Method for obtaining antibodies that bind to transmembrane proteins and cells that produce the antibodies

Abstract

Disclosed are methods for obtaining cells expressing antibodies that bind to transmembrane proteins, methods for producing antibodies from such cells, antibodies against transmembrane proteins and fragments thereof, and nucleic acids encoding the antibodies. More specifically, the disclosure relates to a method for obtaining antibody-producing cells expressing antibodies that bind to transmembrane proteins based on the use of a lipid bilayer membrane scaffold protein complex to present transmembrane protein antigens to cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 130,044, filed December 23, 2020, the entire contents of which are incorporated herein by reference. Incorporation by reference of sequence listing

[0002] A sequence listing in the form of a 30 KB ASCII text file entitled 36526_10465WO01_SequenceListing.txt, created on December 7, 2021 and submitted to the U.S. Patent and Trademark Office via EFS-Web, is incorporated herein by reference.

[0003] The present disclosure generally relates to cells expressing antibodies that bind to transmembrane proteins, methods for producing the same, antibodies against transmembrane proteins and fragments thereof, and nucleic acids encoding the antibodies. More specifically, the present disclosure relates to a method for obtaining antibody-producing cells expressing antibodies that bind to transmembrane proteins based on the use of lipid bilayer membrane scaffold protein complexes to present transmembrane protein antigens to cells. [Background technology]

[0004] Transmembrane proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are the targets of nearly half of all FDA-approved small molecule drugs, yet so far, very few antibodies have been approved for therapy. See Santo et al., Nat. Rev. Drug Disc. 16:19 (2017). Typically, methods utilized to screen for antibodies or antibody-producing cells are inefficient or fail to obtain antibodies that bind to transmembrane proteins. For example, isolation and packaging of multispan transmembrane proteins into carriers such as exosomes, virus-like particles, and proteoliposomes leaves uncertainty as to whether sufficient amounts of biologically active or conformationally correct transmembrane proteins are present. Furthermore, GPCR-expressing cells have been less successful as screens for reagents, and small peptides derived from transmembrane proteins have had little success in obtaining relevant antibodies due to the lack of conformational context. Therefore, new methods are needed to obtain and generate antibodies against transmembrane proteins. Summary of the Invention

[0005] Disclosed herein is a method for obtaining antibodies against transmembrane proteins that utilizes a lipid bilayer membrane scaffold protein complex to present the transmembrane protein antigen to the antibody. The method utilizes a complex that includes a transmembrane protein of interest and lipids and membrane scaffold proteins commonly present in the membrane of a natural cell to present the transmembrane protein in its native conformation to the antibody. Thus, the lipid bilayer membrane scaffold protein complex is used in the disclosed method to identify and collect from a population of antibodies (or cells expressing antibodies) a specific subset of antibodies that bind to an epitope on a transmembrane protein that is accessible in nature, such as an extracellular domain or a portion thereof. Thus, the method circumvents the need for time-consuming screening, identification and selection of epitope-specific antibodies by site-directed mutagenesis and other well-known techniques to confirm whether a particular antibody recognizes a desired portion of a transmembrane protein of interest.

[0006] In one aspect, a method is provided for obtaining an antibody or a cell population expressing an antibody against a transmembrane protein of interest, comprising contacting a population of antibody-producing cells with a lipid bilayer membrane scaffold protein complex that displays the transmembrane protein of interest or a portion thereof.

[0007] In some embodiments, the method comprises contacting a population of antibody-producing cells with a lipid bilayer membrane scaffold protein complex that includes a transmembrane protein of interest or a portion thereof to allow binding between the transmembrane protein antigen presented by the complex and an antibody on the surface of the cells, and collecting the bound antibody-producing cells.

[0008] In some embodiments, the antibody-producing cell population is a homogenous population of cells made up of one particular type of tissue, organ, or cell. In other embodiments, the antibody-producing cell population is a heterogenous population of cells made up of two or more types of tissue, organ, or cell. In certain embodiments, the antibody-producing cell population comprises tissue-derived cells from one or more of the spleen, lymph nodes, bone marrow, or other organs. In some embodiments, the antibody-producing cell population comprises lymphocytes. In certain embodiments, the antibody-producing cell population comprises blood cells. In certain embodiments, the antibody-producing cell population comprises peripheral blood cells, B cells, plasma cells, plasma cell myelomas, or combinations thereof. In certain embodiments, the antibody-producing cell population is a B cell population. In one embodiment, the antibody-producing cell population consists of memory B cells. In one embodiment, the antibody-producing cell population comprises recombinant cells, such as, for example, hybridomas.

[0009] In some embodiments, the method includes obtaining a population of antibody-producing cells from an animal. In certain embodiments, the population of antibody-producing cells is obtained from an animal that produces antibodies against a transmembrane protein of interest after immunization with the transmembrane protein of interest or a nucleic acid immunogen encoding same. In certain embodiments, the animal or immunized animal is a mammal. In some embodiments, the mammal is a mouse, rat, goat, human, hamster, pig, monkey, or guinea pig. In some embodiments, the mammal is not a human. In certain embodiments, the non-human mammal is a mouse, rat, or goat. In certain embodiments, the mammal is a mouse. In another embodiment of the method, the mammal is a human, such as, for example, a human that has been exposed to the immunogen.

[0010] In some examples, the animal is immunized. In certain embodiments, the immunized animal is genetically engineered. For example, the animal can be genetically engineered so that it does not express the transmembrane protein of interest from its endogenous locus. In certain embodiments, the genetically engineered animal is a non-human mammal, such as, for example, a mouse, goat, or rat, and the animal comprises a nucleic acid sequence encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain (IgL) variable region. In some embodiments, the genetically engineered animal comprises a nucleic acid sequence encoding a human immunoglobulin heavy chain and a human immunoglobulin light chain variable region, and also lacks an endogenous gene encoding the transmembrane protein of interest. In certain embodiments, the immunized genetically engineered animal is a mouse or a rat, such as, for example, a VELOCIMMUNE® mouse, comprising a humanized IgH locus and / or a humanized Igκ light chain locus. In some embodiments, the immunized genetically engineered animal is a mouse comprising a humanized IgH locus and a humanized Igκ light chain locus, and the mouse lacks an endogenous mouse gene encoding a transmembrane protein of interest. In one embodiment, the genetically engineered mouse comprises DNA encoding a human immunoglobulin heavy chain and an immunoglobulin lambda light chain (Igλ) variable region. In a particular embodiment, the genetically engineered mouse comprises DNA encoding a human immunoglobulin heavy chain and an immunoglobulin lambda light chain (Igλ) variable region, and lacks an endogenous mouse gene encoding a transmembrane protein of interest.

[0011] In some embodiments, the antibody producing cells are obtained from an animal immunized with an immunogenic transmembrane protein of interest. In certain embodiments, the animal is immunized with a nucleic acid encoding at least a portion of the transmembrane protein of interest, or at least a portion of the transmembrane protein of interest. In some embodiments, the animal is immunized with a nucleic acid encoding a full-length transmembrane protein of interest. In other embodiments, the animal is immunized with a nucleic acid encoding a transmembrane protein of interest. In certain embodiments, the nucleic acid does not encode the amino-terminal and / or carboxy-terminal portion of the full-length transmembrane protein of interest. In other embodiments, the animal is immunized with a nucleic acid encoding a transmembrane protein of interest or a portion thereof that is contained in a carrier capable of expressing the nucleic acid, such as, for example, a plasmid, an expression vector, a virus-like particle (VLP), a cell, an exosome, and a liposome. In some embodiments, the animal is immunized with a transmembrane protein of interest or a portion thereof. In certain embodiments, the animal is immunized with a full-length transmembrane protein of interest. In certain embodiments, the immunogenic transmembrane protein of interest is truncated and does not include the amino-terminal and / or carboxy-terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the transmembrane protein or nucleic acid immunogen of interest is modified to include one or more detectable elements, such as labels, markers, or features. In some embodiments, the immunogen includes a detectable label. In certain embodiments, the detectable label is a FLAG-tag, a histidine tag (His-tag), an Avi-tag, a BirA-tag, or a combination thereof. In some embodiments, the transmembrane protein of the immunogen of interest has a FLAG tag and a His tag. In certain embodiments, the detectable label is located at the amino-terminus or carboxy-terminus of the immunogen. In certain embodiments, an animal is immunized with the transmembrane protein of interest or a portion thereof that is incorporated into a lipid bilayer membrane scaffold protein complex.

[0012] In certain embodiments, animals are immunized with a nucleotide sequence encoding a chimeric transmembrane protein of interest, comprising a portion of a human transmembrane protein of interest operably linked to a portion of a non-human homologue of the transmembrane protein of interest. The non-human homologue may be from, for example, a human, a chimpanzee, a rhesus monkey, a rabbit, a horse, a sheep, a rat, a mouse, a dog, a chicken, or a goat. In certain embodiments, the nucleotide sequence encoding the chimeric transmembrane protein of interest also comprises a nucleotide sequence encoding a detectable element, such as, for example, a His tag, a FLAG tag, an Avi tag, or a Bir-A tag. In other embodiments, animals are immunized with a chimeric transmembrane protein of interest or a portion thereof, comprising a portion of a human transmembrane protein of interest operably linked to a portion of a non-human homologue of the transmembrane protein of interest. In one embodiment, the chimeric transmembrane protein of interest comprises a detectable element, such as, for example, a His tag, a FLAG tag, an Avi tag, or a Bir-A tag.

[0013] In some examples, animals are immunized with two or more immunogens, such as, for example, a protein or peptide, a nucleic acid sequence such as DNA or RNA, a modified protein or encoding DNA, a VLP and a lipid bilayer membrane scaffold protein complex that includes a transmembrane protein of interest or a portion thereof.

[0014] The method includes contacting a population of antibody-producing cells with a lipid bilayer membrane scaffold protein complex that includes a transmembrane protein of interest or a portion thereof, to obtain a population of antibody-producing cells that expresses an antibody that binds to the transmembrane protein of interest.

[0015] The lipid bilayer membrane scaffold protein complex comprises at least one membrane scaffold protein (MSP) and a lipid. In some embodiments, the lipid bilayer membrane scaffold protein complex comprises at least one MSP and a plurality of lipids. In certain embodiments, the lipid bilayer membrane scaffold protein complex comprises at least two or exactly two MSPs. In other embodiments, the lipid bilayer membrane scaffold protein complex comprises three or more MSPs. In some examples, the lipid bilayer membrane scaffold protein comprises at least one membrane scaffold protein, such as MSP1E3D1, MSP1D1, MSP2N3, and MSP2N2. In one embodiment, the lipid bilayer membrane scaffold protein complex comprises two MSP1E3D1 proteins. In some embodiments, the MSPs are the same. In other embodiments, the MSPs in the lipid bilayer membrane scaffold protein complex are different.

[0016] In some embodiments, the lipid bilayer membrane scaffold protein complex contains at least one labeled MSP that includes a detectable element, such as a label, marker, or feature. In certain embodiments, the lipid bilayer membrane scaffold protein complex contains two labeled MSPs and a lipid bilayer. In some embodiments, the MSP protein includes a detectable label, such as a fluorophore. In certain embodiments, the detectable element is a BirA tag or an Avi tag located on one or more MSPs of the complex. In exemplary embodiments, one or more MSPs are biotinylated by chemically biotinylating the MSPs or by genetically introducing an Avi-tag into the MSP coding sequence.

[0017] The lipid bilayer membrane scaffold protein complex also includes multiple lipids, such as sphingolipids and / or phospholipids. The lipid bilayer of the complex may be composed of a single type of lipid or multiple types of lipids. In some embodiments, the lipid bilayer membrane scaffold protein complex includes lipids that form a "disc-shaped" phospholipid bilayer in the shape of a disk around the membrane scaffold protein. In certain embodiments, the lipid bilayer is composed of one or more of sphingomyelin, phosphatidylcholine, and derivatives thereof. In certain embodiments, the lipid bilayer is composed of 1-dioleoylphosphatidylcholine (DOPC), 1-palmitoyl 2-oleoylphosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), and phosphatidylinositol (PI), or combinations thereof. In an exemplary embodiment, the lipid bilayer includes multiple POPC phospholipids.

