Method for selecting an antibody specific to a composite membrane antigen

Abstract

The present disclosure provides compositions and methods for high-throughput screening, selection, and identification of antibodies or antibody-like molecules that bind to target endogenous membrane proteins. TIFF2025524372000023.tif138159

Description

Technical Field

[0001] Sequence Listing This application has been electronically filed in XML format and includes a Sequence Listing, which is hereby incorporated by reference in its entirety. The XML copy created on June 12, 2023, is named 8555_042_SL.xml and is 61,440 bytes in size.

Background Art

[0002] Background Many important targets for therapeutic antibodies are endogenous membrane proteins (IMPs), such as multi-pass transmembrane proteins (GPCRs, ion channels, etc.) that are difficult to express and purify in a structurally intact state. Identifying and selecting antibodies against these targets is difficult because there is no appropriately folded target protein in an isolated state. Certain IMPs can be expressed on the surface of cells, such as mammalian cells, but the whole cell is a complex mixture of antigens, the target expression can be at low levels, and certain display packages used to construct antibody libraries (e.g., vaccinia virus antibody libraries) can bind non-specifically to the whole cell, so there are problems with using whole cells in antibody discovery. Screening for antibodies against a desired antigen within a selected cell line can be difficult because there is a large amount of irrelevant organic molecules that can obscure the antigen of interest in some cases.

[0003] As disclosed in US10,577,427 (Patent Document 1), which is hereby incorporated by reference in its entirety, native membrane protein display on poxvirus extracellular envelope virions, such as vaccinia virus, enables the expression and display of target IMPs of interest in their native three-dimensional structures, at sufficient concentrations, and with minimal competition with other cellular proteins, to enable the identification and selection of therapeutic antibodies and antibody-like molecules. However, both in vitro and in vivo antibody screening methods are labor-intensive, costly, and inefficient. For example, monoclonal antibodies (Mabs) are often selected from antigen-specific single B cells derived from various hosts, and these B cells are extremely short-lived under ex vivo culture conditions and are therefore difficult to investigate. Alternatively, Mabs can also be generated using display technology without relying on antigen-specific B cells. Cell sorting enables the investigation of large-scale library pools or B cell repertoires, but this technique requires soluble antigens. Therefore, there is still a need for an efficient high-throughput antibody screening method for selecting and identifying antibodies against IMPs.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

[0005] Summary The present disclosure provides compositions and methods for high-throughput screening, selection, and identification of antibodies or antibody-like molecules that bind to a target IMP of interest.

[0006] In one aspect, the present disclosure provides a method for selecting a binding molecule that binds to a target integral membrane protein (IMP) comprising the following steps: (a) To form linked antigen virions, attaching antigen virions comprising a poxvirus comprising an endogenous membrane protein (IMP) or a fragment thereof fused to an extracellular envelope virion (EEV) protein or a fragment thereof of the poxvirus to form an IMP-EEV fusion protein to a solid support, wherein the poxvirus expresses the target IMP or a fragment thereof in its native conformation as part of the outer envelope membrane of the EEV, and the IMP or a fragment thereof comprises at least one extracellular region, at least one transmembrane region, and at least one intracellular region; (b) Contacting the linked antigen virions with an antibody display library, wherein the library comprises display packages presenting a plurality of antigen-binding domains; (c) Selecting from the display packages a gene or a fragment thereof of an antibody variable portion that binds to the linked antigen virions such that the sequences encoding the variable light chain (VL), variable heavy chain (VH), and a poxvirus anchor protein are co-expressed as a single polypeptide, and cloning the variable light chain (VL) gene or a fragment thereof and the variable heavy chain (VH) gene or a fragment thereof derived from the display package into a plasmid vector in-frame with a polynucleotide sequence encoding a poxvirus anchor protein, such as an extracellular envelope virion protein, or a functional fragment thereof; (d) Transfecting mammalian cells with the plasmid vector of step (c) such that the transfected cells express the VL antigen-binding domain and the VH antigen-binding domain on the mammalian cell surface; (e) Screening the transfected cells using antigen virions linked to a detectable solid support; and (f) Recovering cells presenting an antigen-binding domain specific for the target.

[0007] In certain embodiments, the solid support in step (a) is magnetic beads labeled with streptavidin. In other embodiments, the poxvirus is fowlpox virus and the biotin label is a biotin - anti - fowlpox antibody. In other embodiments, the poxvirus is biotinylated modified vaccinia virus Ankara (MVA). In some embodiments, the poxvirus anchor protein is the vaccinia virus A56R protein. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, the IMP is a multi - pass IMP such as an ion channel or a G - protein. The multi - pass IMP can have either an even or an odd number of transmembrane domains.

[0008] In another aspect, the present disclosure provides a method for selecting a binding molecule that binds to a target integral membrane protein (IMP) comprising the following steps: (a) Isolating plasma cells from an animal such as a mammal, shark, or chicken immunized with an antigen comprising a target integral membrane protein (IMP) or a fragment thereof; (b) Seeding the plasma cells in a pooled state comprising a plurality of plasma cells and growing them to a desired cell density in a nutrient medium; (d) Performing one or more assays on the plasma cells to identify cells that express a binding molecule that binds to the target IMP protein; and (e) Recovering the plasma cells that express a binding molecule that binds to the target IMP protein.

[0009] In certain embodiments, the isolated plasma cells are seeded in a pooled state comprising a plurality of plasma cells, e.g., 1000 cells or less, e.g., 100 cells or less. In some embodiments of this aspect, a first ELISA is performed to identify cells that express a binding molecule that binds to the target IMP protein, followed by at least one additional ELISA assay. In this additional ELISA assay, the cells identified by the first ELISA assay are diluted before performing an additional ELISA assay to identify cells that express a binding molecule that binds to the target IMP protein. In some embodiments, the recovered cells are used to generate an antibody display library, which library comprises a display package that presents a plurality of antigen-binding domains. In certain embodiments, an animal is immunized with an antigen virion comprising a poxvirus that expresses the target IMP in its native conformation as part of the outer envelope membrane of the EEV.

[0010] In another aspect of the present disclosure, a method for selecting a binding molecule that specifically binds to a target integral membrane protein (IMP) is provided, comprising the following steps: (a) isolating B cells from an animal, such as a mammal, shark, chicken, etc., immunized with the target integral membrane protein (IMP) or a fragment thereof; (b) sorting the B cells to isolate antigen-specific B cells that express IgG that specifically binds to the target IMP; and (c) performing single cell analysis to identify the immunoglobulin variable region genes expressed by the sorted B cells.

[0011] In some aspects of this aspect, single-cell analysis includes RT-PCR. In some aspects of this aspect, the method further includes the step of isolating and cloning the variable heavy chain gene and / or the variable light chain gene from individual sorted B cells. In some aspects of this aspect of the present disclosure, the B cells are sorted using a target IMP linked to a detectable solid support such as streptavidin fluorescent beads. In certain aspects, a phage Fab display library is generated from variable heavy chain (VH) cDNA and variable light chain (VL) cDNA generated from RNA isolated from antigen-specific B cells that express IgG that binds to the target IMP. In certain aspects, the phage Fab display library is panned to exclude anti-poxvirus binding molecules and enrich anti-target IMP binding molecules. In other aspects, the method includes the further step of isolating VH genes and VL genes (V genes) from a phage Fab display library and subcloning the V genes into an expression vector such as a mammalian expression vector while maintaining the pairs of VH and VL present in individual phages as a mini-library (ML). In a particular aspect of this aspect, the IMP is a multi-pass IMP. In this aspect and any aspect of the present disclosure, the multi-pass IMP can have either an even or an odd number of transmembrane domains.

[0012] In another aspect of the present disclosure, a library produced by the methods disclosed herein is provided.

[0013] In another aspect of the present disclosure, an antibody that specifically binds to CD20 and is defined by its VH chain sequence and VL chain sequence is disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

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Mode for Carrying Out the Invention

[0015] Detailed Description The present disclosure provides methods and compositions for the high-throughput screening, selection, and identification of antibodies or antibody-like molecules that bind to a target endogenous membrane protein (IMP), such as a multi-pass IMP, or a fragment thereof.

[0016] Definitions The term "a" or "an" entity means one or more of that entity; for example, "a binding molecule" is understood to represent one or more binding molecules. Thus, the terms "a" (or "an"), "one or more", and "at least one" may be used synonymously herein.

[0017] As used herein, the term "and / or" shall be regarded as specifically disclosing each of the two specified features or components, regardless of the presence or absence of the other. Thus, when the term "and / or" is used in an expression such as "A and / or B" herein, it is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in an expression such as "A, B, and / or C", it is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0018] Unless otherwise defined, technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide general dictionaries for many of the terms used in this disclosure to those of ordinary skill in the art.

[0019] Units, prefixes, and symbols are shown in their internationally recognized form of the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise specified, amino acid sequences are written in the amino- to carboxy- direction, from left to right. The items provided herein are not intended to limit various aspects or aspects of the present disclosure, which can be obtained by referring to the present specification as a whole. Accordingly, the terms defined immediately below are more fully defined by referring to the present specification as a whole.

[0020] As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptides" and means a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" means any one or more chains consisting of two or more amino acids and does not mean a product of a specific length. Thus, the terms peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to mean one or more chains consisting of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" may be used in place of or synonymously with any of these terms. The term "polypeptide" is also intended to mean, non-limitingly, products of post-expression modification of polypeptides, including glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting / blocking groups, protein cleavage, or modification by non-natural amino acids. A polypeptide may be derived from a biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. A polypeptide may be produced in any manner, including chemical synthesis.

