenously.

236. The method of any one of claims 174-232, wherein the B cell is transduced ex vivo.

237. The method of claim 236, wherein the B cell is a primary B cell.

238. The method of claim 236, wherein the B cell is a B cell from a B cell line.

239. The method of any one of claims 174-232, wherein the B cell is transduced in vitro.

240. The method of claim 174-239, wherein the B cell is a primary B cell.

241. The method of claim 239, wherein the B cell is a B cell from a B cell line.

242. The method of any one of claims 174-241, further comprising producing the antibody, or an antigen-binding fragment thereof, by culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell.

243. A pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of claim 242, and a pharmaceutically acceptable carrier or excipient.

244. A method of preventing, treating, and / or ameliorating an infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, of claim 242 or the pharmaceutical composition of claim 243.

245. A method of manufacturing a medicament for preventing, treating, and / or ameliorating an infection, the method comprising culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody of claim 242 under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell.

246. The method of claim 244 or claim 245, wherein the infection is a viral, bacterial, fungal, or parasitic infection.

247. A method of preventing, treating, and / or ameliorating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, of claim 242 or the pharmaceutical composition of claim 243.

248. A method of manufacturing a medicament for preventing, treating, and / or ameliorating cancer, the method comprising culturing a host cell comprising a vector comprising the nucleic acid sequence encoding the antibody of claim 242 under a condition wherein the antibody, or antigen-binding fragment thereof, is expressed by the host cell.

249. The method of claim 247 or claim 248, wherein the cancer is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer.

250. The antibody, or antigen binding fragment thereof, of claim 242, for use in the treatment of at least one sign and / or symptom of an infection, disease, or condition.

251. The antibody, or antigen binding fragment thereof, of claim 250, wherein the infection, disease, or condition is viral, bacterial, fungal, or parasitic infection.

252. The antibody, or antigen binding fragment thereof, of claim 250, wherein the infection, disease, or condition is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer.

253. A kit for the treatment of an infection, disease, or condition, comprising a therapeutically effective amount of the antibody, or antigen binding fragment thereof, of claim 242.

254. The kit of claim 253, wherein the infection, disease, or condition is viral, bacterial, fungal, or parasitic infection.

255. The kit of claim 253, wherein the infection, disease, or condition is prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancer.

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