[0018] The lipid bilayer membrane scaffold protein complex used in the method also includes at least one transmembrane protein of interest or a portion thereof, which is presented by the complex to the cell population as an antigen capable of binding to the antibody generated by the antibody-producing cell. The transmembrane protein of interest presented by the complex can be a native protein having at least one extracellular domain and at least one transmembrane domain. In some embodiments, the transmembrane protein of interest presented by the complex is a human protein. In other embodiments, the transmembrane protein of interest is a non-human protein, such as a mouse, rat, primate, hamster, bacterial, or viral protein. In certain embodiments, the transmembrane protein of interest is modified from its native form. Exemplary modified transmembrane proteins of interest may include one or more changes to their native amino acid sequence, such as amino acid substitutions, amino acid deletions, or amino acid insertions. In one embodiment, the transmembrane protein of interest is modified to delete or "truncate" a portion of the transmembrane protein, such as, for example, the N-terminal and / or C-terminal domains of the full-length protein. In some embodiments, the transmembrane protein of interest incorporated into the lipid bilayer membrane scaffold protein complex comprises a stabilizing mutation at the amino terminus, one or more extracellular loop domains, one or more transmembrane domains, one or more intracellular domains, the C-terminus, or a combination thereof. In one embodiment, the transmembrane protein of interest is a ligand-activated protein, whereby the transmembrane protein of interest changes conformation in the presence or absence of a ligand (i.e., has an active state and an inactive state). In another embodiment, the transmembrane protein of interest incorporated into the lipid bilayer membrane scaffold protein complex is a chimeric protein, comprising a portion of a human transmembrane protein of interest operably linked to a portion of a non-human homologue of the transmembrane protein of interest. In one embodiment, the transmembrane protein of interest comprises a detectable element, such as, for example, a His tag, a FLAG tag, an Avi tag, a Bir-A tag, or a combination thereof. In certain embodiments, the transmembrane protein of interest presented by the complex comprises a His tag and a FLAG tag.

[0019] In certain examples, the transmembrane protein of interest is a GPCR protein, a tetraspanin protein, or an ion channel protein.In some embodiments, the transmembrane protein of interest is a GPCR protein, such as, for example, CCR5, ADORA2A, ADRB3, C3AR1, ADRA2A, GLP1R, CCR4, CCR8 and CXCR4.In some embodiments, the transmembrane protein of interest is CCR5 or a part thereof.In some embodiments, the transmembrane protein of interest is ADRA2A or a part thereof.In certain embodiments, the transmembrane protein of interest is ADORA2A or a part thereof.In some embodiments, the transmembrane protein of interest is C3AR1 or a part thereof.

[0020] In some embodiments, the transmembrane protein of interest is a tetraspanin, such as, for example, TSPAN 1 to TSPAN19, TSPAN21, TSPAN23, TSPAN 31, TSPAN 32, TSPAN 33, UPK1B, PRPH2, CD151, CD53, CD37, CD82, CD63, CD81, CD9, CD82, CD63, CLND6 and CLND9. In some embodiments, the transmembrane protein of interest is CD63 or a portion thereof. In other embodiments, the transmembrane protein of interest is an ion channel protein, such as, for example, a voltage-gated ion channel protein. In certain embodiments, the voltage-gated ion channel protein is a voltage-gated calcium channel or a voltage-gated potassium channel protein. In some embodiments, the ion channel protein is a calcium-activated potassium channel protein, a sodium channel protein, a calcium channel protein, or a chloride channel. In certain embodiments of the method, the transmembrane protein of interest is an ion channel protein, such as, for example, BKCa, MaxiK, Sk, NaV1, CACNG1, CAV, CIC, or transient receptor potential channel (TRP).In some embodiments, the transmembrane protein of interest is NaV1 or a portion thereof.In some embodiments, the transmembrane protein of interest is NaV1.7 or a portion thereof.In some embodiments, the transmembrane protein of interest is CACNG1 or a portion thereof.

[0021] In certain embodiments of the method, the transmembrane protein of interest presented by the complex binds to an antibody on the cell surface of the antibody-producing cell. In some embodiments, the antibody binds to an epitope (a binding domain present on the transmembrane protein of interest) and is located in a particular domain of the transmembrane protein of interest, such as the extracellular domain of the transmembrane protein of interest. In certain embodiments, the antibody binds to an epitope located in the extracellular portion of the N-terminal domain of the transmembrane protein of interest. In certain embodiments, the antibody binds to an epitope located in an extracellular loop of the transmembrane protein of interest or in the extracellular portion of the C-terminal domain of the transmembrane protein of interest. In certain embodiments, the antibody binds to an epitope located in the C-terminal domain of the transmembrane protein of interest. In some embodiments, the antibody binds to an epitope located in an extracellular loop of the transmembrane protein of interest. In some embodiments, the antibody binds to an epitope located in the intracellular domain of the transmembrane protein of interest.

[0022] The method may also include contacting a population of antibody-producing cells with a detectable element that binds to a cell surface protein or biomarker of interest. For example, a heterogeneous population of antibody-producing cells obtained from an immunized animal may be contacted with a fluorescently labeled antibody that binds to a B cell surface protein, such as IgG. A subset of antibody-producing B cells can then be obtained from the population by detecting the binding between the antibody and the B cells and isolating the bound cells from the population. In certain embodiments, the population of antibody-producing cells can be contacted with a fluorescently labeled antibody that binds to a B cell surface protein at the same time that the cells are contacted with a lipid bilayer membrane scaffold protein complex containing a transmembrane protein of interest, or the cells can be contacted at different times.

[0023] In certain embodiments, the method may also include contacting the population of antibody-producing cells with a blocking agent. For example, the population of antibody-producing cells may be contacted with a molecule, such as a peptide or compound, that recognizes or binds a portion of a transmembrane protein of interest, a portion of an MSP protein, or a detectable marker, such as a His tag or a FLAG tag. In such embodiments, the antibody-producing cells are incubated with one or more blocking agents to allow binding between the blocking agent and the antibody produced by the antibody-producing cells, and the antibody-producing cells bind to the epitope located on the blocking agent.

[0024] The method may also include washing the cell population, such as antibody-producing cells, for a period of time to remove unbound material or cells from the bound cells.

[0025] Binding between the antibodies produced by the antibody-producing cells and the binding domains (epitopes) present on the transmembrane protein of interest presented by the lipid bilayer membrane scaffold protein complex can be detected, and the antibody-producing cells bound to the transmembrane protein can be collected. For example, in some embodiments, binding is detected by a conformational change in the transmembrane protein of interest, activation or inactivation of the transmembrane protein of interest in the cell, or the use of one or more detectable markers. In certain embodiments, antibody-producing cells presenting antibodies bound to the transmembrane protein antigen of interest can be detected and isolated from other antibody-producing cells in the population using high-throughput techniques for single-cell isolation, such as fluorescence-activated cell sorting (FACS). In one embodiment, FACS is used to identify and isolate single antibody-producing cells bound to the lipid transmembrane protein of interest presented by the lipid bilayer membrane scaffold protein complex by detecting a signal emitted by a detectable label attached to the complex or transmembrane protein of interest contained therein. In certain embodiments, the signal is emitted by one or more detectable labels of biotin / streptavidin-PE complexes or fluorescent molecules.

[0026] The method may also include obtaining or isolating the antibody or nucleic acid encoding the antibody from an antibody-producing cell.

[0027] In certain embodiments, the nucleic acid encoding the antibody (e.g., gene) or portion thereof is isolated from an antibody-producing cell. In some embodiments, the nucleic acid encodes a variable domain of the antibody. In certain embodiments, the nucleic acid encodes an antibody heavy chain or a fragment thereof. In other embodiments, the nucleic acid encodes an antibody light chain or a fragment thereof. In particular examples, the nucleic acid isolated from the antibody-producing cell encodes a full-length antibody. In some embodiments, the method comprises isolating from the antibody-producing cell, a nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region of the antibody expressed by the cell, and a nucleic acid comprising a nucleotide sequence encoding a light chain variable region of the antibody expressed by the cell.

[0028] In certain embodiments of the method, a nucleic acid encoding the antibody is expressed in a host cell. In some embodiments, the host cell containing the nucleic acid is cultured under conditions to express the full-length antibody, which can then be produced and isolated for further use. In certain embodiments, the host cell contains a nucleic acid encoding a variable domain of the antibody, and the cell is cultured under conditions to express the variable domain. In some embodiments, the host cell expresses the variable heavy chain (V H ) domain, and the cell comprises a nucleic acid encoding the V H In some embodiments, the host cells are cultured under conditions to express the variable light chain (V L ) domain, and the cell comprises a nucleic acid encoding the V L In certain embodiments, the host cell is cultured under conditions to express the V domain of the antibody. H Nucleic acids encoding domains and V of antibodies L and a nucleic acid encoding a V domain. H Domains and V L The cells are cultured under conditions that allow expression of the domain.

[0029] Thus, in one aspect of the disclosure, a cell is provided that comprises a nucleic acid molecule encoding an antibody specific for a transmembrane protein of interest isolated using the methods of the disclosure. In some embodiments, the variable heavy chain (VH) of the antibody specific for the transmembrane protein of interest is H In some embodiments, a cell is provided that comprises a nucleic acid encoding a variable light chain (V) domain of an antibody specific for a transmembrane protein of interest. L In some embodiments, a cell is provided that comprises a nucleic acid encoding a variable heavy chain (VH) domain of an antibody specific for a transmembrane protein of interest. H Nucleic acids encoding the variable light chain (V L ) domain. In some embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In one embodiment, the cell may be any one or more of the following cell types: Chinese Hamster Ovary (CHO) cells (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epithelial), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, and MMT cells and tumor cells. In certain embodiments, the cell is a CHO cell.

[0030] These and other objects, features and advantages of the method of the present disclosure will become apparent from the following detailed description of various aspects of the method taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES [Brief description of the drawings]

[0031] [Figure 1]1A-1B demonstrate that a transmembrane protein of interest presented by a lipid bilayer membrane scaffold protein complex can be specifically bound to an antibody that recognizes the transmembrane protein of interest and is detectable in vitro using the disclosed methods. A lipid bilayer membrane scaffold protein complex having at least two membrane scaffold proteins, each containing a detectable Bir-A label, contains a first exemplary transmembrane protein of interest (in complex with GPCR1) and was compared to a control lipid bilayer membrane scaffold protein complex without the transmembrane protein of interest (empty complex) for its ability to bind to an exemplary antibody known to bind to GPCR1 (positive control AB) and an isotype control mab that does not bind to the GPCR protein (negative control AB). (A) Histograms show that a transmembrane protein of interest presented by a lipid bilayer membrane scaffold protein complex specifically binds to a positive control antibody (0.6 nm) but not to an anti-GPCR1 antibody and an empty complex (-0.05 nm), a negative control antibody and a lipid bilayer membrane scaffold protein complex containing a GPCR1 transmembrane protein (-0.06 nm), or a negative control antibody and an empty complex (-0.03 nm). (B) The ability to detect biotinylated lipid bilayer membrane scaffold protein complexes presenting a transmembrane protein of interest (complexed with GPCR1) and separate them from biotinylated lipid bilayer membrane scaffold protein complexes not containing the transmembrane protein of interest (empty complex) was analyzed by providing biotinylated lipid bilayer membrane scaffold protein complexes with and without the transmembrane protein to a plate with streptavidin tethered to it, followed by incubation with an antibody known to bind GPCR1 (positive control AB) or a negative control antibody. The histograms show that a transmembrane protein of interest presented by a lipid bilayer membrane scaffold protein complex specifically binds to the positive control antibody but not to the negative control antibody.