[0021] The polypeptides disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. A polypeptide can have a defined three-dimensional structure, but it is not necessarily required to have such a structure. A polypeptide having a defined three-dimensional structure is called folded. A polypeptide that does not have a defined three-dimensional structure and rather can adopt a number of different conformations is called unfolded. As used herein, the term glycoprotein means a protein linked to at least one carbohydrate moiety that is attached to the protein via a side chain containing oxygen or a side chain containing nitrogen of an amino acid, such as serine or asparagine.

[0022] An “isolated” polypeptide or fragment, variant, or derivative thereof is intended to mean a polypeptide that is not in its natural environment. A specific level of purification is not required. For example, a polypeptide to be isolated can be removed from its native or natural environment. Recombinant polypeptides and proteins produced by expression in a host cell are considered to be isolated as disclosed herein, in the same manner as native polypeptides or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0023] As used herein, the term "non-naturally occurring" polypeptide, or any grammatical variation thereof, is a conditional term that expressly excludes, but only excludes, polypeptides in a form well understood by one of ordinary skill in the art as "naturally occurring", or as determined or construed by a judge or administrative or judicial body as being "naturally occurring", or as may be determined or construed at any point in time.

[0024] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. As used herein, the terms "fragment", "variant", "derivative", and "analog" include any polypeptide that retains at least some of the properties of the corresponding native antibody or polypeptide, such as specific binding to an antigen. Fragments of a polypeptide include, for example, proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein. Variants of a polypeptide include, for example, fragments as described above, and also polypeptides having an amino acid sequence that has been changed due to an amino acid substitution, deletion, or insertion. In certain aspects, a variant can be non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions. A derivative is a polypeptide that has been modified so as to exhibit additional features not found in the original polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a polypeptide can also mean a subject polypeptide having one or more amino acids that have been chemically derivatized by reaction of a functional side group. Peptides containing one or more derivatives of the 20 standard amino acids are also included as "derivatives". For example, 4-hydroxyproline can be used in place of proline, 5-hydroxylysine can be used in place of lysine, 3-methylhistidine can be used in place of histidine, homoserine can be used in place of serine, and ornithine can be used in place of lysine.

[0025] As used herein, the terms "intrinsic membrane protein" or "IMP" mean a protein or polypeptide that is associated with a biological membrane. An example of an IMP is a transmembrane protein that spans the lipid bilayer of a biological membrane one or more times. A single-pass membrane protein crosses the membrane only once, while a multi-pass membrane protein goes in and out and proceeds in a sewing-like manner, crossing the membrane several times. A type I single-pass protein is positioned such that its amino terminus is on the outside of the membrane, i.e., "extracellular," and its carboxyl terminus is on the inside of the membrane, i.e., "intracellular." A type II single-pass protein has its amino terminus on the inside of the membrane. A multi-pass transmembrane protein passes through the membrane two or more times and can have various different topologies. A protein having an even number of transmembrane domains will have both its amino terminus and carboxyl terminus on the same side of the membrane. An example of such a protein is CD20, which is expressed on B cells. A protein having an odd number of transmembrane domains will have its amino terminus and carboxyl terminus on opposite sides of the membrane. Examples include G protein-coupled receptors, which typically have seven transmembrane domains, with their amino terminus on the outside of the membrane and their carboxyl terminus on the inside of the membrane. Certain IMPs do not have transmembrane domains but instead are anchored to the membrane via a lipid, such as a glycosylphosphatidylinositol or palmitoyl group, for example. IMPs have numerous biological functions, including but not limited to transporters, linkers, channels, receptors, enzymes, energy conversion, or cell adhesion.

[0026] The term "polynucleotide" is intended to encompass single nucleic acids and plural nucleic acids, and means an isolated nucleic acid molecule or nucleic acid construct, such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). Polynucleotides can include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNA)). The term "nucleic acid" or "nucleic acid sequence" means any one or more nucleic acid segments, e.g., a DNA fragment or an RNA fragment present in a polynucleotide.

[0027] An "isolated" nucleic acid or polynucleotide is intended to mean any form of nucleic acid or polynucleotide that is separated from its native environment. For example, a gel-purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector, would be considered to be "isolated". Also, a polynucleotide segment, e.g., a PCR product that has been engineered to have restriction sites for cloning, is considered to be "isolated". Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or (partially or substantially) purified polynucleotides in a non-native solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not found naturally. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. Additionally, a polynucleotide or nucleic acid can be or can include regulatory elements such as a promoter, ribosome binding site, or transcription terminator.

[0028] As used herein, the term “non-naturally occurring” polynucleotide, or any grammatical variation thereof, is a conditional definition that expressly excludes, but only excludes, polynucleotides in a form well understood by one of ordinary skill in the art as “naturally occurring,” or as determined or construed by a judge or administrative or judicial body as “naturally occurring,” or as may be determined or construed at any point in time.

[0029] As used herein, a “coding region” is a portion of a nucleic acid consisting of codons that are translated into amino acids. A “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid but can be considered part of the coding region. However, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, and introns, are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Further, any vector can contain a single coding region or can contain two or more coding regions, e.g., a single vector can encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region separately. Further, a vector, polynucleotide, or nucleic acid can also contain a heterologous coding region, which can be fused or unfused to another coding region. Heterologous coding regions include, without limitation, those encoding special elements or motifs such as a secretion signal peptide or a heterologous functional domain.

[0030] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide can typically include a promoter and / or other transcriptional or translational control elements operably linked to one or more coding regions. An operable linkage exists when a coding region for a gene product, such as a polypeptide, is linked to one or more regulatory sequences in such a way that the expression of the gene product is placed under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter linked thereto) are "operably linked" if transcription of mRNA encoding the desired gene product occurs upon induction of promoter function and the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product nor with the ability of the DNA template to be transcribed. Thus, a promoter region is considered to be operably linked to a nucleic acid encoding a polypeptide if the promoter can effect transcription of the nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of DNA in a given cell. In addition to the promoter, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to the polynucleotide to direct cell-specific transcription.

[0031] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, for example, promoter segments and enhancer segments derived from cytomegalovirus (used in combination with the immediate early promoter, intron A), those derived from simian virus 40 (early promoter), and those derived from retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcription control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0032] Poxvirus promoters (e.g., p7.5 or H5) or bacteriophage T7 promoters can also be used as transcription control regions. When using the T7 promoter, an inducible vaccinia expression system can be utilized. The vaccinia expression system can include, but is not limited to, a first recombinant vaccinia virus encoding the entire bacteriophage T7 gene 1 coding region for T7 RNA polymerase, and a second recombinant vaccinia virus encoding a gene of interest flanked by a T7 promoter and termination regulatory elements. When eukaryotic cells are coinfected with both recombinant vaccinia viruses, synthesis of T7 RNA polymerase and expression of the gene of interest controlled by the T7 promoter occur.

[0033] Similarly, a variety of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation codons and translation termination codons, and elements derived from picornaviruses (in particular, the internal ribosome entry site, also referred to as the CITE sequence).

[0034] In other embodiments, the polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

[0035] The coding regions of the polynucleotide and nucleic acid can be ligated to additional coding regions that encode a secretory peptide or signal peptide that directs the secretion of the polypeptide encoded by the polynucleotides disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide sequence or secretory leader sequence that is cleaved from the mature protein once the elongating protein chain begins to be translocated across the rough endoplasmic reticulum. Polypeptides secreted from vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide, and those skilled in the art are aware that the signal peptide is cleaved from the full-length or "full-length" polypeptide to yield the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy chain signal peptide or immunoglobulin light chain signal peptide, or a functional derivative of this sequence that retains the ability to direct the secretion of the polypeptide to which it is operably linked, is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can also be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0036] As used herein, a "library" is a collection of polynucleotides related by, for example, derivation from a representative genus of polynucleotides, such as a single animal species, tissue type, organ, or cell type, and a library collectively includes at least two different species within a given genus of polynucleotides. A library of polynucleotides can include, for example, at least 2, at least 5, at least 10, 100, 103, 104, 105, 106, 107, 108, or 109 different species within a given genus of polynucleotides. In certain aspects, a library of polynucleotides provided herein can encode a plurality of polypeptides including a polypeptide of interest. In certain aspects, a library of polynucleotides provided herein can encode a plurality of immunoglobulin subunit polypeptides, such as a heavy chain subunit polypeptide or a light chain subunit polypeptide. In this context, a "library" provided herein includes polynucleotides of a common genus, the genus being polynucleotides encoding a particular type and class of immunoglobulin subunit polypeptide, for example, a library can encode a human μ, γ-1, γ-2, γ-3, γ-4, α-1, α-2, ε, or δ heavy chain, or a human κ or λ light chain. Each member of any one library constructed according to the methods provided herein can encode the same heavy chain constant region or light chain constant region and / or membrane anchor domain, but the library can collectively include at least 2, at least 5, or at least 10, 100, 103, 104, 105, 106, 107, 108, or 109 different variable regions linked to a common constant region.

[0037] In other aspects, the library can be a plurality of species of immunoglobulin single-chain fragments, such as ScFv fragments, that include variable regions such as a light chain variable region or a heavy chain variable region and / or both a light chain variable region and a heavy chain modified region.

[0038] As used herein, a "display library" is a library of polynucleotides, each of which is contained in a "display package" that expresses on its surface a polypeptide encoded by the polynucleotide of the library. For example, an antibody display library can contain a plurality of display packages, each of which presents an antigen-binding domain of an antibody on its surface. When a display library is allowed to interact with an antigen of interest immobilized on, for example, a solid surface, the display packages that bind to the antigen can be separated and recovered from the other parts of the library. Subsequently, the polynucleotide encoding the antigen-binding domain presented on the surface of the display package can be isolated. Display libraries include, without limitation, phage display libraries in bacteria, or libraries in eukaryotic systems, such as yeast display, mammalian cell display, such as CHO cells, retroviral display, or expression in DNA viruses such as poxviruses. See, for example, U.S. Patent No. 7,858,559 and U.S. Patent Application Publication No. 2013-028892, which are hereby incorporated by reference in their entirety. In certain aspects, an antibody display library can be prepared in a poxvirus, such as a vaccinia virus vector, as a fusion protein with an EEV-specific protein such that the "display package" is an EEV particle. See U.S. Patent Application Publication No. 2013-028892.