[0032] [Figure 2-1]2A-2F show detection and isolation of antibody-producing B cells expressing antibodies specific for exemplary transmembrane proteins of interest by flow cytometry. Splenocytes were harvested and isolated from control mice (A, C, E) and genetically engineered mice that were not immunized by injection of DNA encoding the first transmembrane protein of interest (GPCR1, B), the second transmembrane protein of interest (GPCR2, D), or the third transmembrane protein of interest (GPCR3, F). To detect populations of B cells (surface IgG positive cells) expressing antibodies specific for each of the exemplary transmembrane proteins of interest (antigen-binding cells), splenocytes were stained with a fluorescent label for a B cell marker (i.e., anti-IgG) and the cell populations were collected using FACS by contacting them with a biotinylated lipid bilayer membrane scaffold protein complex presenting one of the exemplary transmembrane proteins of interest. (A) Only 2 B cells out of 1 million splenocytes bound non-specifically to the first transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex shown (rectangle). (B) 11 out of 1 million B cells expressing an antibody specific for the first exemplary GPCR transmembrane protein of interest (GPCR1) were detected using the method (rectangle). (C) Only 2 B cells out of 1 million splenocytes obtained from the subject mouse bound non-specifically to the second transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex shown (rectangle). (D) 78 out of 1 million B cells expressing an antibody specific for the second exemplary GPCR transmembrane protein of interest (GPCR2) were detected using the method (rectangle). (E) Shown (rectangle) are only 11 B cells non-specifically bound to a third transmembrane protein of interest presented by a biotinylated lipid bilayer membrane scaffold protein complex obtained from 1 million splenocytes from a control mouse. (F) 65 of 1 million B cells expressing an antibody specific for a third exemplary GPCR transmembrane protein of interest (GPCR3) were detected using the method (rectangle). [Figure 2-2]2A-2F show detection and isolation of antibody-producing B cells expressing antibodies specific for exemplary transmembrane proteins of interest by flow cytometry. Splenocytes were harvested and isolated from control mice (A, C, E) and genetically engineered mice that were not immunized by injection of DNA encoding the first transmembrane protein of interest (GPCR1, B), the second transmembrane protein of interest (GPCR2, D), or the third transmembrane protein of interest (GPCR3, F). To detect populations of B cells (surface IgG positive cells) expressing antibodies specific for each of the exemplary transmembrane proteins of interest (antigen-binding cells), splenocytes were stained with a fluorescent label for a B cell marker (i.e., anti-IgG) and the cell populations were collected using FACS by contacting them with a biotinylated lipid bilayer membrane scaffold protein complex presenting one of the exemplary transmembrane proteins of interest. (A) Only 2 B cells out of 1 million splenocytes bound non-specifically to the first transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex shown (rectangle). (B) 11 out of 1 million B cells expressing an antibody specific for the first exemplary GPCR transmembrane protein of interest (GPCR1) were detected using the method (rectangle). (C) Only 2 B cells out of 1 million splenocytes obtained from the subject mouse bound non-specifically to the second transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex shown (rectangle). (D) 78 out of 1 million B cells expressing an antibody specific for the second exemplary GPCR transmembrane protein of interest (GPCR2) were detected using the method (rectangle). (E) Shown (rectangle) are only 11 B cells non-specifically bound to a third transmembrane protein of interest presented by a biotinylated lipid bilayer membrane scaffold protein complex obtained from 1 million splenocytes from a control mouse. (F) 65 of 1 million B cells expressing an antibody specific for a third exemplary GPCR transmembrane protein of interest (GPCR3) were detected using the method (rectangle). [Figure 2-3]2A-2F show detection and isolation of antibody-producing B cells expressing antibodies specific for exemplary transmembrane proteins of interest by flow cytometry. Splenocytes were harvested and isolated from control mice (A, C, E) and genetically engineered mice that were not immunized by injection of DNA encoding the first transmembrane protein of interest (GPCR1, B), the second transmembrane protein of interest (GPCR2, D), or the third transmembrane protein of interest (GPCR3, F). To detect populations of B cells (surface IgG positive cells) expressing antibodies specific for each of the exemplary transmembrane proteins of interest (antigen-binding cells), splenocytes were stained with a fluorescent label for a B cell marker (i.e., anti-IgG) and the cell populations were collected using FACS by contacting them with a biotinylated lipid bilayer membrane scaffold protein complex presenting one of the exemplary transmembrane proteins of interest. (A) Only 2 B cells out of 1 million splenocytes bound non-specifically to the first transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex shown (rectangle). (B) 11 out of 1 million B cells expressing an antibody specific for the first exemplary GPCR transmembrane protein of interest (GPCR1) were detected using the method (rectangle). (C) Only 2 B cells out of 1 million splenocytes obtained from the subject mouse bound non-specifically to the second transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex shown (rectangle). (D) 78 out of 1 million B cells expressing an antibody specific for the second exemplary GPCR transmembrane protein of interest (GPCR2) were detected using the method (rectangle). (E) Shown (rectangle) are only 11 B cells non-specifically bound to a third transmembrane protein of interest presented by a biotinylated lipid bilayer membrane scaffold protein complex obtained from 1 million splenocytes from a control mouse. (F) 65 of 1 million B cells expressing an antibody specific for a third exemplary GPCR transmembrane protein of interest (GPCR3) were detected using the method (rectangle).

[0033] [Diagram 3]FIG. 3 shows that antibodies isolated from antibody-producing cells using a biotinylated lipid bilayer membrane scaffold protein complex to present a transmembrane protein antigen specifically bind to a second exemplary GPCR transmembrane protein of interest, independent of the type of immunogen used. Antibody-producing cells were obtained from genetically engineered mice immunized with either DNA (DNA) encoding a transmembrane protein (TPP) or DNA encoding a purified transmembrane protein of interest using a biotinylated lipid bilayer membrane scaffold protein complex to present the transmembrane protein of interest and antibody, and generated for screening. Full-length transmembrane protein of the antigen of interest was expressed in cells (TMB overexpression), and the antigen-expressing cells were compared to control cells that were not transfected with DNA encoding any of the TMB (parental cells). The cells were then incubated with antibodies to identify antibodies that specifically bind to the exemplary transmembrane protein of interest. Antibodies above the dashed line were able to bind to the transmembrane protein of interest. In contrast, antibodies below the dashed line were weak binders or were unable to bind to the transmembrane protein of interest, as shown by comparison with positive (squares) and negative (triangle) control antibodies.

[0034] [Figure 4]FIG. 4 shows antibodies isolated from antibody-producing cells using the disclosed method, which antibodies bound to transmembrane proteins of an antigen of interest expressed on the cell surface for screening. Antibody-producing cells were obtained from immunized, genetically engineered mice using a biotinylated lipid bilayer membrane scaffold protein complex that represents an n-terminal truncated form of the transmembrane protein of the antigen of interest, and antibodies were generated for screening. The antigen-expressing cells were compared to control cells transfected with DNA encoding either the truncated TMB antigen or full-length TMB (parent cells), where the truncated form of the transmembrane protein of the antigen of interest was expressed in the cells (overexpressed truncated TMB) or the full-length form of the transmembrane protein of the antigen of interest was expressed in the cells (overexpressed full-length TMB), and the cells were incubated with antibodies to identify antibodies that specifically bind to epitopes located in the extracellular loop domains (boxed) of the exemplary transmembrane protein of interest. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Table 1: Isolation of antibodies from cells expressing cell surface antibodies against various transmembrane proteins from immunized mice. Mice genetically engineered to prevent expression of a transmembrane protein of interest from an endogenous gene (antigen-free genetically modified mice) or genetically engineered to express a transmembrane protein of interest from an endogenous gene (genetically modified mice) were immunized with DNA (DNA) encoding the transmembrane protein of interest, DNA encoding a modified form of the transmembrane protein (modified DNA), purified transmembrane protein (protein), a lipid bilayer membrane scaffold protein complex encompassing the transmembrane protein of interest (complex with antigen), or a combination thereof. Antibody-producing cells and antibodies collected using the sorting method disclosed herein (cell sorting and complex with antigen) were isolated from cells and compared to antibodies isolated from cells obtained using standard hybridoma technology (hybridoma). For each of the transmembrane proteins analyzed, a higher percentage of antibodies bound to the transmembrane protein of interest were obtained from antibody production by sorting cells with lipid bilayer membrane scaffold protein complexes displaying the transmembrane protein of interest than standard hybridoma technology.

[0036] Table 2: Comparison of cell sorting strategies for the ability to obtain antibody-producing cells expressing antibodies that specifically bind to a transmembrane protein of interest. Genetically engineered mice with (VI) or without (VI-KO) an endogenous mouse gene encoding an exemplary transmembrane protein of interest, i.e., GPCR1 or ion channel 2, were immunized by injection of one or more of the following immunogens: DNA (DNA) encoding the transmembrane protein of interest, purified transmembrane protein (protein), virus-like particles (VLPs) capable of expressing the transmembrane protein of interest, or a combination thereof (VLPs and DNA). B cells obtained from the immunized mice were then sorted using one of the sorting agents: biotinylated lipid bilayer membrane scaffold protein complexes (complexes containing TMB) presenting the transmembrane protein, VLPs or purified transmembrane protein (protein). Cells that produced antibodies that bound to the sorting agent were then collected, and antibodies were generated from each cell for comparison.

[0037] Table 3: Comparison of antibodies generated from antibody-producing cells obtained from genetically engineered mice immunized with either DNA (DNA) encoding a human transmembrane protein of interest or purified human transmembrane protein (protein) when cells obtained from each mouse were sorted using a biotinylated lipid bilayer membrane scaffold protein complex (complex containing TMB) that presented the transmembrane protein of interest. The data presented are the results of five representative immunization campaigns.

[0038] Table 4: Isolation of antibody-producing cells and generation of antibodies from genetically modified mice that do not express the mouse homologs of two different exemplary transmembrane proteins of interest (VI-KO), comparison of different immunization strategies showing that a biotinylated lipid bilayer membrane scaffold protein complex can be used to obtain cross-reactive antibodies that bind to the mouse homolog of a transmembrane protein of interest (mouse TMB) and the human homolog of the transmembrane protein of interest. Data provided are a combination of four representative immunization campaigns against TMB1 and four representative immunization campaigns against TMB2.

[0039] Table 5: Isolation of antibody-producing cells and generation of antibodies from genetically modified mice (VI-KO) that do not express a mouse homolog of an exemplary transmembrane protein of interest, immunization of genetically modified mice with a lipid bilayer membrane scaffold protein complex encompassing a human TMB2 protein (human TMB2), sorting of antibody-producing B cells with lipid bilayer membrane scaffold protein complexes presenting human TMB2 protein (complexes containing human TMB2) and / or with lipid bilayer membrane scaffold protein complexes presenting mouse TMB2 (complexes containing mouse TMB2), identification of antibody-producing B cells expressing cross-reactive antibodies capable of binding mouse TMB2 and human TMB2 protein homologs as well as antibodies specific for the human TMB2 protein. Data provided is a combination of two representative immunization campaigns.

[0040] Disclosed herein is a method of utilizing lipid bilayer membrane scaffold protein complexes to present transmembrane protein antigens to antibodies produced by cells.The lipid bilayer membrane scaffold protein complexes comprise a transmembrane protein of interest or a portion thereof, and generally the lipid and membrane scaffold protein are found in the membrane of native cells, presenting the transmembrane protein antigen to antibodies in its native conformation.Thus, the lipid bilayer membrane scaffold protein complexes are used in the methods of the present invention to identify and collect a specific subset of antibodies (or cells expressing antibodies) in a population that bind to an epitope on a transmembrane protein that is accessible in nature, such as an extracellular domain.

[0041] Without being limited to any one theory, the methods disclosed herein demonstrate that by immunizing an animal and isolating antibody-producing cells from the immunized animal using a lipid bilayer membrane scaffold protein complex that presents a transmembrane protein of an antigen of interest, it is possible to identify cells that produce antibodies specific to an epitope on a conformationally correct transmembrane protein.

[0042] Furthermore, antibodies and nucleic acids encoding the antibodies can be directly isolated from antibody-producing cells, for example, by single cell isolation and collection techniques such as FACS.Thus, the present disclosure also provides an effective and efficient method for obtaining antibodies with affinity for transmembrane proteins directly from a population of antibody-producing cells.The present method circumvents the need for time-consuming screening, identification and selection of epitope-specific antibodies by site-directed mutagenesis and other well-known techniques to confirm whether a particular antibody recognizes the desired portion of the transmembrane protein of interest.

[0043] In one aspect of the present disclosure, a method is provided for obtaining an antibody or a cell population expressing an antibody against a transmembrane protein of interest, comprising contacting a population of antibody-producing cells with a lipid bilayer membrane scaffold protein complex that displays the transmembrane protein of interest or a portion thereof. In one embodiment, the method comprises contacting a population of antibody-producing cells obtained from an animal with a lipid bilayer membrane scaffold protein complex that encompasses the transmembrane protein of interest or a portion thereof to allow binding between the transmembrane protein (i.e., antigen) and the antibody on the surface of the cell, and collecting the bound antibody-producing cells in the cell population.

[0044] In the following description, certain conventions are followed with regard to the use of terms.Generally, the terms used herein are intended to be interpreted consistently with the meanings of the terms known to those skilled in the art.In implementing this disclosure, those skilled in the art will use many conventional techniques in molecular biology, microbiology, cell biology, biochemistry and immunology. These techniques are described in more detail, for example, in Molecular Cloning: a Laboratory Manual, 4th edition, edited by JF Sambrook and DW Russell, Cold Spring Harbor Laboratory Press 2012; Recombinant Antibodies for Immunotherapy, edited by Melvyn Little, Cambridge University Press 2009; "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Animal Cell Culture" (edited by RIFreshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by FMA Russubel et al., 1987, regularly updated); "PCR: The Polymerase Chain Reaction", (edited by Mullis et al., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001). The contents of these references, and other references containing standard protocols commonly known and relied upon by those skilled in the art, including manufacturer's instructions, are incorporated herein by reference as part of this disclosure.

[0045] Immunization to generate antibody-producing cells Immunization of animals can be accomplished by any method known in the art. See, for example, E. Harlow and D. Lane "Antibodies A Laboratory Manual, Cold Spring Harbor" (1988); Malik and Lillehoj, Antibody techniques: Academic Press, CA, 1994. For example, immunogens may be administered directly to animals, such as mammals, via a variety of routes, including, but not limited to, intravenous or intraperitoneal injection, with or without adjuvants, which can help stimulate the immune response. Adjuvants known in the art include, but are not limited to, complete and incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptide). See O'Hagan, Vaccine Adjuvant, by Human Press, (2000) NJ. The term "immunogen" refers to a composition that includes an antigen (e.g., a transmembrane protein of interest or a nucleic acid encoding it) against which an antigen-specific antibody is generated by the host's immune response. The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a binding agent, such as an antibody or a fragment thereof. Antigens can also be used to generate antibodies that can bind to the epitope of the respective antigen.

[0046] The immunogen can be administered to the host animal as a protein, a nucleic acid sequence encoding a protein or fragment thereof, a peptide fragment, a protein fusion, or by a carrier containing the immunogen-encoding gene or protein immunogen or a peptide fragment thereof of interest. The immunization process induces an immune response from the host and can express the antigen (e.g., a transmembrane protein of interest) in vivo using the host's cellular expression machinery.

[0047] A variety of immunization techniques are known in the art and can be used in carrying out the subject methods. For example, an animal can be immunized by injecting an immunogen, such as a nucleic acid encoding at least a portion of a transmembrane protein of interest, at least a portion of a transmembrane protein of interest, or a carrier comprising such a nucleic acid, a transmembrane protein of interest or a portion thereof.

[0048] In some examples, the animal is immunized with a nucleic acid encoding a transmembrane protein of interest or a portion thereof. In certain embodiments, the animal is immunized with a nucleic acid encoding a full-length transmembrane protein of interest. In certain embodiments, the animal is immunized with a nucleic acid encoding a full-length human transmembrane protein of interest. In other embodiments, the animal is immunized with a nucleic acid encoding a full-length non-human transmembrane protein of interest. In certain embodiments, the animal is immunized with a nucleic acid encoding a full-length mouse transmembrane protein of interest.

[0049] The term "nucleic acid" or "nucleic acid sequence" or "nucleotide sequence" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner as the reference nucleotide. The term "nucleic acid code" or "encoding nucleic acid" refers to a DNA or RNA sequence that codes for a sequence of amino acids, such as a peptide, a protein, a detectable element or label, and / or a regulatory element. A "gene" refers to a DNA nucleic acid sequence that codes for a sequence of amino acids, including all or a portion of one or more polypeptides, proteins, or enzymes, and may or may not include introns, as well as regulatory DNA sequences, such as promoter or enhancer sequences, 5' untranslated regions, or 3' untranslated regions, that influence the conditions under which the gene or gene product is expressed.

[0050] The terms "polypeptide", "peptide" and "protein" are used herein to refer to polymers of amino acid residues linked via peptide bonds. The terms include amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers. The term "protein" refers to a large polypeptide. For example, in this disclosure, a protein can be a full-length protein or an endogenous protein. The term "peptide" generally refers to a short polypeptide, such as, for example, a fragment or portion of a protein or polypeptide. As used herein, a "fragment" or "portion" of a polypeptide or protein refers to any portion of a polypeptide that is smaller than the full-length polypeptide or protein expression product. A fragment or portion may be a "truncated" or deletion analog of a full-length protein in which one or more amino acid residues have been removed from the full-length protein. For example, in this disclosure, a peptide may be described as a "truncated protein" or a "portion of a protein" or a "fragment of a protein". In certain examples, a "portion" of a transmembrane protein of interest includes at least the entire membrane portion of the transmembrane protein of interest. A "truncated protein" may be a portion of a transmembrane protein of interest that does not include the amino-terminal and / or carboxy-terminal portions of the full-length protein. Synthetic polypeptides, peptides, and proteins can be synthesized, for example, using an automated polypeptide synthesizer or by recombinant techniques known to those of skill in the art.

[0051] The terms "transmembrane protein" and "transmembrane protein of interest" are used interchangeably herein and refer to a protein or polypeptide portion thereof that is attached to and embedded in the membrane of a cell or organelle. Thus, a transmembrane protein is a protein that traverses a membrane and is therefore composed of at least one membrane spanning domain. In some examples, a transmembrane protein also comprises at least one cytoplasmic domain and / or at least one extracellular domain. The cytoplasmic domain may be one or more of an amino-terminal domain, a carboxy-terminal domain, and an intracellular loop domain. The extracellular domain may be one or more of an amino-terminal domain, a carboxy-terminal domain, and an extracellular loop domain. In some examples, the extracellular domain of a transmembrane protein of interest comprises an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of the transmembrane protein of interest. A transmembrane protein may be a naturally occurring protein from any organism, including, but not limited to, prokaryotes, eukaryotes, and viruses. In certain embodiments, the transmembrane protein may be a human, chimpanzee, rhesus monkey, rabbit, horse, sheep, rat, mouse, dog, chicken, or goat protein. In some examples, the transmembrane protein is a mammalian protein, such as a human, mouse, rat, or primate transmembrane protein. In certain embodiments, the transmembrane protein of interest is a human protein. In certain embodiments, the transmembrane protein of interest is a non-human protein. In one embodiment, the transmembrane protein of interest is a mouse protein.

[0052] In some embodiments, the transmembrane protein of interest is modified as described herein. Exemplary modified transmembrane proteins of interest may include one or more changes to their native amino acid sequence, such as amino acid substitutions, deletions, and insertions. In some embodiments, the transmembrane protein of interest is modified to delete or "truncate" a portion of the transmembrane protein, such as, for example, the N-terminal domain and / or the C-terminal domain of the full-length protein. In some embodiments, the transmembrane protein of interest includes stabilizing mutations at the amino terminus, one or more extracellular loop domains, one or more transmembrane domains, one or more intracellular domains, the C-terminus, or a combination thereof. In certain embodiments, the transmembrane protein is a chimeric transmembrane protein. In certain embodiments, the modified transmembrane protein of interest includes one or more detectable elements, such as, for example, a His tag, a FLAG tag, an Avi tag, or a Bir-A tag. In some embodiments, the transmembrane protein of the immunogen of interest includes a His tag and a FLAG tag.

[0053] In one embodiment, the transmembrane protein is a ligand-activated protein, whereby the transmembrane protein changes conformation in the presence or absence of a ligand (i.e., has an active state and an inactive state). For example, a ligand-activated transmembrane protein can bind to a ligand on its intracellular or extracellular domain, whereby the binding induces a conformational change in one or more domains of the transmembrane protein that regulates signal transduction within the cell. In some examples, the transmembrane protein of interest is a solute carrier transporter (SLC), a receptor, a receptor with kinase activity, a class I growth factor receptor, a G protein-coupled receptor (GPCR), an ion channel protein, or a tetraspanin. In particular examples, the transmembrane protein of interest is a GPCR protein, a tetraspanin protein, or an ion channel protein. In one embodiment, the transmembrane protein of interest is an SLC protein.

[0054] In one embodiment, the transmembrane protein of interest is GPCR protein.The GPCR for use in this method is well known in the art.See, for example, Foord et al., Pharmacol.Rev.(2005)57:279-288, the entire contents of which are incorporated herein by reference. Thus, GPCRs are involved in the functions of adenosine receptors, beta-adrenergic receptors, neurotensin receptors, muscarinic receptors, 5-hydroxytryptamine receptors, adrenergic receptors, anaphylatoxin receptors, angiotensin receptors, apelin receptors, bombesin receptors, bradykinin receptors, cannabinoid receptors, chemokine receptors, cholecystokinin receptors, dopamine receptors, endothelin receptors, free fatty acid receptors, bile acid receptors, galanin receptors, motilin receptors, ghrelin receptors, glycoprotein hormone receptors, GnRH receptors, histamine receptors, KiSS1-derived peptide receptors, leukotriene and lipoxin receptors, lysophospholipid receptors, melanin-concentrating hormone receptors, melanocortin receptors, melatonin receptors, neuromedin U receptors, neuropeptide receptors, The receptor may be any of N-formyl peptide family receptors, nicotinic acid receptors, piodol receptors, opsin-like receptors, orexin receptors, P2Y receptors, peptide P518 receptors, platelet activating factor receptors, prokineticin receptors, prolactin releasing peptide receptors, prostanoid receptors, protease activated receptors, relaxin receptors, somatostatin receptors, SPC / LPC receptors, tachykinin receptors, trace amino receptors, thyrotropin releasing hormone receptors, urotensin receptors, vasopressin / oxytocin receptors, orphan GPCRs, calcitonin receptors, corticotropin releasing factor receptors, glucagon receptors, parathyroid receptors, VIP / PACAP receptors, LNB7TM receptors, GABA receptors, metabotropic glutamate receptors, and calcium sensor receptors.

[0055] In certain embodiments, the transmembrane protein of interest is a GPCR protein, such as, for example, CCR5, ADORA2A, ADRB3, C3AR1, ADRA2A, GLP1R, CCR4, CCR8 and CXCR4. In certain embodiments, the transmembrane protein is a GPCR protein selected from the following GPCR proteins: CCR5, ADORA2A, ADRB3, C3AR1, ADRA2A, GLP1R, CCR4, CCR8 or CXCR4. In one embodiment, the transmembrane protein is CCR5. In another embodiment, the transmembrane protein is ADORA2A. In another embodiment, the transmembrane protein is C3AR1. In yet another embodiment, the transmembrane protein is ADRA2A. In another embodiment, the transmembrane protein is GLP1R.

[0056] In some examples, the transmembrane protein is a tetraspanin. In certain examples, the transmembrane protein of interest is a tetraspanin, such as, for example, TSPAN 1 to TSPAN19, TSPAN21, TSPAN23, TSPAN 31, TSPAN 32, TSPAN 33, UPK1B, PRPH2, CD151, CD53, CD37, CD82, CD81, CD9, CD63, TCD63, CLND6 and CLND9. In some embodiments, the transmembrane protein is CD63. In another embodiment, it is CLDN6. In other embodiments, the transmembrane protein is CLDN9.

[0057] In other embodiments, the transmembrane protein of interest is an ion channel protein. In one embodiment, the ion channel protein is a voltage-gated ion channel protein. In certain embodiments, the voltage-gated ion channel protein is a voltage-gated calcium channel gamma-1 subunit (CACNG1), or a voltage-gated potassium channel protein, such as KVS or KIRS. In other embodiments, the ion channel protein is a calcium-activated potassium channel protein (e.g., BKCa, MaxiK, or Sk), a voltage-gated sodium channel protein, such as NaV1 protein (e.g., voltage-gated channel alpha subunit 9 (NaV1.7), voltage-gated channel alpha subunit 2 (NaV1.2)), a calcium channel protein (e.g., CAV) or a chloride channel protein. In some embodiments, the ion channel protein is a transient receptor potential channel (TRP) protein. In certain embodiments, the TRP is a canonical transmembrane protein (TRPC), vanilloid receptor (TRPV), melastatin (TRPM), polycystin (TRPP), mucolipin (TRPML), or anankyrin transmembrane protein 1 (TRPA1), such as a canonical TRP (TRPC), vanilloid receptor (TRPV), melastatin (TRPM), polycystin (TRPP), mucolipin (TRPML), ankyrin transmembrane protein 1 (TRPA1). In certain embodiments, the transmembrane protein of interest is BKCa, MaxiK, Sk, NAV1.7, CACNG1, CAV, CIC, or TRP.