[0039] Such a display library can be screened against the IMP fusion protein presented on the surface of the EEV provided herein.

[0040] "Recipient cell", "host cell", or "cell" means a cell or cell population in which a recombinant protein can be expressed, a virus can be propagated, or a polynucleotide library provided herein can be constructed and / or expanded. Host cells provided herein are typically eukaryotic cells or cell lines, such as vertebrate, mammalian, rodent, mouse, primate, or human cells or cell lines. "Population of host cells" means a group of cultured cells in which the "libraries" provided herein can be constructed, expanded, and / or expressed. Any host cell that is permissive to the infectivity of vaccinia virus or fowlpox virus is suitable for the methods provided by this disclosure. Host cells for use in the methods provided herein can be adherent, i.e., host cells that grow attached to a solid substrate, or the host cells can be in suspension.

[0041] The host cells provided herein can include a constitutive secretory pathway. In this pathway, proteins, such as proteins of interest expressed by a cell or library, are secreted from the interior of the cell and expressed on the cell or viral membrane surface or are fully secreted as soluble polypeptides. In certain aspects, a protein of interest expressed on or in a biological membrane, such as an IMP, is expressed on the surface of an enveloped virus produced by the host cell, such as extracellular enveloped vaccinia virus or EEV. IMPs can follow the same pathway as fully secreted or fully secreted proteins, passing through the ER lumen, except that they can remain in the ER membrane due to the presence of one or more trafficking stop signals or “transmembrane domains”. A transmembrane domain is a hydrophobic stretch of about 20 amino acids that assumes an α-helical conformation when crossing the membrane. Membrane-embedded proteins are anchored in the phospholipid bilayer of the plasma membrane. Transmembrane forms of polypeptides of interest, such as membrane-bound immunoglobulin heavy chain polypeptides, typically utilize an amino-terminal signal peptide as utilized by fully secreted forms.

[0042] Signal peptides, transmembrane domains, and cytoplasmic or “intramembrane” domains are known for a wide variety of membrane-bound proteins and / or fully secreted proteins.

[0043] Suitable transmembrane domains can include, but are not limited to, the TM domains of the HA protein A56R specific for vaccinia virus EEV or the vaccinia virus transmembrane proteins A33R, A34R, A36R, or B5R specific for EEV. See, for example, U.S. Patent Application Publication No. 2013 / 0288927, published October 31, 2013, which is hereby incorporated by reference in its entirety. In certain aspects, an EEV-specific protein, such as the vaccinia virus protein F13L, can be anchored to the inner surface of the viral envelope via a palmitoyl group. In some embodiments, the transmembrane domain is referred to herein as an "anchor protein," such as a "poxvirus anchor protein."

[0044] As used herein, the term "binding molecule" means, in its broadest sense, a molecule that specifically binds to a receptor, such as an epitope or antigenic determinant. As further described herein, a binding molecule can include one or more "antigen-binding domains" as described herein. Non-limiting examples of binding molecules are antibodies, or fragments thereof that retain antigen-specific binding.

[0045] The terms "binding domain" and "antigen-binding domain" are used interchangeably herein and mean the region of a binding molecule that is necessary and sufficient to specifically bind to an epitope. For example, the "Fv," e.g., the variable heavy and variable light chains of an antibody, either as two separate polypeptide subunits or as a single chain, are considered "binding domains."

[0046] Other antigen-binding domains include, without limitation, the variable heavy chain (VHH) of an antibody derived from a camelid species, or six immunoglobulin complementarity-determining regions (CDRs) expressed in a fibronectin scaffold.

[0047] The terms "antibody" and "immunoglobulin" can be used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof disclosed herein) comprises at least the variable region of the heavy chain (e.g., in the case of camelid species), or at least the variable regions of the heavy and light chains. The basic immunoglobulin structure in the vertebrate system is relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise specified, the term "antibody" encompasses anything from small antigen-binding fragments of an antibody to a normally sized antibody, e.g., an IgG antibody comprising two complete heavy chains and two complete light chains.

[0048] The term "immunoglobulin" includes a wide variety of biochemically distinguishable classes of polypeptides. One of ordinary skill in the art will understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and there are several subclasses within each (e.g., γ1-γ4 or γ1-γ4 or α1-α2). Depending on the nature of this chain, the "class" of the antibody is determined to be IgG, IgM, IgA, IgG, or IgE, respectively. The subclasses (isotypes) of immunoglobulins, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc., are well characterized and are known to confer functional specializations.

[0049] Light chains are classified into either kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or a lambda light chain. Generally, when immunoglobulins are produced by any of hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are joined to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the branched end of the Y shape to the C-terminus at the bottom of each chain. The basic structure of certain antibodies, such as IgG antibodies, forms a "Y" structure, also referred to herein as the "H2L2" structure, and includes two heavy chain subunits and two light chain subunits covalently bonded via disulfide bonds.

[0050] The term "epitope" includes any molecular determinant that can bind specifically to an antibody. In certain aspects, an epitope can include a chemically active grouping of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls that gather on the surface, and in some aspects, can have three-dimensional structural features and / or specific charge characteristics. An epitope is the region of a target to which an antibody binds.

[0051] The term "target" is used in the broadest sense and includes a substance to which a binding molecule can bind. A target can be, for example, a polypeptide, nucleic acid, carbohydrate, lipid, or other molecule. Further, a "target" can be, for example, a cell, organ, or organism that includes an epitope to which a binding molecule can bind.

[0052] Both the light and heavy chains are divided into structurally and functionally homologous regions. The terms "constant" and "variable" are used in relation to function. In this regard, it is considered that antigen recognition and specificity are determined by the variable regions of both the variable light (VL) chain portion and the variable heavy (VH) chain portion (which may be referred to synonymously as "variable domains" herein). As used herein, the term "gene or fragment thereof of the antibody variable portion" means a gene or a portion thereof that encodes the VL or VH of an antibody or a fragment thereof. Conversely, the constant domain of the light chain (CL) and the constant domains of the heavy chain (e.g., CH1, CH2, or CH3) confer biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement binding. By convention, in numbering the constant region domains, the numbers increase as the domains become more distant from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion has the constant region; the CH3 (or CH4 in the case of IgM) domain and the CL domain are at the carboxy termini of the heavy and light chains, respectively.

[0053] The six "complementary determining regions" or "CDRs" present in the antibody antigen-binding domain are discontinuous short amino acid sequences that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining portion of the amino acids in the antigen-binding domain, called the "framework" region, exhibits lower intermolecular variability. The framework region predominantly adopts a β-sheet structure, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, the framework region serves to form a scaffold that enables the positioning of the CDRs in the correct orientation by non-covalent intermolecular interactions. The antigen-binding domain formed by these positioned CDRs defines the shape of a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. The amino acids that constitute the CDRs and the framework regions are defined in various different ways, so that those skilled in the art can readily identify them for any given heavy chain variable region or light chain variable region (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are hereby incorporated by reference in their entirety).

[0054] Where two or more definitions exist for terms used and / or accepted within the art, the definitions of terms used herein are intended to include all such meanings unless the contrary is explicitly stated. A specific example is the use of the term "complementary determining region" ("CDR") to describe non - contiguous antigen - binding sites found within the variable regions of both heavy - chain polypeptides and light - chain polypeptides. These particular regions are described, for example, by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901 - 917 (1987), which are incorporated herein by reference. Immunoglobulin variable domains can also be analyzed, for example, using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V - Quest) to identify variable - region segments that contain CDRs. (See, for example, Brochet et al., Nucl. Acids Res., 36:W503 - 508, 2008).

[0055] Kabat et al. also defined a numbering scheme for variable - domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" scheme to any variable - domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" means the numbering scheme described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). However, unless the use of the Kabat numbering scheme is explicitly stated, sequential numbering is used for all amino - acid sequences in the present disclosure.

[0056] Binding molecules, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, include, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-bonded Fv (sdFv), single-domain antibodies such as camelid VHH antibodies, fragments containing either a VL domain or a VH domain, and fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Immunoglobulin or antibody molecules encompassed by the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Also contemplated are immunoglobulin new antigen receptors (IgNAR) isotypes that are bivalent and contain single chains that include IgNAR variable domains (VNAR). (See Walsh et al., Virology 411:132-141, 2011).

[0057] As used herein, "specifically binds" generally means that a binding molecule, such as an antibody, or a fragment, variant, or derivative thereof, binds to an epitope via its antigen-binding domain and that the binding requires some complementarity between the antigen-binding domain and the epitope. In accordance with this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it would bind to a random unrelated epitope. The term "specificity" is used herein to describe the relative affinity of a particular binding molecule for a particular epitope. For example, binding molecule "A" can be considered to have a higher specificity for a given epitope than binding molecule "B", or it can be said that binding molecule "A" binds to epitope "C" with a higher specificity than it has for related epitope "D".