[0058] In certain embodiments, the transmembrane protein of interest is a voltage-gated sodium channel protein. The family of voltage-gated sodium channels has nine known members, with over 50% amino acid identity in the transmembrane segments and extracellular loop regions. The proteins of these channels are named NaV1.1 to NaV1.9, and are generally referred to as "NaV1 proteins," and the gene names encoding NaV1.1 to NaV1.9 are referred to as Scn1a to Scn11a. Each of the NaV1 proteins has four repeat domains, each containing six transmembrane segments. The fourth segment is highly conserved and functions as the voltage sensor of the channel. The voltage sensitivity of this channel is due to the positive amino acids located at every third position in the fourth segment (Nicholls et al., (2012) "From Neuron to Brain," 5th Edition, pg. 86, incorporated herein by reference in its entirety). When stimulated by a change in transmembrane voltage, this segment moves towards the extracellular side of the cell membrane, making the channel permeable to ions. Ions are conducted through a pore, which can be divided into two regions. The outer (i.e., more extracellular) part of the pore is formed by the region between the fifth and sixth transmembrane segments (also known as the "P-loop") of each of the four domains. This region is the narrower part of the pore and is responsible for its ion selectivity. The inner (i.e., more cytoplasmic) part of the pore is formed by the combination of the fifth and sixth transmembrane segments of the four domains.More specifically, the human NaV1.1 protein corresponds to the amino acid sequence set forth in accession number P35498.2; the human NaV1.2 protein corresponds to the amino acid sequence set forth in accession number Q99250.3; the human NaV1.3 protein corresponds to the amino acid sequence set forth in accession number Q9NY46.2; the human NaV1.4 protein corresponds to the amino acid sequence set forth in accession number P35499.4; the human NaV1.5 protein corresponds to the amino acid sequence set forth in accession number Q14524.2; the human NaV1.6 protein corresponds to the amino acid sequence set forth in accession number Q9UQD0.1; the human NaV1.7 protein corresponds to the amino acid sequence set forth in accession number Q15858.3; the human NaV1.8 protein corresponds to the amino acid sequence set forth in accession number Q9Y5Y9.2; and the human NaV1.9 protein corresponds to the amino acid sequence set forth in accession number Q9UI33.2.

[0059] In one such embodiment, the transmembrane protein of interest is NaV1 protein. In a particular embodiment, the transmembrane protein of interest is NAV1.7. NaV1.7 is expressed in nociceptive (pain) neurons in dorsal root ganglia, sympathetic neurons, Schwann cells, and neuroendocrine cells. NaV1.7 is a key component of membrane excitability and is important for pain sensation. Gain-of-function mutations in human SCN9A gene are associated with pain syndromes, and loss-of-function mutations are associated with severe insensitivity to pain. NaV1.7 is highly conserved across species, as evidenced by the alignment of exemplary sequences of NaV1.7 proteins from the following 14 animal species: The human NaV1.7 protein corresponds to the amino acid sequence set forth in accession number Q15858.3; the chimpanzee NaV1.7 homolog corresponds to the amino acid sequence set forth in accession number XP_016804947.1; the rhesus macaque NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_014965766.1; the Malayan flying lemur NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_008588371.1; the bovine NaV1.7 protein corresponds to the amino acid sequence set forth in accession number NP_001104257.2; the ovine NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_004004679.1; and the dromedary NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_010980767.1. the killer whale NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_004267302.1; the horse NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_001496473.1; the dog NaV1.7 protein corresponds to the amino acid sequence set forth in accession number XP_022270547.1; the mouse NaV1.7 protein corresponds to the amino acid sequence set forth in accession number Q62205.2; the rat NaV1.7 protein corresponds to the amino acid sequence set forth in accession number O08562.1; the rabbit NaV1.7 protein corresponds to the amino acid sequence set forth in accession number Q28644.1; and the chicken NaV1.7 protein corresponds to the amino acid sequence set forth in accession number NP_001280211.1.

[0060] In a particular embodiment, the transmembrane protein of interest is human NaV1.7. In another embodiment, the transmembrane protein of interest is mouse NaV1.7.

[0061] In certain embodiments, the transmembrane protein of interest is NAV1.2.NaV1.2 is expressed in central and peripheral neurons.Mutations in human SCN2A gene (encoding NaV1.2) are associated with some seizure disorders and autism spectrum disorders.NaV1.2 is highly conserved across species, as revealed by the alignment of exemplary sequences of NaV1.2 protein from 14 animal species. The accession numbers of exemplary sequences included in the alignment are: the human NaV1.2 homolog corresponds to the amino acid sequence set forth in accession number Q99250.3; the chimpanzee NaV1.2 homolog corresponds to the amino acid sequence set forth in accession number XP_003820970.1; the rhesus macaque NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_001100368.1; the Malayan flying lemur NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_008582720.1; the bovine NaV1.2 protein corresponds to the amino acid sequence set forth in accession number NP_001137581.1; the ovine NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_014948870.1; and the dromedary NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_010980763.1. the killer whale NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_004283641.1; the horse NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_014588001.1; the mouse NaV1.2 protein corresponds to the amino acid sequence set forth in accession number NP_001092768.1; the rat NaV1.2 protein corresponds to the amino acid sequence set forth in accession number P04775.1; the rabbit NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_008256915.1; the chicken NaV1.2 protein corresponds to the amino acid sequence set forth in accession number NP_001280210.1; and the green sea turtle NaV1.2 protein corresponds to the amino acid sequence set forth in accession number XP_007056690.1.

[0062] In a particular embodiment, the transmembrane protein of interest is a human NaV1.2 homolog. In another embodiment, the transmembrane protein of interest is a mouse NaV1.2 homolog.

[0063] In some embodiments, the transmembrane protein of interest is a CACNG1 protein. In one embodiment, the transmembrane protein of interest is a human CACNG1 protein. In another embodiment, the transmembrane protein of interest is a non-human CACNG1 protein.

[0064] In certain examples, the transmembrane protein of interest is an SLC protein. SLC proteins have been characterized and are well known to those skilled in the art. For example, hundreds of human transmembrane SLC proteins have been identified and are organized into many families of SLC proteins, as described, for example, in Lin, L et al. Nat.Rev.Drug Disc.(2015)14:8 pp.543-560, the entire contents of which are expressly incorporated herein by reference. Thus, in certain embodiments, the transmembrane protein of interest is an SLC protein, for example, SLCO1A2, SLCO1B1, SLCO1B3, SLCO1B7, SLCO1C1, SLCO2A1, SLCO2B1, SLCO3A1, SLCO4A1, SLCO4C1, SLCO5A1, SLCO6A1, SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7, SLC2A1, SLC2A2, SLC2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14, SLC 3A1, SLC3A2, SLC4A1, SLC4A2, SLC4A3, SLC4A4, SLC4A5, SLC4A7, SLC4A8, SLC4A9, SLC4A10, SLC4A11, SLC5A1, SLC5A2, SLC 5A3, SLC5A4, SLC5A5, SLC5A6, SLC5A7, SLC5A8, SLC5A9, SLC5A10, SLC5A11, SLC5A12, SLC6A1, SLC6A2, SLC6A3, SLC6A4, S LC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, SLC6A10P, SLC6A11, SLC6A12, SLC6A13, SLC6A14, SLC6A15, SLC6A16, SLC6A17, S LC6A18, SLC6A19, SLC6A20, SLC6A21P, SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A9, SLC7 A10, SLC7A11, SLC7A13, SLC7A14, SLC7A15P, SLC8A1, SLC8A2, SLC8A3, SLC8B1, SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5,<h2 style=";text-align:left;direction:ltr">SLC9A6、SLC9A7、SLC9A8、SLC9A9、SLC9B1、SLC9B2、SLC9C1、SLC9C2、SLC10A 1、SLC10A2、SLC10A3、SLC10A4、SLC10A5、SLC10A6、SLC10A7、SLC11A1、SLC11 A2, SLC12A1, SLC12A2, SLC12A3, SLC12A4, SLC12A5, SLC12A6, SLC12A7, SLC12A8, SLC12A9, SLC13A1, SLC13A2, SLC13A3, SLC13A4, SLC13A5, SLC14A1, SL C14A2、SLC15A1、SLC15A2、SLC15A3、SLC15A4、SLC15A5、SLC16A1、SLC16A2、 SLC16A3、SLC16A4、SLC16A5、SLC16A6、SLC16A7、SLC16A8、SLC16A9、SLC16A1 0, SLC16A11, SLC16A12, SLC16A13, SLC16A14, SLC17A1, SLC17A2, SLC17A3, SLC17A4, SLC17A5, SLC17A6, SLC17A7, SLC17A8, SLC17A9, SLC18A1, SLC18A2 SLC18A3, SLC18B1, SLC19A1, SLC19A2, SLC19A3, SLC20A1, SLC20A2, SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLC2 2A9、SLC22A10、SLC22A11、SLC22A12、SLC22A13、SLC22A14、SLC22A15、SLC2 2A16、SLC22A17、SLC22A18、SLC22A20P、SLC22A23、SLC22A24、SLC22A25、SLC 22A31, SLC23A1, SLC23A2, SLC23A3, SLC23A4P, SLC24A1, SLC24A2, SLC24A3, SLC24A4, SLC24A5, SLC25A1, SLC25A2, SLC25A3, SLC25A4, SLC25A5, SLC25A 6, UCP1, UCP2, UCP3, SLC25A10, SLC25A11, SLC25A12, SLC25A13, SLC25A14, SLC25A15, SLC25A16, SLC25A17, SLC25A18, SLC25A19, SLC25A20, SLC25A21,<h2 style=";text-align:left;direction:ltr">SLC25A22, SLC25A23, SLC25A24, SLC25A25, SLC25A26, SLC25A27, SLC25A28, SLC25A29, SLC25A30, SLC25A31, SLC25A32, SLC25A33, SLC25A34, SLC25A3 5、SLC25A36、SLC25A37、SLC25A38、SLC25A39、SLC25A40、SLC25A41、SLC25A42、SLC25A43、SLC25A44、SLC25A45、SLC25A46、SLC25A47、SLC25A48、MTCH1、 MTCH2, SLC25A51, SLC25A52, SLC25A53, SLC26A1, SLC26A2, SLC26A3, SLC26A4, SLC26A5, SLC26A6, SLC26A7, SLC26A8, SLC26A9, SLC26A10, SLC26A11, SL C27A1, SLC27A2, SLC27A3, SLC27A4, SLC27A5, SLC27A6, SLC28A1, SLC28A2, SLC28A3, SLC29A1, SLC29A2, SLC29A3, SLC29A4, SLC30A1, SLC30A2, SLC30A3 SLC30A4, SLC30A5, SLC30A6, SLC30A7, SLC30A8, SLC30A9, SLC30A10, SLC31A1, SLC31A2, SLC32A1, SLC33A1, SLC34A1, SLC34A2, SLC34A3, SLC35A1, SLC 35A2, SLC35A3, SLC35A4, SLC35A5, SLC35B1, SLC35B2, SLC35B3, SLC35B4, SLC35C1, SLC35C2, SLC35D1, SLC35D2, SLC35D3, SLC35E1, SLC35E2A, SLC35E2 B、SLC35E3、SLC35E4、SLC35F1、SLC35F2、SLC35F3、SLC35F4、SLC35F5、SLC35F6、SLC35G1、SLC35G2、SLC35G3、SLC35G4、SLC35G5、SLC35G6、SLC36A1、SLC 36A2, SLC36A3, SLC36A4, SLC37A1, SLC37A2, SLC37A3, SLC37A4, SLC38A1, SLC38A2, SLC38A3, SLC38A4, SLC38A5, SLC38A6, SLC38A7, SLC38A8, SLC38A9SLC38A10, SLC38A11, SLC39A1, SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A 8, SLC39A9, SLC39A10, SLC39A11, SLC39A12, SLC39A13, SLC39A14, SLC40A1, SLC41A1, SLC41A2, S LC41A3, RHAG, RHBG, RHCG, SLC43A1, SLC43A2, SLC43A3, SLC44A1, SLC44A2, SLC44A3, SLC44A4, SL C44A5, SLC45A1, SLC45A2, SLC45A3, SLC45A4, SLC46A1, SLC46A2, SLC46A3, SLC47A1, SLC47A2, SL C48A1, FLVCR1, FLVCR2, SLC49A3, SLC49A4, SLC50A1, SLC51A, SLC51B, SLC52A1, SLC52A2, SLC52A3, XPR1, MPC1, MPC2, MPC1L, LETM1, LETM2, LETMD1, SFXN1, SFXN2, SFXN3, SFXN4, SFXN5, NIPA1, NIPA2, NIPAL1, NIPAL2, NIPAL3, NIPAL4, MAGT1, TUSC3, MFSD2A, MFSD2B, MFSD4A, MFSD4B, MFSD5, ANKH, SPNS1, SPNS2, SPNS3, TMEM165, NPC1, NPC1L1, SLC66A1, SLC66A2, SLC66A3, CTNS and MPDU1. ,

[0065] The term "endogenous" refers to a polypeptide, polynucleotide, or other composition that is naturally expressed in a host organism or that originates from a cell, tissue, or organism. "Exogenous" refers to a polypeptide, polynucleotide, or other composition that originates outside a cell, tissue, or organism, or that is foreign to a particular host organism.

[0066] In some embodiments of the method, the animal is immunized with a nucleic acid encoding a transmembrane protein of interest or a portion thereof. In certain embodiments, the nucleic acid encodes a truncated version of the full-length transmembrane protein of interest. In some embodiments, the truncated transmembrane protein of interest does not include one or more extracellular loop domains, the amino terminal portion and / or the carboxy terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not include the amino terminal portion of the full-length transmembrane protein of interest. In other embodiments, the truncated transmembrane protein does not include the carboxy terminal portion of the full-length transmembrane protein of interest. In some embodiments, the truncated transmembrane protein does not include the extracellular loop domain of the full-length transmembrane protein of interest.

[0067] In some embodiments, animals are immunized with a nucleic acid encoding a transmembrane protein of interest or a portion thereof that is contained in a carrier capable of expressing the nucleic acid. Non-limiting examples of carriers for use in immunization include vectors, such as plasmids, expression vectors, and cells, such as virus-like particles (VLPs), irradiated cells, exosomes, and liposomes. The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. One example of a vector is an "expression vector," which is a recombinantly or synthetically produced nucleic acid construct that has a set of specific nucleic acid elements that allow for the transcription of a specific nucleic acid in a host cell. An expression vector may be part of a plasmid, a virus, or a nucleic acid fragment. In certain examples, an expression vector comprises a nucleic acid to be transcribed that is operably linked to a promoter. For example, "operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., an array of promoter transcription factor binding sites) with a second nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0068] In some embodiments, the animal is immunized with a vector comprising a nucleic acid encoding a transmembrane protein of interest or a portion thereof. In certain embodiments, the animal is immunized with an expression vector comprising a nucleic acid encoding a transmembrane protein of interest or a portion thereof. In one embodiment, the animal is immunized with a plasmid comprising a nucleic acid encoding a transmembrane protein of interest or a portion thereof.

[0069] In some examples, the immunogen is administered to the host animal by one or more injections over time, such as intravenous or intraperitoneal injection.In certain embodiments, the immunogen is administered to the host mammal by one, two, three, four or more injections.In particular, the immunogen is administered by three separate injections.

[0070] The amount of immunogen to be administered to an animal can be readily determined by one of skill in the art using known methods.

[0071] In some examples, the nucleic acid immunogen is injected into the animal at a concentration of at least 0.1 mg / mL, at least 0.5 mg / mL, at least 1.0 mg / mL, at least 1.5 mg / mL, at least 2.0 mg / mL, at least 3.0 mg / mL, at least 4.0 mg / mL, at least 5.0 mg / mL, at least 6.0 mg / mL, at least 7.0 mg / mL, at least 8.0 mg / mL, at least 9.0 mg / mL, at least 9.5 mg / mL, at least 10.0 mg / mL, or at least 10.5 mg / mL. In certain embodiments, the nucleic acid immunogen is injected into the animal at a concentration of 0.5 mg / mL to 20 mg / mL, 0.5 mg / mL to 15 mg / mL, 0.5 mg / mL to 12 mg / mL, 0.5 mg / mL to 11 mg / mL, 1.0 mg / mL to 15 mg / mL, 1.0 mg / mL to 5 mg / mL, 1.0 mg / mL to 4 mg / mL, 1.0 mg / mL to 3 mg / mL, 1.0 mg / mL to 2 mg / mL, 2.0 mg / mL to 12 mg / mL, 5.0 mg / mL to 12 mg / mL, 7.0 mg / mL to 12 mg / mL, 8.0 mg / mL to 12 mg / mL, 8.0 mg / mL to 11 mg / mL, 9.0 mg / mL to 11 mg / mL, or 9.5 mg / mL to 10.5 mg / mL. In certain embodiments, the nucleic acid immunogen is at a concentration of 0.5 mg / mL, 0.7 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, The nucleic acid immunogen is injected into the animal at a concentration of 7.5mg / mL, 8.0mg / mL, 8.5mg / mL, 9.0mg / mL, 9.5mg / mL, 10.0mg / mL, 10.5mg / mL, 11.0mg / mL, 11.5mg / mL, 12.0mg / mL, 12.5mg / mL, 13.0mg / mL, 13.5mg / mL, 14.0mg / mL, 14.5mg / mL, 15.0mg / mL, 15.5mg / mL or more. In a particular embodiment, the nucleic acid immunogen is injected into the animal at a concentration of 1.6mg / mL. In one embodiment, the nucleic acid immunogen is injected into the animal at a concentration of 10mg / mL.

[0072] In some embodiments, the animal is immunized with the transmembrane protein of interest or a portion thereof. In certain embodiments, the transmembrane protein of interest or a portion thereof is of human origin. In other embodiments, the animal is immunized with a non-human transmembrane protein of interest. In some embodiments, the animal is immunized with a nucleic acid encoding a full-length mouse transmembrane protein of interest. In some embodiments, the animal is immunized with a full-length transmembrane protein of interest. In certain embodiments, the transmembrane protein of interest is truncated. In some embodiments, the truncated transmembrane protein of interest does not include one or more extracellular loop domains, the amino terminal portion and / or the carboxy terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not include the amino terminal portion of the full-length transmembrane protein of interest. In other embodiments, the truncated transmembrane protein does not include the carboxy terminal portion of the full-length transmembrane protein of interest. In one embodiment, the truncated transmembrane protein does not include the extracellular loop domain of the full-length transmembrane protein of interest.

[0073] In particular examples, the protein or peptide immunogen is administered to an animal at a concentration of at least 0.1 mg / mL, at least 0.2 mg / mL, at least 0.3 mg / mL, at least 0.4 mg / mL, at least 0.5 mg / mL, at least 0.6 mg / mL, at least 0.7 mg / mL, at least 0.8 mg / mL, at least 0.9 mg / mL, at least 1.0 mg / mL, at least 2.0 mg / mL, at least 3.0 mg / mL, at least 4.0 mg / mL, at least 5.0 mg / mL, at least 10.0 mg / mL or more. In certain embodiments, the protein or peptide immunogen is from 0.1 mg / mL to 20 mg / mL, 0.1 mg / mL to 15 mg / mL, 0.1 mg / mL to 10 mg / mL, 0.1 mg / mL to 8.0 mg / mL, 0.1 mg / mL to 7.0 mg / mL, 0.1 mg / mL to 6.0 mg / mL, 0.1 mg / mL to 5.0 mg / mL, 0.1 mg / mL to 3.0 mg / mL, 0.1 mg / mL to 2.0 mg / mL, 0.1 mg / mL to 1.0 mg / mL, 0.2 mg / mL to 10.0 mg / mL, 0.2 mg / mL to 7.0 mg / mL, 0.2 mg / mL to 6.0 mg / mL, 0.2 mg / mL to 5.0 mg / mL The following concentrations are injected into animals: 0.2 mg / mL to 3 mg / mL, 0.2 mg / mL to 2 mg / mL, 0.2 mg / mL to 1 mg / mL, 0.5 mg / mL to 10.0 mg / mL, 0.5 mg / mL to 7.0 mg / mL, 0.5 mg / mL to 5.0 mg / mL, 0.5 mg / mL to 3.0 mg / mL, 0.5 mg / mL to 2.0 mg / mL, 0.5 mg / mL to 1.0 mg / mL, 1.0 mg / mL to 5.0 mg / mL, 1.0 mg / mL to 3.0 mg / mL, 1.0 mg / mL to 2.0 mg / mL, 2.0 mg / mL to 10.0 mg / mL, and 5.0 mg / mL to 10.0 mg / mL, inclusive.In certain embodiments, the peptide or peptide immunogen is at 0.1 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3.0 mg / mL, 3.1 mg / mL, 3.2 mg / mL, 3.3 mg / mL, 3.4 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 ... The protein or peptide immunogen is injected into the animal at a concentration of 0.9 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, 12.0 mg / mL, 15.0 mg / mL, 20.0 mg / mL or more. In a particular embodiment, the protein or peptide immunogen is injected into the animal at a concentration of 0.9 mg / mL. In one embodiment, the protein or peptide immunogen is injected into the animal at a concentration of 0.5 mg / mL. In another embodiment, the protein or peptide immunogen is injected into the animal at a concentration of 1.9 mg / mL. In a specific embodiment, the protein or peptide immunogen is injected into the animal at a concentration of 2.5 mg / mL.

[0074] In certain embodiments, animals are immunized with a chimeric transmembrane protein of interest or a nucleotide sequence encoding the chimeric transmembrane protein of interest. As used herein, the term "chimera" refers to a protein or a nucleic acid encoding a protein having portions derived from different species. For example, a chimeric protein may have one or more human domains and at least one non-human domain, or vice versa. A nucleic acid encoding a chimeric protein may, for example, have a human gene encoding one or more domains of a protein operably linked to a non-human gene encoding at least one domain of the protein.

[0075] In one non-limiting example, the chimeric nucleic acid comprises a portion of a human gene encoding a first portion of a transmembrane protein operably linked to a portion of a mouse gene encoding a different portion of the transmembrane protein. In some embodiments, an animal is immunized with DNA encoding a chimeric transmembrane protein of interest, the DNA comprising a human gene sequence encoding one or more extracellular loop domains of the human transmembrane protein, and a mouse gene sequence encoding the amino-terminal domain and / or the carboxy-terminal domain from a mouse homolog of the transmembrane protein. In other embodiments, an animal is immunized with DNA encoding a chimeric transmembrane protein of interest, the DNA comprising a DNA encoding the amino-terminal domain and / or the carboxy-terminal domain from a human transmembrane protein, and a DNA encoding one or more extracellular loop domains from a mouse homolog of the transmembrane protein. In one embodiment, an animal is immunized with DNA encoding a chimeric transmembrane protein of interest, the DNA comprising a gene sequence encoding one or more extracellular loop domains from a human transmembrane protein, and a sequence encoding the amino-terminal domain from a mouse homolog of the transmembrane protein. In certain embodiments, animals are immunized with DNA encoding a chimeric transmembrane protein of interest, which comprises a genetic sequence encoding an amino-terminal domain from a human transmembrane protein and a genetic sequence encoding one or more extracellular loop domains from a mouse homolog of the transmembrane protein. In certain embodiments, the nucleotide sequence encoding the chimeric transmembrane protein of interest is also modified to include a nucleotide sequence encoding a detectable element, such as, for example, a His tag, a FLAG tag, an Avi tag, or a Bir-A tag.

[0076] In some embodiments, animals are immunized with a chimeric transmembrane protein of interest, comprising a portion of a human transmembrane protein of interest operably linked to a portion of a non-human homologue of the transmembrane protein of interest. In some embodiments, animals are immunized with a chimeric transmembrane protein of interest, comprising one or more extracellular loop domains from a human transmembrane protein of interest, and an amino-terminal domain and / or a carboxy-terminal domain from a mouse homologue of the transmembrane protein. In some embodiments, animals are immunized with a chimeric transmembrane protein of interest, comprising one or more extracellular loop domains from a human transmembrane protein of interest, and an amino-terminal domain from a mouse homologue of the transmembrane protein. In some embodiments, animals are immunized with a chimeric transmembrane protein of interest, comprising one or more extracellular loop domains from a human transmembrane protein of interest, and an amino-terminal domain from a mouse homologue of the transmembrane protein. In some embodiments, animals are immunized with a chimeric transmembrane protein of interest, which comprises an amino-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a mouse homologue of the transmembrane protein. In certain embodiments, the chimeric transmembrane protein of interest also comprises a detectable element, such as, for example, a His tag, a FLAG tag, an Avi tag, or a Bir-A tag. In one embodiment, the chimeric transmembrane protein of interest comprises a His tag and a FLAG tag.

[0077] In certain embodiments, the transmembrane protein or nucleic acid immunogen of interest is modified. In some embodiments, the immunogen is a modified transmembrane protein of interest or a DNA encoding it that contains one or more stabilizing amino acid substitutions. In some embodiments, the transmembrane protein or nucleic acid immunogen of interest is modified to include one or more detectable elements, such as a label, marker, or feature. In one embodiment, the modified immunogen is a transmembrane protein of interest that includes a detectable label. In certain embodiments, the detectable label is a FLAG-tag, an Avi-tag, a histidine tag (His-tag), a Bir-A-tag, or a combination thereof. In certain embodiments, the detectable label is located at the amino-terminus or carboxy-terminus of the transmembrane protein immunogen. In one embodiment, the transmembrane protein immunogen has a FLAG-tag and a His-tag attached to the carboxy-terminus.

[0078] Proteins for use in the present methods can be produced by methods known in the art. For example, the transmembrane protein of interest or a portion thereof can be encoded by a suitable nucleic acid and expressed in a cell. Suitable nucleic acid molecules encoding the transmembrane protein of interest can be produced using standard cloning techniques, site-directed mutagenesis, and PCR, as is well known in the art. Suitable expression systems include, for example, constitutive or inducible expression systems in bacteria or yeast, viral expression systems such as baculovirus, Semliki Forest virus, and lentivirus, or transient transfection in insect or mammalian cells. Suitable host cells include Escherichia coli, Lactococcus lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Spodoptera frugiperda, and Urticaria gracilis cells. Suitable mammalian host cells include HEK 293, COS, CHO, NSO, DT40. Suitable insect host cells include Sf9 and S2 cells.