[0058] As used herein, the term "affinity" means the degree of strength of binding between an individual epitope and one or more antigen-binding domains of, for example, an immunoglobulin molecule. See, e.g., pages 27-28 of Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). As used herein, the term "avidity" means the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., pages 29-34 of Harlow. Avidity is related to both the affinity of the individual antigen-binding domains in the population for specific epitopes and also the valency of the immunoglobulin and the antigen. For example, the interaction of a bivalent monoclonal antibody with an antigen having a repetitive epitope structure, such as a polymer, is considered to be one of high avidity. The interaction of a bivalent monoclonal antibody with a receptor present at high density on the cell surface is also considered to be of high avidity.

[0059] As used herein, the terms "heavy chain subunit" or "heavy chain domain" include amino acid sequences derived from immunoglobulin heavy chains. For example, an antibody comprising a heavy chain subunit can include at least one of a VH domain, a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant or fragment thereof.

[0060] As used herein, the terms "light chain subunit" or "light chain domain" include amino acid sequences derived from immunoglobulin light chains. A light chain subunit can include at least one of a VL domain or a CL (e.g., Cκ or Cλ) domain.

[0061] Binding molecules, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be described or characterized in terms of the epitope or portion of an antigen that they recognize or specifically bind to. A portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant". A target antigen can include a single epitope or at least two epitopes, and can include any number of epitopes depending on the size, conformation, and type of the antigen.

[0062] As used herein, the terms "linked," "fused," or "fusion," or other grammatical equivalents may be used synonymously. These terms mean connecting two or more elements or components by any means, including chemical conjugation or recombinant means. "In-frame fusion" means the ligation of two or more polynucleotide open reading frames (ORFs) to form a longer continuous ORF in a manner that maintains the translational reading frame of the original open reading frame (ORF). Thus, a recombinant fusion protein is a single protein that contains two or more segments corresponding to the polypeptides encoded by the original ORFs (these segments are not normally linked in that way in nature). The reading frame is thus continuous throughout the fused segments, although each segment may be physically or spatially separated, for example, by an in-frame linker sequence. For example, polynucleotides encoding IMP and a vaccinia virus EEV-specific protein may be fused in-frame, but may be separated by a polynucleotide encoding a linker or spacer as long as the "fused" open reading frame is co-translated as part of a continuous polypeptide.

[0063] As used herein, the term "hemagglutinin tag" or "HA tag" is a protein corresponding to amino acids 98 - 106 derived from the human influenza hemagglutinin surface glycoprotein (HA). The HA tag is widely used as a common epitope tag in expression vectors. Recombinant proteins can be engineered to express the HA tag, and the HA tag does not appear to interfere with the biological activity or in vivo distribution of the recombinant protein. This tag facilitates the detection, isolation, and purification of the protein of interest.

[0064] In the context of a polypeptide, a "linear sequence" or "sequence" is the arrangement of amino acids from the amino terminus or N-terminus to the carboxyl terminus or C-terminus in a polypeptide, where adjacent amino acids in the sequence are contiguous in the primary structure of the polypeptide.

[0065] A portion of a polypeptide that is the "amino terminus" or "N-terminus" relative to another portion of the polypeptide is the portion that comes earlier in a continuous polypeptide chain. Similarly, a portion of a polypeptide that is the "carboxyl terminus" or "C-terminus" relative to another portion of the polypeptide is the portion that comes later in a continuous polypeptide chain.

[0066] As used herein, the term "expression" means the process by which a gene produces a biochemical substance, such as a polypeptide. This process includes any manifestation of the functional influence of the gene within a cell and includes, without limitation, gene knockdown as well as both transient and stable expression. This includes, without limitation, transcription of the gene into messenger RNA (mRNA) and translation of such mRNA into a polypeptide. When the final desired product is a biochemical substance, expression includes creating that biochemical substance and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, such as messenger RNA produced by transcription of the gene, or a polypeptide translated from the transcript. Gene products described herein further include nucleic acids that have undergone post-transcriptional modifications, such as polyadenylation, or polypeptides that have undergone post-translational modifications, such as methylation, glycosylation, lipid addition, binding to other protein subunits, and proteolytic cleavage.

[0067] The term "eukaryote" or "eukaryotic organism" is intended to encompass all organisms belonging to the animal kingdom, plant kingdom, and protist kingdom, including protozoa, fungi, yeast, green algae, single-celled plants, multi-cellular plants, and all animals, i.e., both vertebrates and invertebrates. This term does not include bacteria or viruses. "Eukaryotic cell" is intended to include both the singular "eukaryotic cell" and the plural "eukaryotic cells" and includes cells derived from eukaryotes.

[0068] As used herein, the term "identify" means a method of distinguishing an antibody or antibody-like molecule that binds to a desired molecule, e.g., a target protein of interest (e.g., an endogenous membrane protein of interest), from a plurality of such molecules or a library of such molecules. Identification methods include "selection" and "screening" or "panning". As used herein, a "selection" method is a method by which a desired molecule can be directly isolated from a library, e.g., using drug resistance. As used herein, a "screening" method or "panning" method is a method of subjecting a pool containing a desired molecule to an assay capable of detecting the desired molecule. An aliquot of the pool in which the molecule is detected is then stepwise divided into smaller pools and assayed similarly until a pool highly enriched for the desired molecule is obtained.

[0069] The IMP fusion proteins provided herein are produced in a poxvirus vector, such as a vaccinia virus vector. The term "poxvirus" includes any member of the family Poxviridae. See, for example, B. Moss in: Virology, 2d Edition, B. N. Fields, D. M. Knipe et al., Eds., Raven Press, p. 2080 (1990). The genus Orthopoxvirus includes, for example, vaccinia virus, variola virus (the virus that causes smallpox), and raccoonpox virus. Vaccinia virus is the prototype orthopoxvirus and has been developed and well-characterized as a vector for expressing heterologous proteins. The IMP fusion proteins can be produced as disclosed in US10,550,199, which is hereby incorporated by reference in its entirety.

[0070] In the aspects in which a poxvirus vector, particularly a vaccinia virus vector, is used to express the IMP fusion proteins provided herein, any suitable poxvirus vector can be used, such as a vaccinia virus vector, a fowlpox vector, or a rabbitpox vector. The polynucleotide encoding the IMP fusion proteins provided herein can be inserted into a poxvirus vector, particularly a vaccinia virus vector or a fowlpox virus vector, under functional linkage with a transcriptional control region that functions in the cytoplasm of poxvirus-infected cells. Suitable poxvirus vectors include wild-type vaccinia virus, such as the Western Reserve strain or the WR strain, or attenuated vaccinia virus, such as modified vaccinia Ankara (MVA) (Mayr, A. et al., Infection 3:6-14 (1975)).

[0071] At least six virus-encoded proteins have been reported as components of the EEV envelope membrane. Of these, four proteins (A33R, A34R, A56R, and B5R) are glycoproteins, one (A36R) is a non-glycosylated transmembrane protein, and one (F13L) is a palmitoylated surface membrane protein. See, for example, Lorenzo et al., Journal of Virology 74 (22):10535 (2000). Upon infection, these proteins localize to the Golgi complex where they are incorporated into infectious virus, which is then transported and released into the extracellular medium. As provided herein, the IMP fusion protein is directed to the EEV membrane and expressed on the EEV membrane as a fusion protein with an EEV-specific protein, such as F13L or A56R.

[0072] The A56R protein is a vaccinia virus hemagglutinin and is a standard type I integral membrane protein that contains an amino-terminal extracellular ("ecto") domain, one transmembrane domain, and a cytoplasmic ("endo") domain. A56R contains an N-terminal signal peptide of approximately 33 amino acids, an Ig-like domain extending from around amino acid 34 to around amino acid 103, a stalk region extending from around amino acid 121 to around amino acid 275, a transmembrane domain extending from around amino acid 276 to around amino acid 303, and a cytoplasmic ("endo") domain extending from around amino acid 304 to around amino acid 314. See DeHaven et al., J. Gen Virol. 92:1971-1980 (2011). A56R is shown as SEQ ID NO: 1. TIFF2025524372000002.tif41128

[0073] As used herein, "solid support" refers to any support capable of binding an EEV, which can be in any of a variety of forms as known in the art. Well-known supports include tissue culture plastics, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamide, rhyolite, and magnetite. The nature of the carrier can be either soluble or insoluble to some extent for the purposes of the present disclosure. The support material can have virtually any structural shape as long as the linked EEV can bind to a presented binding molecule such as an antibody. Thus, the support shape can be spherical as in the case of beads, or cylindrical as in the case of the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as in the case of a sheet or a test strip. Typical supports include beads, such as magnetic polystyrene beads like DYNABEADS® which can be removed from a suspension by a magnet. Support shapes can include tubes, beads, microbeads, wells, plates, tissue culture plates, Petri dishes, microplates, microtiter plates, flasks, sticks, strips, vials, paddles, and the like. The solid support can be magnetic or non-magnetic. Those skilled in the art will know or be able to readily appreciate many other suitable carriers for binding the EEV provided herein. In certain aspects, the EEV provided herein can be bound to a solid support, for example, via reaction with a tosyl group, epoxy group, carboxylic acid group, or amino group bound to the surface. For example, the EEV can be bound to the surface of tosyl group-activated magnetic beads, such as MYONE™ tosyl group-activated beads. Alternatively, the EEV can be biotinylated and bound to a streptavidin solid surface, such as magnetic beads coated with streptavidin. Any solid support can be used in the methods of the present disclosure.

[0074] The poxvirus antigen constructs of the present disclosure can be used, for example, in phage panning or cell sorting applications to provide native conformations of complex antigens such as GPCRs and ion channels for antibody discovery. The present disclosure provides a method for selecting a binding molecule that binds to a complex antigen of the present disclosure, such as a multi-pass transmembrane protein of interest, such as an antibody, an antibody fragment that binds to an antigen, or an antibody-like binding molecule.