[0079] In certain embodiments, a protein immunogen comprising a transmembrane protein of interest or a portion thereof is expressed in cells and the immunogen protein is produced by the cells and purified to provide a purified protein immunogen for immunization.

[0080] In certain embodiments, animals can be immunized with a transmembrane protein of interest or a portion thereof contained within a carrier, such as, for example, a lipid bilayer membrane scaffold protein complex, a VLP, a cell, an exosome, or a liposome.

[0081] "Lipid bilayer membrane scaffold protein complex" refers to a composition comprising a lipid bilayer in complex with at least one membrane scaffold protein (MSP), i.e. bound by at least one MSP. Lipid bilayer membrane scaffold protein complexes have been described. See, for example, Inagaki, S. et al., Biophysical Characterization of Membrane Proteins in Nanodiscs. Methods (2013) 59(3):287-300, the entire contents of which are expressly incorporated herein by reference. A lipid bilayer membrane scaffold protein complex can be formed, for example, as a disc-shaped phospholipid bilayer surrounded and stabilized by two membrane scaffold proteins. Without being bound to any one theory, the formation and function of the complex is regulated by the lipid ratio of MSP and the length of MSP. For example, the lipid to MSP ratio can be adjusted to generate a homogeneous or non-homogeneous population of lipid bilayer membrane scaffold protein complexes. Furthermore, to provide a native-like environment, the MSP protein should be large enough to form a complex with a transmembrane protein or protein of interest, providing space for both lipid bilayer formation and .

[0082] As used herein, the term "membrane scaffolding protein" or "MSP" refers to a class of amphipathic helical proteins derived from the amphipathic apolipoprotein AI (Apo-AI), as described, for example, in Schuler, M. et al., Methods Mol Biol. 2013; 974: 415-433, the entire contents of which are incorporated herein by reference. Membrane scaffolding proteins include an amphipathic helix that contains hydrophobic residues on the inside of the helix that contact the lipid hydrophobic tail, and hydrophilic residues facing outward (away from the lipid). Membrane scaffolding proteins may be of different sizes due to the deletion or insertion of one or more helical domains in a portion of the MSP amino acid sequence, thereby allowing the formation of lipid bilayer-MSP complexes of different sizes. It is understood that the structure and function of MSP is different from the saposin family of lipid-binding proteins.

[0083] In some examples, the lipid bilayer membrane scaffold protein complex comprises at least one membrane scaffold protein selected from the following exemplary MSPs: MSP1, MSP2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP1D1, MSP1D2, MSP2N1, MSP2N3, and MSP2N2.

[0084] In some embodiments, the membrane scaffold protein is MSP1 comprising the structure FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 12], where FX is the N-terminal domain of the membrane scaffold protein having the amino acid sequence MGHHHHHHIEGR [SEQ ID NO: 1], H1 is Helix 1 having the amino acid sequence LKLLDNWDSVTSTFSKLREQLG [SEQ ID NO: 2], H2 is Helix 2 having the amino acid sequence PVTQEFWDNLEKETEGLRQEMS [SEQ ID NO: 3], H3 is Helix 3 having the amino acid sequence KDLEEVKAKVQ [SEQ ID NO: 4], H4 is Helix 4 having the amino acid sequence PYLDDFQKKWQEEMELYRQKVE [SEQ ID NO: 5], and H5 is Helix 5 having the amino acid sequence PLRAELQEGARQKLHELQEKLS [SEQ ID NO: 6]. 5, H6 is Helix 6 having the amino acid sequence PLGEEMRDRARAHVDALRTHLA [SEQ ID NO: 7], H7 is Helix 7 having the amino acid sequence PYSDELRQRLAARLEALKENGG [SEQ ID NO: 8], H8 is Helix 8 having the amino acid sequence ARLAEYHAKATEHLSTLSEKAK [SEQ ID NO: 9], H9 is Helix 9 having the amino acid sequence PALEDLRQGLL [SEQ ID NO: 10], and H10 is Helix 10 having the amino acid sequence PVLESFKVSFLSALEEYTKKLNTQ [SEQ ID NO: 11]. In some embodiments, the membrane scaffold protein is MSP2 comprising the structure FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 13]. In some embodiments, the membrane scaffold protein is an extended membrane scaffold protein comprising one or more 22 amino acid amphipathic helices inserted into the MSP1 amino acid sequence. In one such embodiment, the extended membrane scaffold protein is MSP1E1 comprising the structure H1-H2-H3-H4-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 14], whereby the amino acid sequence of MSP1 is extended by having a duplicated "H4".In another embodiment, the extended membrane scaffold protein is MSP1E2 comprising the structure FX-H1-H2-H3-H4-H5-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 15], whereby the amino acid sequence of MSP1 is extended by having a duplicated "H4-H5". In yet another embodiment, the extended membrane scaffold protein is MSP1E3 comprising the structure FFX-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 16], whereby the amino acid sequence of MSP1 is extended by having a duplicated "H4-H5-H6".

[0085] In certain embodiments, the membrane scaffold protein is MSP1D1 comprising the structure TEV-H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 17], whereby TEV is a modified N-terminal domain having the amino acid sequence MGHHHHHHH DYDIPTTENLYFQG [SEQ ID NO: 18] and H1Δ(1-11) is a truncated H1 helical domain having the amino acid sequence STFSKLREQLG [SEQ ID NO: 19]. In some embodiments, the membrane scaffold protein is MSP1D2 comprising the structure TEV-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 20]. In some embodiments, the membrane scaffold protein is MSP2N1 having the structure TEV-H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 21]. In another embodiment, the membrane scaffold protein is MSP2N2 having the structure TEV-H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 22]. In yet another embodiment, the membrane scaffold protein is MSP2N3 having the structure TEV-H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-17)-H2-H3-H4-H5-H6-H7-H8-H9-H10 [SEQ ID NO: 24], whereby H1Δ(1-17) is a truncated H1 helical domain having the amino acid sequence REQLG [SEQ ID NO: 23].

[0086] In certain embodiments, the lipid bilayer membrane scaffold protein complex comprises two MSPs. In one embodiment, the lipid bilayer membrane scaffold protein complex is a disc-shaped phospholipid bilayer surrounded by two MSPs (e.g., two molecules of MSP) like a belt. In one embodiment, the lipid bilayer membrane scaffold protein complex is a disc-shaped phospholipid bilayer surrounded by two molecules of MSP1E3D1.

[0087] In some embodiments, the lipid bilayer membrane scaffold protein complex comprises a detectable label. In certain embodiments, the lipid bilayer membrane scaffold protein complex contains at least two labeled membrane scaffold proteins and a plurality of lipids. In certain embodiments, the lipid bilayer membrane scaffold protein complex contains two labeled membrane scaffold proteins and a lipid bilayer. In some embodiments, the labeled membrane scaffold proteins are the same or different. The detectable label can be any detectable label known in the art. For example, the detectable label includes a fluorescent molecule, a His-tag, and a FLAG-tag. In exemplary embodiments, one or more membrane scaffold proteins are labeled with a detectable marker, such as a Bir-A tag or an Avi-tag for biotinylation. In certain embodiments, all MSPs in the lipid bilayer membrane scaffold protein complex are biotinylated. Biotinylation can be performed, for example, by chemically biotinylating the membrane scaffold protein or by genetically introducing an Avi-tag or a Bir-A tag into the MSP nucleic acid coding sequence and producing the modified protein using known techniques.

[0088] The lipid bilayer portion of the lipid bilayer membrane scaffold protein complex is reconstituted. The lipids can form a bilayer of lipids, such as, for example, sphingolipids and / or phospholipids. The lipid composition of the complex can vary, for example, based on the native cell type of the particular transmembrane protein of interest included in the complex. For example, E. coli membranes contain 70-80% phosphatidylethanolamine, 15-20% phosphatidylglycerol, and 5% cardiolipin, while rat liver cell membranes contain 14-20% phosphatidylethanolamine, 32-47% phosphatidylcholine, 8% phosphatidylinositol, 4-8% phosphatidylserine, and 13-14% sphingomyelin. See Inagaki, S. et al., Methods (2013) 59(3):287-300. Thus, the lipid composition of the lipid bilayer membrane scaffold protein complex can include a single type of lipid, or more than a single type of lipid. In certain embodiments, the lipids that form the lipid bilayer membrane scaffold-protein complex are synthetic lipids or recombinantly produced lipids.

[0089] In some embodiments, the lipid bilayer is comprised of one or more of sphingomyelin, phosphatidylcholine, and derivatives thereof. In certain embodiments, the lipid bilayer is comprised of 1-dioleoylphosphatidylcholine (DOPC), 1-palmitoyl 2-oleoylphosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and phosphatidylinositol (PI), or combinations thereof. In certain embodiments, the lipid bilayer comprises a plurality of POPC phospholipids.

[0090] In some embodiments, the lipid bilayer membrane scaffold protein complex includes a lipid that forms a disc-shaped phospholipid bilayer around a membrane scaffold protein that includes a plurality of lipids of one or more of POPC, POPG, and POPS. In certain embodiments, the lipid bilayer membrane scaffold protein complex includes a plurality of POPC lipids that form a disc-shaped phospholipid bilayer around two membrane scaffold proteins. In one such embodiment, the ratio of MSP to lipid is 1:100 to 1:150, 1:110 to 1:140, 1:120 to 1:140, 1:120 to 1:140, 1:120 to 1:130, and 1:125 to 1:135, inclusive. In other embodiments, the ratio of MSP to lipid is 1:100, 1:110, 1:111, 1:112, 1:113, 1:114, 1:115, 1:116, 1:117, 1:118, 1:119, 1:120, 1:121, 1:122, 1:123, 1:124, 1:125, 1:126, 1:127, 1:128, 1:129, 1:200, 1:201, 1:202, 1:203, 1:204, 1:205, 1:206, 1:207, 1:208, 1:209, 1:210, 1:211, 1:212, 1:213, 1:214, 1:215, 1:216, 1:217, 1:218, 1:219, 1:300, 1:301, 1:302, 1:303, 1:304, 1:305, 1:306, 1:307, 1:308, 1:309, 1:310, 1:311, 1:312, 1:313, 1:314, 1:315, 1:316, 1:317, 1:318, 1:319, 1:400, 1:401, 1:402, 1:403, 1:404, 1:405, 1:406, 1:407, 1:408, 1:409, 1 1:129, 1:130, 1:131, 1:132, 1:133, 1:134, 1:135, 1:136, 1:137, 1:138, 1:139, 1:140, 1:141, 1:142, 1:143, 1:144, 1:145, 1:146, 1:147, 1:148, 1:149 or 1:150, inclusive.

[0091] In certain examples, the lipid bilayer membrane scaffold protein complex also includes at least one antigen. In certain embodiments, the antigen is a transmembrane protein of interest or a portion thereof. In some embodiments, the transmembrane protein of interest is a full-length transmembrane protein of interest, a truncated transmembrane protein of interest, or a chimeric transmembrane protein of interest. In some embodiments, the truncated transmembrane protein of interest does not include one or more extracellular loop domains, the amino terminal portion and / or the carboxy terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein of interest does not include the amino terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein of interest includes at least the entire transmembrane portion of the transmembrane protein of interest. In other embodiments, the truncated transmembrane protein does not include the carboxy terminal portion of the full-length transmembrane protein of interest. In one embodiment, the truncated transmembrane protein does not include the extracellular loop domain of the full-length transmembrane protein of interest. In some embodiments, the truncated protein comprises an extracellular domain of a transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of a transmembrane protein of interest. In other embodiments, the antigen is a chimeric transmembrane protein of interest or a portion thereof as described herein.

[0092] In some embodiments, a lipid bilayer membrane scaffold protein complex comprising an antigen, such as a transmembrane protein or portion thereof of interest, comprises a single molecule of the antigen (e.g., one copy of the transmembrane protein or portion thereof per lipid bilayer MSP complex or per Nanodisc). In some embodiments, a lipid bilayer membrane scaffold protein complex comprising an antigen comprises multiple molecules of the antigen (e.g., multiple copies of the transmembrane protein or portion thereof per lipid bilayer MSP complex or per Nanodisc).