[0075] In certain aspects of the present disclosure, a display library is used in the method for selecting a binding molecule. Any display library that contains a plurality of binding domains, such as antibodies, antibody-like molecules, or other binding molecules, is suitable for use in the methods of the present disclosure. For example, the display library can be a phage display library, a yeast display library, or a library constructed in a vaccinia virus vector as described elsewhere herein.

[0076] In certain aspects of the present disclosure, animals such as chickens, sharks, mammals, for example mice, are immunized one or more times with a target antigen to produce antibodies specific to the target antigen, for example, a target IMP. In some aspects, the target antigen comprises antigen virions comprising a fusion protein of an endogenous membrane protein (IMP) and an extracellular enveloped virion (EEV), expressed in its native conformation as part of the outer envelope membrane of the EEV, the poxvirus. In some aspects, B cells are isolated from the immunized animal, for example, a chicken, shark, mammal, and sorted to isolate antigen-specific B cells that express IgG that specifically binds to the target IMP. The variable heavy chain gene and / or variable light chain gene can be isolated and cloned from individual sorted B cells. Platforms used to discover and isolate target-specific antibodies from single B cells include, for example, hybridoma technology, culture techniques for memory B cells and adipose stem cells (ASCs), staining of membrane-bound B cell receptors (BCRs) of B cells with antigen, single B cell screening methods, B cell replica methods, single B cell repertoire analysis, and clone expansion-based identification, including but not limited to. (Pedrioli et al., Trends in Immunology, Single B cell technologies for monoclonal antibody discovery, 2021, vol. 42, no. 12; which is incorporated herein by reference in its entirety).

[0077] Unless otherwise defined, the present disclosure uses conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art. Such techniques are well described in the literature. (For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.See 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0078] General principles regarding antibody engineering are described in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles regarding protein engineering are described in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles regarding antibodies and antibody-hapten binding are described in Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). Furthermore, standard methods of immunology known in the art and not specifically described, such as those described in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.); and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY), can be followed.

[0079] Standard reference works that explain general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th ed.; H. Freeman & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press) are included.

[0080] All of the references cited above, as well as all references cited in this specification, are hereby incorporated by reference in their entirety into this specification. The following examples are provided for illustration purposes only and not for limitation.

Example

[0081] Example 1: Preparation of Streptavidin Fluorescent Polystyrene Beads Conjugated with EEV As described in US10,577,427, which is hereby incorporated by reference in its entirety, extracellular enveloped virions (EEVs) expressing antigens of interest (e.g., CD20, CD39, SEMA4D) were prepared. To conjugate the EEVs of fowlpox virus (FPV) to polystyrene beads, the virus was captured using purified and biotinylated chicken anti-FPV antibody (LSBio). In the case of the EEVs of modified vaccinia virus Ankara (MVA), the virus was directly biotinylated using FSL biotin (Sigma), and then conjugated to beads coated with streptavidin. Fluorescent polystyrene beads coated with streptavidin (Spherotech catalog numbers SVFP-1068-5 and SVFP-0552-5) were vortexed, sonicated, an appropriate volume of beads was taken out and added to 1 mL of 1×PBS (pH 7.2) containing 1% BSA. The beads were pelleted by centrifugation at 13,000×g for 20 minutes and the supernatant was removed. The beads were resuspended in 200 ul of 1×PBS (pH 7.2) containing 1% BSA, and 25 ug of biotinylated capture antibody per bead was added. The beads were then rotated at room temperature for 2 hours. These beads were washed twice with 1% BSA, 1×PBS (pH 7.2), and the beads were pelleted using centrifugation at 13,000×g. These beads were resuspended in 400 uL of 1×PBS (pH 7.2) containing 1% BSA, and the antigen EEV was added to the beads at a bead-to-virus ratio of 5:1 or 1:1. The beads + EEV were rotated overnight at room temperature in the dark. The beads were washed once with 1×PBS (pH 7.2) containing 1% BSA and then blocked with 1 mL of 1×PBS (pH 7.2) containing 1% BSA and 2% FBS. To completely block, the beads were rotated overnight at room temperature in the dark. The beads were then pelleted by centrifugation at 13,000×g for 20 minutes and resuspended in the original starting volume in 1×PBS (pH 7.2) containing 1% BSA. The beads + EEV were stored wrapped in foil at 4°C until use. 8 25 ug of biotinylated capture antibody per bead was added. The beads were then rotated at room temperature for 2 hours. These beads were washed twice with 1% BSA, 1×PBS (pH 7.2), and the beads were pelleted using centrifugation at 13,000×g. These beads were resuspended in 400 uL of 1×PBS (pH 7.2) containing 1% BSA, and the antigen EEV was added to the beads at a bead-to-virus ratio of 5:1 or 1:1. The beads + EEV were rotated overnight at room temperature in the dark. The beads were washed once with 1×PBS (pH 7.2) containing 1% BSA and then blocked with 1 mL of 1×PBS (pH 7.2) containing 1% BSA and 2% FBS. To completely block, the beads were rotated overnight at room temperature in the dark. The beads were then pelleted by centrifugation at 13,000×g for 20 minutes and resuspended in the original starting volume in 1×PBS (pH 7.2) containing 1% BSA. The beads + EEV were stored wrapped in foil at 4°C until use.

[0082] V genes for CD20, CD39, and Sema4D (CD100) cloned into an A56R vector V genes derived from sorted B cells were cloned and sequenced for display library generation. The following vectors were constructed. TIFF2025524372000003.tif198151TIFF2025524372000004.tif93151TIFF2025524372000005.tif199151TIFF2025524372000006.tif92151TIFF2025524372000007.tif205151TIFF2025524372000008.tif92152

[0083] Generation of stable CHO cell lines expressing membrane-bound antibodies One day prior to transfection, CHO cells were seeded into 6-well plates (0.8×10 6 cells / well). 4 μg of vector DNA was transfected into the cells using Lipofectamine 2000 reagent according to the manufacturer's instructions. Cells without vector were included as a control. The next day, the cells were harvested and dispensed into a T175 flask containing DMEM medium supplemented with 10% FBS, 1 mM HEPES, and G418 (0.6 mg / ml) for drug selection. To maintain the selection pressure, the medium containing the drug was changed every 2 - 3 days. When the cells without vector had all died, the transfectants were stained with an anti-Fab antibody for fluorescence-activated cell sorting (FACS) on a BD FACS Aria sorter. Cells highly expressing the antigen were collected, cultured, and the enrichment after sorting was measured by flow cytometry.

[0084] Staining with fluorescent yellow / blue beads Cells were harvested using Accutase dissociation reagent, counted, pelleted at 300×g for 5 minutes, and resuspended in blocking buffer (1×PBS (pH 7.2) containing 1% BSA, 2% FBS) at 1 million cells / ml. The cells were stored on ice for 1 hour. Next, the cells were pelleted and resuspended in 1 ml of 1% BSA, 100 units of heparin, 1×PBS (pH 7.2) per 1 million cells and incubated at room temperature for 20 minutes. Then, the cells were dispensed into flow staining tubes (0.2 ml each), and fluorescent beads conjugated with EEV were added to each tube. The cells were incubated with the beads for 1 hour at room temperature in the dark, mixing occasionally. Next, the cells were washed twice with 1×PBS, pH 7.2, 1% BSA and resuspended in 1×PBS, pH 7.2, 1% BSA containing anti-Fab-FITC or anti-Fab-APC. The cells were incubated at 4°C for 30 minutes, then washed twice with 1×PBS, pH 7.2, 1% BSA, fixed with 0.5% paraformaldehyde in 1×PBS, pH 7.2, 1% BSA, and then measured on a BD FACS Canto II using propidium iodide to discriminate between live and dead cells.

[0085] Example 3: Flow Staining and Sorting of CHO Cells Stably Expressing a Membrane-Bound IgG Library A membrane-bound Fab-A56 plasmid library was generated from the product of phage panning against the antigen EEV.

[0086] Preparation of a Membrane-Bound Mini Library (MBML) by Phage Panning: Bacterial pellets containing phagemids were obtained by panning and plasmid DNA was extracted (Qiagen HiSpeed Maxiprep kit, catalog number 12662). An expression cassette containing the linked heavy and light chains (variable light chain / constant light chain - RBS element - variable heavy chain) was subcloned as a pool into the mammalian expression double gene vector pEFDGV3-Mab-A56R(Kan) using the BsrG1 restriction site and NheI restriction site as well as standard protocols for ligation and transformation (pEFDGV3 contains the γI CH1 constant domain to complete the antibody cassette during cloning). The library was plated on standard 150 mm LB AGAR plates containing 50 mg / mL kanamycin (LB-Kan50) and incubated overnight at 37°C. Plates containing only the control vector were included. Colonies were counted and the background was determined. At least 5000 colonies were recovered from the plates (10 ML LB / glycerol was added to each plate and the colonies were gently lifted from the agar surface using a sterile cell scraper), and plasmid DNA was extracted using the Qiagen plasmid DNA kit. Subsequently, this pool was digested with SalI / BssHI to remove the RBS element and replaced it with an IRES element for co-expression in mammals. The transformation was plated on 150 mm LB-Kan50 plates and incubated overnight at 37°C. At least 5000 colonies were collected together and plasmid DNA was isolated. The DNA concentration was measured by nanodrop and the DNA was turned over for transfection.

[0087] To cover the diversity of the plasmid library, approximately 10 million CHO cells were transfected in large quantities using Lipofectamine 2000 and incubated for 24 hours. Subsequently, the cells were harvested and dispensed into three T300 flasks containing DMEM medium supplemented with 10% FBS, 1 mM HEPES, and G418 (0.6 mg / ml). To maintain the selection pressure, the medium containing the drug was replaced every 2 - 3 days. After 2 - 3 weeks of drug selection culture, the cells were harvested using Accutase dissociation reagent and counted. At least 6 million cells were used for sorting. The cells were pelleted at 300 g for 5 minutes. The medium was aspirated, and then the cells were resuspended in blocking buffer (1×PBS (pH 7.2) containing 1% BSA and 2% FBS) on ice for 1 hour. The cells were pelleted and resuspended in 1 ml of 1% BSA, 100 U heparin, 1×PBS, pH 7.2 per 2.5 million cells and incubated at room temperature for 20 minutes. Subsequently, the cells were dispensed into flow staining tubes (0.4 ml each), and fluorescent yellow beads conjugated with EEV were added to each tube (40 μl / tube). The cells with beads added were incubated in the dark at room temperature for 45 minutes to 1 hour with occasional mixing. The cells were washed twice with 1×PBS, pH 7.2, 1% BSA, and then resuspended in 0.4 ml of the same solution containing anti-Fab-APC. The cells were incubated at 4°C for 30 minutes, then washed twice with 1×PBS, pH 7.2, 1% BSA, resuspended in 1 ml of the same solution, filtered through a 40 μm filter mesh, and sorted. A gate was created using a control to capture antigen EEV+ / anti-Fab+ cells. The sorted cells were pelleted and washed with 1×PBS. After storing the cell pellet at -20°C, genomic DNA extraction (Qiagen QiaAmp BloodDNA Mini) was performed, and the mab expression cassette was amplified using PCR (Takara Advantage cDNA polymerase 2) to contain restriction sites for cloning.

[0088] For the library with human constant regions, the mab expression cassette was amplified using the following primers. TIFF2025524372000009.tif12128

[0089] For libraries with full mouse expression, the mab expression cassette was amplified using the following primer pairs. TIFF2025524372000010.tif29128

[0090] Cycle number adjustments were performed to identify appropriate amplification conditions to minimize bias. The PCR product (approx. 2000 bp) was digested with either BsrG1 / Nhe (human V gene) or BsiW1 / Nhe1 for full mouse, and the cassette was subcloned into the inventors' mammalian dual gene expression vectors EVDGV3 (human), EFDGVmVKG1 or EFDGVmVKG2a (mouse).

[0091] Transformations were plated onto 100 mm LB-Kan50 plates at various densities to ensure proper colony separation and incubated overnight at 37°C. 94 colonies were picked into 96-well deep well growth plates containing 1.6 mL / well of LB / Kan50 and grown at 37°C for 22 hours. Spot plates were arranged to allow for future growth of individual clones. Plasmid DNA was isolated in this format using the Qiagen turbo 96 kit. DNA concentration was measured by nanodrop and a single plate concentration was assigned by averaging, and the DNA was taken forward for transfection.

[0092] DNA was sequenced at Genewiz using two primers, namely the Ef1F forward primer for the light chain variable region TIFF2025524372000011.tif4128 and the cGS reverse primer for the human heavy chain variable region TIFF2025524372000012.tif4128.

[0093] ECMVIRESFWD primer (SEQ ID NO:15) for a part of the mouse heavy chain variable region and the constant region TIFF2025524372000013.tif4128

[0094] Example 4: Flow cytometry staining of mouse B cells immunized with antigen EEV Balb / c mice were immunized 3 or 4 times with EEV MVA-T7-CD20-G-F. Six days after the last immunization, the mice were sacrificed and the spleens were removed to isolate B cells according to the Miltenyi Biotec B cell CD19+ positive isolation protocol. The B cells were blocked with 1×PBS (pH 7.2) containing 1% BSA and 2% FBS for 1 hour on ice. The B cells were pelleted and resuspended in 0.5 ml of 1×PBS (pH 7.2) containing 1% BSA. Fluorescent blue beads (25 μl) conjugated with FPV-H5-CD20-F EEV were added to the B cells and incubated in the dark at room temperature for 45 minutes to 1 hour with gentle mixing every 15 minutes. The B cells were washed twice with 2 ml of 1×PBS (pH 7.2) containing 1% BSA and resuspended in 0.5 ml of the same solution. Secondary antibodies, namely anti-B220-FITC, anti-mIgG1-BV421, anti-mIgG 2a and 2b-BV421, and anti-mIgM-PerCP-eFluor 710 were added and incubated at 4°C for 30 minutes. The B cells were washed twice with 2 ml of 1×PBS (pH 7.2) containing 1% BSA, resuspended in the same solution, and then sorted by passing through a 40 μm filter mesh. Gating was set to collect B220+ / IgM- / IgG+ / CD20 antigen-specific B cells. The sorted cells were pelleted, washed with PBS, and stored at 4°C before cDNA processing.

[0095] Construction of a phage library from sorted B cells 1700 sorted B cells (related to Y189) were stored in RNAlater (trademark) (ThermoFisher catalog number AM7020). RNA was extracted using the RNeasy Micro Kit (Qiagen 74004), treated with DNase, and quantified by Nanodrop. cDNA was prepared using standard protocols and subsequently treated with RNase. For cDNA synthesis, reactions were initiated using primers specific to the constant domain of the mouse γ-chain constant region 1 and IgG2a constant region genes as well as the mouse κ-chain constant region. Using standard methods and a mixture of primers for the mouse VH gene and JH gene containing the restriction sites BssHII and BsteII, the heavy chain variable region was PCR amplified. This PCR product was gel purified. Using standard methods and a mixture of primers for the mouse VK gene and JK gene containing the restriction sites ApaL1 and Xho1, the light chain variable region was PCR amplified. The heavy chain PCR product was gel purified and the V gene was cloned together into the phagemid pool (pAD14huGLlights) at the BssHII / BsteII site using NxGen T4 DNA ligase (Lucigen 3024-1) (the pAD phagemid backbone in the pool contains 14 human germline variable light chains fused to the human constant region, which are separated by the ribosome binding site (RBS)). The ligation reaction was used to transform TG1 electrocompetent cells (Lucigen 60502-2) by electroporation, grown for 1 hour, added to 2YXT buffer containing glucose and ampicillin, and the culture was expanded by shaking at 37°C for 5 hours. The phagemid library was recovered by centrifugation at 4°C, 6200 rpm for 15 minutes. The pellet was resuspended in freezing medium (containing 2×YT, glycerol, glucose, and Amp). The bacteria were plated to titer the library, a subset of the phagemids was miniprepped, and sequenced for quality control of the library. Glycerol was used for expansion and maxipreps were performed by standard procedures using the Qiagen HiSpeed Plasmid Maxi Kit (catalog number 12662).This DNA was used as a source of heavy chain V genes for the final library.

[0096] The light chain PCR products were gel purified. The V genes were pooled and cloned into the phagemid pool (pApAD) at the ApaL1 / Xho1 sites using NxGen T4 DNA ligase (Lucigen 3024-1). (The pApAD phagemid backbone in the pool contains a human light chain constant region fused to a human constant region with a stuffer sequence for cloning variable regions, and these regions are separated by a ribosome binding site (RBS)). The ligation reaction was used to transform TG1 electrocompetent cells (Lucigen 60502-2) by electroporation, grown for 1 hour, added to 2YXT buffer containing glucose and ampicillin, and cultured with shaking at 37°C for 5 hours to expand the culture. The phagemid library was recovered by centrifugation at 6200 rpm for 15 minutes at 4°C. The pellet was resuspended in freezing medium (containing 2×YT, glycerol, glucose, and Amp). The bacteria were plated to titer the library, a subset of phagemids was miniprepped, and sequenced for quality control of the library. Glycerol was expanded and maxiprepped by standard procedures. This DNA was used as the vector backbone for the final library. The DNA obtained from the maxipreps from the heavy chain library and the light chain library was digested with BssHII and Nhe1. The heavy chain was cloned into the stuffer region of the light chain library. NxGen T4 DNA ligase (Lucigen 3024-1). The ligation reaction was used to transform TG1 electrocompetent cells (Lucigen 60502-2) by electroporation, grown for 1 hour, added to 2YXT buffer containing glucose and ampicillin, and cultured with shaking at 37°C for 5 hours to expand the culture. The phagemid library was recovered by centrifugation at 6200 rpm for 15 minutes at 4°C. The pellet was resuspended in freezing medium (containing 2×YT, glycerol, glucose, and Amp). The bacteria were plated to titer the library (390 million clones), a subset of phagemids was miniprepped, and sequenced for quality control of the library.

[0097] Primer used for cDNA, heavy chain TIFF2025524372000014.tif12128

[0098] Primer used for PCR amplification, heavy chain TIFF2025524372000015.tif45141

[0099] Primer used for cDNA, light chain TIFF2025524372000016.tif4128

[0100] Primer used for PCR amplification, light chain TIFF2025524372000017.tif101142

[0101] A phage library was prepared from a bacterial glycerol stock by diluting 100-fold in 2×YT medium containing 2% glucose and 100 μg / mL ampicillin at a volume necessary to achieve 3-fold coverage of library diversity. The library was grown with shaking at 37 °C until an OD600 of approximately 0.5 was reached, and at this point, hyperphage was added at an MOI of 20. After 1 hour of growth, the bacteria were pelleted by centrifugation, resuspended in 2×YT medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin, and grown overnight for phage amplification. After centrifuging the overnight culture, the concentrated phage was precipitated from the supernatant, concentrated by centrifugation, and resuspended in PBS for later use in panning.

[0102] Phage display selection was performed with some modifications for panning in viruses according to the standard method (Toxins, 2018, Basics of Antibody Phage Display Technology, 10, 236). To select antibodies by phage panning, 3×10 in 1 mL of PBS at a final volume8 By combining 50 μL of the pfu EEV with beads, the EEV was ligated to MyOne tosyl group-activated Dynabeads (Invitrogen, 6550) and rotated overnight at 37°C. The beads were attracted to a magnet and washed to remove unbound EEV. The beads were then blocked with PBS + 10% FBS / 1% BSA for 2 hours at 37°C. For the first selection, a phage Fab display library containing approximately 1 × 10 8 unique clones was added to the ligated EEV for 2 hours at room temperature. After thorough washing, the phage bound to the beads was added directly to TG1 cells for overnight amplification. After centrifuging the overnight culture of TG1, the concentrated phage was precipitated from the supernatant, concentrated by centrifugation, and resuspended in PBS for subsequent panning. In each additional round of panning, the amount of input phage was decreased by a factor of 10 and further depleted multiple times with control EEV to remove anti-viral binding antibodies. The EEV ligation for depletion was performed in the same manner as for selection. After rounds of concentration, the V genes derived from the phage pool were subcloned into a mammalian expression vector while maintaining the VH and VL pairs present in each phage as a mini-library (ML).

[0103] Preparation of mini-library (ML) by phage panning: Bacterial pellets containing phagemids were obtained by phage panning. Plasmid DNA was extracted (Qiagen HiSpeed Maxiprep kit, catalog number 12662). An expression cassette containing the linked heavy and light chains (variable light chain / constant light chain - RBS element - variable heavy chain) was subcloned as a pool into the mammalian expression dual gene vector pEFDGV3ApaL1(Kan) for mammalian expression using the ApaL1 restriction site and NheI restriction site as well as standard protocols for ligation and transformation (pEFDGV3ApaL1 contains the heavy chain constant region for completing the antibody cassette during cloning). The library was plated on 4 standard 150 mm LB AGAR plates containing 50 mg / mL kanamycin (LB-Kan50) and incubated overnight at 37°C. Include plates with only the control vector. Colonies were counted and the background was determined. Approximately 5000 colonies were recovered from the plates (add 10 mL LB / glycerol per plate to each plate and gently lift the colonies from the agar surface using a sterile cell scraper), and plasmid DNA was extracted using the Qiagen plasmid DNA kit. Subsequently, this pool was digested with SalI / BssHI to remove the RBS element and replaced it with an IRES element for co-expression in mammals. The transformants were plated on 100 mm LB-Kan50 plates at various densities to ensure proper separation of the colonies and incubated overnight at 37°C. 94 colonies were picked into 96-well deep well growth plates containing 1.6 mL / well of LB / Kan50 and grown at 37°C for 22 hours. Spot plates were arranged to enable future growth of individual clones. Plasmid DNA was isolated in this format using the Qiagen turbo 96 kit. The DNA concentration was measured by nanodrop, and a single plate concentration was assigned by averaging, and the DNA was used for transfection and antibody testing by flow cytometry.

[0104] The DNA was two primers, namely the Ef1F forward primer for the variable region of the light chain TIFF2025524372000018.tif4128 and cGS reverse primer for the heavy chain variable region Sequenced at Genewiz using TIFF2025524372000019.tif4128.

[0105] Example 5: Flow Cytometry Staining of Immunized Mouse B Cells CD20 and CD39 Yellow and blue fluorescent streptavidin polystyrene beads (1 - 1.5×10 8 particles) were coated with 25 μg of biotinylated chicken anti - FPV antibody, washed, and incubated overnight at room temperature (RT) with FPV - H5 - CD20 - F or FPV - H - CD39 - F at a bead - to - virus ratio of 5:1. The beads were then washed and blocked overnight at room temperature with 1% BSA, 2% FBS in PBS. The beads were resuspended in 100 μl of 1×PBS 1% BSA and used for staining. CHO stable cell lines (2×10 5 cells) expressing membrane - bound human anti - CD20 antibody (mab271) or anti - CD39 antibody (mab26086) were blocked on ice for 1 hour with 1×PBS 1% BSA 2% FBS, washed, treated with 100 units of heparin in 1×PBS, 1% BSA at room temperature for 20 minutes, aliquoted into flow cytometry tubes, and incubated at room temperature for 1 hour with 40 μl of yellow (Figure 1A and 1B) or 25 μl of blue (Figure 1C and 1D) FPV - coated beads with occasional mixing. The cells were then washed twice with 1×PBS 1% BSA, resuspended in goat anti - human Fab - APC secondary antibody or goat anti - human Fab - FITC secondary antibody, and incubated at 4°C for 30 minutes. The cells were pelleted, washed twice, fixed with 0.5% paraformaldehyde in 1% BSA PBS, and then measured on a BD FACS Canto II using propidium iodide to discriminate between live and dead cells. Cells expressing the antibody and binding to FPV - coated beads were identified as double - positive events (Figure 1A - 1D). Good signal - to - noise was observed using control antibody transfectants and two different fluorescent virus constructs.

[0106] SEMA4D Fluorescent yellow and fluorescent blue streptavidin polystyrene beads (1 - 1.5×10 8 particles) were incubated overnight at room temperature in the dark at a bead-to-virus ratio of 5:1 with FSL-biotinylated MVA-SEMA-ECD EEV and MVA-CXCR5-G-SL EEV. The beads were then washed and blocked overnight at room temperature with 1% BSA, 2% FBS in PBS. The beads were resuspended in 100 μl of 1×PBS 1% BSA and used for staining. CHO stable cell lines (2×10 5 cells) expressing membrane-bound human anti-SEMA4D antibody (mab67) were blocked on ice for 1 hour with 1×PBS 1% BSA 2% FBS, washed, treated with 100 units of heparin in 1×PBS,  1% BSA at room temperature for 20 minutes, dispensed into flow staining tubes, and incubated at room temperature for 1 hour with 40 μl of yellow (Figure 2A, left panel) or 25 μl of blue (Figure 2B, right panel) MVA-coated beads with occasional mixing. The cells were then washed twice with 1×PBS, 1% BSA, resuspended in goat anti-human Fab secondary antibody conjugated with APC or FITC, and incubated at 4°C for 30 minutes. The cells were pelleted, washed twice, fixed with 0.5% paraformaldehyde in 1% BSA PBS, and then measured on a BD FACS Canto II using propidium iodide to discriminate between live and dead. Cells expressing the antibody and binding to MVA-coated beads were identified as double-positive events. (Figure 2A - 2D). Good signal-to-noise was observed using control antibody transfectants and two different fluorescent virus constructs.

[0107] SEMA4D and SEMA4D IgG library CHO cells stably expressing membrane-bound human anti-SEMA4D (Figure 3A) or the SEMA4D IgG library were blocked on ice for 1 hour with 1×PBS 1% BSA 2% FBS, washed, treated with 100 units of heparin in 1×PBS, 1% BSA at room temperature for 20 minutes, dispensed into flow staining tubes (2×105 Individual cells / tubes) were incubated with fluorescent yellow streptavidin beads coated with FPV-SEMA4D or FPV-CXCR5 for 1 hour in the dark at room temperature. For SEMA4D IgG library selection, 9×10 6 cells were resuspended in 3.6 ml of 1×PBS 1% BSA, dispensed into flow staining tubes (0.4 ml / tube), and incubated with fluorescent yellow streptavidin beads coated with FPV-SEMA4D (40 μl / tube). The cells were then washed twice with 1×PBS 1% BSA, resuspended in secondary anti-human Fab-APC, and incubated at 4°C for 30 minutes. The cells were pelleted and washed twice. For flow cytometry analysis and pre-selection evaluation, the cells were fixed with 0.5% paraformaldehyde in 1% BSA PBS and then measured on a BD FACS Canto II using propidium iodide to discriminate between live and dead cells. For selection of the SEMA4D IgG library, the cells were resuspended in 1×PBS 1% BSA and filtered through a 40-μm filter. The selected cells were pelleted and washed with 1×PBS. After storing the cell pellet at -20°C, genomic DNA extraction and PCR were performed. These results are shown in Figures 3A and 3B. As can be confirmed in Figure 3C, the recovery rate of the anti-SEMA4D antibody after selection was 5% positive clones.

[0108] B cells selected based on B220+, IgM(-), IgG1(+), IgG2ab(+): FPV-CD20 and FPV-CD39 Balb / c mice were immunized three times with EEV MVA-T7-CD20-G-F. Six days after the last immunization, the mice were sacrificed and the spleens were removed to isolate B cells according to the Miltenyi Biotech B cell negative selection protocol. The B cells were blocked with 1×PBS (pH 7.2) containing 1% BSA and 2% FBS for 1 hour on ice. Next, the B cells were pelleted and resuspended at 1×10 7Cells were resuspended in 0.4 ml of 1×PBS (pH 7.2) containing 1% BSA per cell. Fluorescent blue beads coated with FPV-H5-CD20-F EEV or FPV-H5-CD39-F EEV (negative control) were added to the B cells (25 ul / 1×10 7 cells), and incubated for 1 hour at room temperature in the dark with gentle mixing every 15 minutes. The B cells were then washed twice with 2 ml of 1×PBS (pH 7.2) containing 1% BSA and resuspended in 0.4 ml of the same solution per 1×10 7 cells. Secondary antibodies, namely anti-B220-FITC, anti-mIgG1-BV421, anti-mIgG2a and 2b-BV421, and anti-mIgM-PerCP-eFluor 710 were added and incubated for 30 minutes at 4°C. The B cells were washed twice with 2 ml of 1×PBS (pH 7.2) containing 1% BSA and resuspended in the same solution. The results are shown in FIGS. 4A-4D.

[0109] A phage display library was prepared from the sorted cells. The phage library was panned 3 times on FPV-CD20 and then tested with a specific anti-CD20 antibody. The newly identified CD20 antibodies are shown in Table 1 below.

[0110]

Table 1

[0111] Example 6: Isolation of Plasma Cells and ELISA for CXCR4 Mice were immunized intraperitoneally 4 times with MVA-CXCR4 intracellular mature virus (IMV). On the 5th day after the final booster immunization, the spleen and bone marrow were separately excised in 1×PBS, pH 7.2. The spleen was cut into small pieces and passed through a 40 μM filter and ground. The spleen cells were washed twice with 1×PBS, pH 7.2. After removing the PBS, red blood cells were lysed using lysis buffer (Biolegend, catalog number 420301) according to the manufacturer's protocol. Separately, the bone marrow was filtered through a 40 μM filter and washed twice with 1×PBS, pH 7.2. Then, plasma cells were isolated separately from the bone marrow and spleen using a CD138+ plasma cell isolation kit (Milentyi, catalog number 30-092-530) according to the manufacturer's protocol. The cells were counted and seeded at a density of 1000 cells / well in plasma cell complete growth medium (growth medium: RPMI, 10% FBS, 1×β-mercaptoethanol, 10 ng / ml CXCL12, 50 ng / ml APRIL, 5 ng / ml IL-6, 5 ng / ml BAFF, 5 ng / ml IL-4) in a V-bottom 96-well plate. After 72 hours, the supernatant was collected and plasma cell ELISA was performed.

[0112] For plasma cell ELISA, the ELISA plate was coated with 2×10 6Coated with either FPV-CXCR4opt-GFP-F13L crude EEV or FPV wt crude EEV at pfu / ml and incubated overnight at 4°C. The next day, the plates were washed three times with 1×PBS (pH 7.2), 0.05% Tween 20, and blocked with 250 μl / well of 1×PBS (pH 7.2), 1% BSA for 1 hour at room temperature. The plates were washed three times again, and after dilution at 1:2, 100 μl of the supernatant was added to each virus-coated plate. The plates were incubated for 1 hour at room temperature. After washing three times, 100 μL / well of 1:40,000 anti-mouse IgG (H+L)-biotin (Jackson catalog number 115-065-166) was added to each well for detection. The plates were incubated for 1 hour at room temperature and then washed three times. 1:8000 streptavidin-HRP (Thermo Fisher, catalog number SNN2004) was added to each well (100 μl) and incubated for 30 minutes at room temperature. The plates were washed three times, and 100 μl of TMB substrate (BioFx, catalog number TMBW-1000-01) was added to develop the color of the plates. After 15 minutes, 100 μl of 2N sulfuric acid was added to stop the color development. The plates were read at 450 - 570 nM using a BioTek PowerWave reader.

[0113] After reading, the positive wells containing plasmablasts were diluted to 100 cells / well with plasmablast complete growth medium. After 72 hours, the supernatant was collected and plasmablast ELISA was performed as described above. The positive plasmablast wells were stored in 100 μl of RNALater (Invitrogen, catalog number AM7021). Plasmablasts were recovered and used to generate a phage display antibody library.

[0114] Example 7: Isolation of Plasmablasts and ELISA for CD20 Mice were immunized three times via intraperitoneal injection using either MVA-CD20 IMV or EEV. Four days after the final booster immunization, spleens and bone marrows were separately harvested in 1×PBS, pH 7.2. Bone marrow was processed as in the previous example, except that plasma cells were diluted to 100 cells / well after isolation.

[0115] For plasma cell ELISA, ELISA plates were coated with either 2×10 6 pfu / ml of either FPV-CD20-F13L crude EEV or FPV wt crude EEV and incubated overnight at 4°C. The next day, ELISA was performed as shown in the previous example (see Figure 5A for strategy).

[0116] After reading, RNALater (Invitrogen, catalog number AM7021) was added to the positive wells containing plasma cells. The recovered plasma cells were used to generate an antibody display library. The phage display library was then panned on FPV CD20 and specific anti-CD20 antibodies were tested. The newly identified CD20 antibodies are shown in Table 2 below. Individual histograms of the antibodies are shown in Figure 5B.

[0117]

Table 2

Claims

**Claim 1** A method for selecting a binding molecule that binds to a target integral membrane protein (IMP), comprising the following steps: (a) attaching an antigen virion comprising a poxvirus comprising an integral membrane protein (IMP) or a fragment thereof fused to an extracellular envelope virion (EEV) protein or a functional fragment thereof to a solid support to form a linked antigen virion, wherein the poxvirus expresses the target IMP or a fragment thereof in its native conformation as part of the outer envelope membrane of the EEV, and the IMP or a fragment thereof comprises at least one extracellular region, at least one transmembrane region, and at least one intracellular region; (b) contacting the linked antigen virion with an antibody display library, wherein the library comprises display packages presenting a plurality of antigen-binding domains; (c) selecting, from the display package, a gene or a fragment thereof of the antibody variable region that binds to the linked antigen virion such that the sequences encoding the variable light chain (VL), variable heavy chain (VH), and poxvirus anchor protein are co-expressed as a single polypeptide, and cloning the variable light chain (VL) gene or a fragment thereof and the variable heavy chain (VH) gene or a fragment thereof derived from the display package into a plasmid vector in-frame with a polynucleotide sequence encoding the poxvirus anchor protein or a fragment thereof; (d) transfecting a mammalian cell with the plasmid vector of step (c) such that the transfected cell expresses the VL antigen-binding domain and the VH antigen-binding domain on the mammalian cell surface; (e) screening the transfected cells using the antigen virion linked to a detectable solid support; and (f) recovering the cells presenting the antigen-binding domain specific to the target. **Claim 2** The method according to claim 1, wherein the solid support in step (a) is magnetic beads labeled with streptavidin. **Claim 3** The method according to claim 2, wherein the poxvirus is fowlpox virus labeled with biotin-anti-fowlpox antibody. **Claim 4** The method according to claim 2, wherein the poxvirus is biotinylated vaccinia virus Ankara (MVA). **Claim 5** The method according to claim 1, wherein the poxvirus anchor protein is the vaccinia virus A56R protein.

6. The method according to claim 1, wherein the mammalian cell is a CHO cell.

7. The method according to claim 1, wherein the IMP is a multi-pass transmembrane protein.

8. The method according to claim 7, wherein the IMP is an ion channel or a G protein.

9. A method for selecting a binding molecule that binds to a target integral membrane protein (IMP), comprising the following steps: (a) isolating plasma cells from an animal immunized with an antigen comprising a target integral membrane protein (IMP) or a fragment thereof; (b) seeding the plasma cells in a pooled state comprising a plurality of cells and growing them to a desired cell density in a nutrient medium; (d) performing one or more assays on the plasma cells to identify cells that express a binding molecule that binds to the target IMP protein; and (e) recovering the plasma cells that express a binding molecule that binds to the target IMP protein.

10. A first ELISA is performed to identify cells that express a binding molecule that binds to the target IMP protein, and subsequently at least one additional ELISA assay is performed, in which the cells identified by the first ELISA assay are diluted before performing the additional ELISA assay for identifying cells that express a binding molecule that binds to the target IMP protein. The method according to claim 9.

11. The method according to claim 9, wherein the recovered cells are used to generate an antibody display library, and the library comprises a display package that presents a plurality of antigen-binding domains.

12. The method according to claim 9, wherein the IMP is a multi-pass transmembrane protein.

13. The method according to claim 9, wherein the mammal is a mouse.

14. A method for selecting a binding molecule that specifically binds to a target integral membrane protein (IMP), comprising the following steps: (a) isolating B cells from an animal immunized with a target integral membrane protein (IMP) or a fragment thereof; (b) sorting the B cells to isolate antigen-specific B cells that express IgG that specifically binds to the target IMP. (c) A step of performing single-cell analysis to identify immunoglobulin variable region genes expressed by the selected B cells.

15. A phage Fab display library is generated from variable heavy chain (VH) cDNA and variable light chain (VL) cDNA generated from RNA isolated from antigen-specific B cells expressing IgG that binds to the target IMP; and the phage Fab display library is panned to exclude anti-poxvirus binding molecules and enrich anti-target IMP binding molecules. The method according to claim 14.

16. The method according to claim 14, further comprising the step of isolating and cloning variable heavy chain genes and / or variable light chain genes from individual selected B cells.

17. The method according to claim 14, wherein the single-cell analysis includes RT-PCR.

18. The method according to claim 14, wherein the B cells are selected using a target IMP linked to a detectable solid support.

19. The method according to claim 18, wherein the detectable solid support is streptavidin-fluorescent beads.

20. The method according to claim 15, further comprising the step of isolating VH genes and VL genes (V genes) from the phage Fab display library and subcloning the V genes into a mammalian expression vector while maintaining pairs of VH and VL present in individual phages as a mini-library (ML).

21. The library according to claim 15.

22. Variable heavy chain (VH) of SEQ ID NO: 38 and variable light chain (VL) of SEQ ID NO: 39; VH of SEQ ID NO: 40 and VL of SEQ ID NO: 41; VH of SEQ ID NO: 42 and VL of SEQ ID NO: 43; VH of SEQ ID NO: 44 and VL of SEQ ID NO: 45; VH of SEQ ID NO: 46 and VL of SEQ ID NO: 47; VH of SEQ ID NO: 48 and VL of SEQ ID NO: 49; VH of SEQ ID NO: 50 and VL of SEQ ID NO: 51; VH of SEQ ID NO: 52 and VL of SEQ ID NO: 53; VH of SEQ ID NO: 54 and VL of SEQ ID NO: 55; VH of SEQ ID NO: 56 and VL of SEQ ID NO: 57; VH of SEQ ID NO: 58 and VL of SEQ ID NO: 59; and VH of SEQ ID NO: 60 and VL of SEQ ID NO: 59 An antibody that specifically binds to CD20, selected from the group consisting of antibodies comprising

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