[0093] Formation of lipid bilayer membrane scaffold protein complexes, including those that include a transmembrane protein of interest or a portion thereof, can be carried out using methods known in the art. See, for example, Bayburt TH et al., Arch Biochem Biophys. (2006) 450: 215-222, the entire contents of which are incorporated herein by reference. In general, formation of lipid bilayer membrane scaffold protein complexes involves self-assembly of the complex by mixing lipids, MSPs, and, if applicable, transmembrane proteins. Typically, the transmembrane protein is purified using one or more detergents. In some embodiments, the purified transmembrane protein is mixed with lipids and MSPs to form a lipid bilayer membrane scaffold protein complex that includes the transmembrane protein. In some embodiments, the purified transmembrane protein is provided in the presence of one or more detergents (e.g., detergent-solubilized fractions), mixed with lipids and MSPs, and then the one or more detergents are removed to induce the formation of the complex structure.

[0094] In some examples, a purification step can be used to separate complexes that contain the protein of interest from those that do not (i.e., empty complexes). Purification can be achieved by incorporating one or more detectable labels on the transmembrane protein of interest contained in the complex, such as affinity tags, and selecting one or more labels. Examples of detectable labels for use as affinity tags include, but are not limited to, His-tags that can be recognized by immobilized metal ion affinity chromatography (IMAC), FLAG-tags for FLAG M1 anti-FLAG immunoaffinity chromatography, and 1D4-tags using 1D4 resin. Molecular sieving chromatography can also be utilized, for example, by performing SDS-PAGE and Western blot analysis to identify and / or confirm the presence of lipid bilayer membrane scaffold protein complexes that contain the transmembrane protein of interest or a portion thereof. In embodiments in which both the transmembrane protein of interest and the MSP contain detectable labels, at least one label on the MSP and one label on the transmembrane protein of interest can be different.

[0095] In certain embodiments, the lipid bilayer membrane scaffold protein complex containing the transmembrane protein of interest or a portion thereof can be formed by first solubilizing the transmembrane protein of interest in a detergent (e.g., N-dodecyl-β-D-maltoside (DDM) or the like), then mixing with cholate-solubilized phospholipids and membrane scaffold protein, and then removing the detergent. In some examples, the detergent can be extracted from the mixture using biobeads, allowing the assembly of a disc-shaped lipid bilayer membrane scaffold protein complex that integrates the purified transmembrane protein of interest. The disc-shaped lipid bilayer membrane scaffold protein complex that incorporates the transmembrane protein of interest can then be isolated from the disc-shaped lipid bilayer membrane scaffold protein complex that does not contain the transmembrane protein using affinity purification beads. For example, if the transmembrane protein of interest contains a corresponding "tag" sequence at its c-terminus or n-terminus, the beads are bearing anti-"tag" antibodies (e.g., FLAG-tag, His-tag, HA-tag, etc., such tags are well known in the art). Further purification can be achieved by molecular sieve chromatography.

[0096] In certain examples, the immunogen incorporated into the lipid bilayer membrane scaffold protein complex is administered to an animal at a concentration of at least 0.1 mg / mL, at least 0.2 mg / mL, at least 0.3 mg / mL, at least 0.4 mg / mL, at least 0.5 mg / mL, at least 0.6 mg / mL, at least 0.7 mg / mL, at least 0.8 mg / mL, at least 0.9 mg / mL, at least 1.0 mg / mL, at least 2.0 mg / mL, at least 3.0 mg / mL, at least 4.0 mg / mL, at least 5.0 mg / mL, at least 10.0 mg / mL or more. In certain embodiments, the immunogen is injected into the animal at a concentration of 0.2 mg / mL to 20 mg / mL, 0.5 mg / mL to 15 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 7.0 mg / mL, 0.5 mg / mL to 5.0 mg / mL, 1.0 mg / mL to 10 mg / mL, 1.0 mg / mL to 5.0 mg / mL, 1.0 mg / mL to 4.0 mg / mL, 1.0 mg / mL to 3.0 mg / mL, 1.0 mg / mL to 2.0 mg / mL or 2.0 mg / mL to 5.0 mg / mL, inclusive. In certain embodiments, the immunogen is at least 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL , 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, 12.0 mg / mL, 15.0 mg / mL, 20.0 mg / mL or more. In one embodiment, the immunogenic transmembrane protein of interest incorporated into a lipid bilayer membrane scaffold protein complex is injected into an animal at a concentration of 0.5 mg / mL.

[0097] In certain embodiments, animals can be immunized with cells expressing an immunogen, such as irradiated cells, hi one particular embodiment, animals can be immunized with cells expressing a transmembrane protein, modified form thereof, or portion thereof of an immunogen of interest described herein.

[0098] In some embodiments, animals can be immunized with VLPs or exosomes that express a transmembrane protein of interest in the animal. In certain embodiments, animals can be immunized with VLPs that contain a nucleic acid encoding a transmembrane protein of interest, a modified version thereof, or a portion thereof.

[0099] In some examples, animals are immunized with lipid bilayer membrane scaffold protein complexes that include, for example, proteins or peptides, modified proteins or peptides, nucleic acids, modified nucleic acids, VLPs and transmembrane proteins of interest or portions thereof described herein.

[0100] The animal to be immunized may be any animal. In certain embodiments, the animal is a mammal. In some embodiments, the mammal is a mouse, a rat, a goat, a human, a hamster, a pig, a monkey, or a guinea pig. In some embodiments, the mammal is not a human. In certain embodiments, the non-human mammal is a mouse, a rat, or a goat. In certain embodiments, the mammal is a mouse. In another embodiment, the mammal is a human, such as, for example, a human that has been exposed to the immunogen.

[0101] In some examples, the immunized animal is genetically engineered. Genetic modification can be performed by known methods, such as those used to delete or block genes encoding proteins. For example, animals can be genetically engineered so that they do not express (e.g., knock out) a transmembrane protein of interest (i.e., antigen) from their endogenous gene locus. As an example, a genetically engineered non-human mammal is a mouse that cannot express an endogenous mouse transmembrane protein of interest, for example, as a result of genetic modification to the locus of the endogenous mouse transmembrane protein or inactivation (e.g., complete or partial deletion) of the endogenous gene.

[0102] In certain embodiments, the genetically engineered animal is a non-human mammal, such as, for example, a mouse, goat, or rat, that includes in its genome (e.g., via breeding or a multiple gene targeting strategy) (i) one or more humanized immunoglobulin heavy chain loci comprising one or more human variable heavy chain gene segments, (ii) one or more human variable light chain gene segments comprising one or more human variable light chain gene segments, and / or (iii) one or more humanized immunoglobulin light chain loci (e.g., kappa and / or lambda) comprising one or more human variable light chain gene segments. As used herein, the term "humanized" includes those that have been modified to include human sequences. For example, a humanized locus is a locus (e.g., an endogenous locus) that has been modified to include human sequences (e.g., gene segments or genes).

[0103] In some embodiments, the genetically engineered animal is a non-human mammal, such as a mouse, goat, or rat, that contains in its genome a humanized immunoglobulin heavy chain locus that comprises one or more human variable heavy chain gene segments.

[0104] In some embodiments, the genetically engineered animal is a non-human mammal, such as a mouse, goat, or rat, that comprises in its genome a humanized immunoglobulin light chain locus that comprises one or more human variable light chain gene segments.

[0105] In some embodiments, the genetically engineered animal is a non-human mammal, such as, for example, a mouse, goat, or rat, that comprises in its genome a humanized immunoglobulin light chain locus (e.g., κ and / or λ) that comprises one or more human variable light chain gene segments. In one embodiment, the genetically engineered animal comprises a humanized immunoglobulin light chain locus that comprises one or more human variable kappa (Vκ) gene segments. In another embodiment, the genetically engineered animal comprises a humanized immunoglobulin light chain locus that comprises one or more human variable lambda (Vλ) gene segments.

[0106] In some embodiments, the human immunoglobulin heavy chain variable region (V H ) and / or a nucleic acid sequence encoding a human immunoglobulin light chain variable region (V L Genetically engineered animals that contain a nucleic acid sequence encoding a transmembrane protein of interest may also lack the endogenous gene encoding the transmembrane protein of interest.

[0107] In other examples, the animals are "wild-type" animals that express an endogenous homolog of the transmembrane protein of interest.

[0108] In some embodiments, the genetically engineered animal (e.g., rat or mouse) produces antibodies that include, among other things, a heavy chain, each heavy chain comprising a human heavy chain variable domain operably linked to a rodent (e.g., rat or mouse) heavy chain constant domain. In some embodiments, the genetically engineered animal (e.g., rat or mouse) produces antibodies that include, among other things, an immunoglobulin chain, each immunoglobulin chain comprising a human light chain variable domain operably linked to a rodent (e.g., rat or mouse) heavy chain constant domain. In some embodiments, the genetically engineered animal (e.g., rat or mouse) produces antibodies that include, among other things, a κ light chain, each κ light chain comprising a human κ light chain variable domain operably linked to a rodent (e.g., rat or mouse) κ light chain constant domain. In some embodiments, the genetically engineered animal (e.g., rat or mouse) produces antibodies that include, among other things, a λ light chain, each λ light chain comprising a human λ light chain variable domain operably linked to a rodent (e.g., rat or mouse) λ light chain constant domain. In some embodiments, the genetically engineered animal (e.g., a rat or a mouse) produces antibodies that include, among other things, a light chain, each light chain comprising a human λ light chain variable domain operably linked to a rodent (e.g., a rat or a mouse) κ light chain constant domain. In some embodiments, the genetically engineered animal (e.g., a rat or a mouse) produces antibodies that include, among other things, a λ light chain, each λ light chain comprising a human λ light chain variable domain operably linked to a rodent (e.g., a rat or a mouse) λ light chain constant domain.

[0109] In some embodiments, the non-human mammal (e.g., rat or mouse) described herein is as described in, for example, U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, 8,791,323, 9,226,484, and WO2019 / 113065, all of which are incorporated herein by reference in their entirety. Breeding (or "mating" or "crossbreeding") can be performed according to protocols readily available in the art. See, for example, JoVE Science Education Database.Lab Animal Research, Fundamentals of Breeding and Weaning, JoVE, Cambridge, MA, (2018) (video article); Breeding Strategies for Maintaining Colonies of Laboratory Mice, A Jackson Laboratory Resource Manual, (copyright) 2007 The Jackson Laboratory (all of which are incorporated herein by reference). Alternatively, an engineered Igλ light chain locus can be engineered into an ES cell containing a humanized IgH locus and / or a humanized Igκ locus and the resulting ES cell can be used to generate a genetically engineered animal, or a genetically engineered animal containing a humanized Igλ light chain locus can be crossed with another genetically engineered animal containing a humanized IgH locus and / or a humanized Igκ locus. Various genetically engineered animals containing a humanized IgH locus and / or a humanized Igκ locus are known, such as the VELOCIMMUNE® strain (see, e.g., U.S. Pat. Nos. 8,502,018 and / or 8,642,835, which are incorporated by reference in their entireties), the XENOMOUSE™ strain (see, e.g., Mendez, MJ et al., 1997, Nat. Genetics 15(2):146-56 and Jakobovits, A. et al., 1995, Ann. NY Acad. Sci. 764:525-35, which are incorporated by reference in their entireties).

[0110] In some embodiments, the genetically engineered animals described herein are disclosed in accordance with U.S. Patent Nos. 9,796,788; 9,969,814; U.S. Patent Publication Nos. 2011 / 0195454A1, 2012 / 0021409A1, 2012 / 0192300A1, 2013 / 0045492A1, 2013 / 0185821A1, 2013 / 0 302836A1; International Patent Application Publication Nos. 2011 / 097603, 2012 / 148873, 2013 / 134263, 2013 / 184761, 2014 / 160179, 2014 / 160202 (all of which are incorporated by reference in their entireties). In some embodiments, the rodents described herein can be any of the methods described in WO2019 / 113065, WO2017214089, US20180125043, and U.S. Pat. Nos. 9,035,128, 9,066,502, 9,163,092, 9,150,662, 9,334,333, 9,006,511, 9,029 ,628, 9,206,261, 9,012,717, 9,394,373, 9,206,262, 9,206,263, 9,226,484, 9,540,452, and 9,399,683, all of which are incorporated herein by reference in their entireties.

[0111] Once an animal is immunized, the animal's immune response to the immunogen is monitored using an antigen-specific immunoassay. The humoral immune response in an animal can be determined based on the titer of antibodies in the serum specific to the transmembrane protein of interest (antigen). Various assays can be used to determine antibody titers, including ELISA and flow cytometry-based assays (see, for example, David H. Margulies, Induction of Immune Responses, Current Protocols in Immunology, 89, 1(2.0.1-2.0.3)(2010)); Henri V. van der Heyde et al., "Analysis of antigen-specific antibodies and their isotypes in experimental malaria," Cytometry, Voltry, Vol. Vol. 71A(4):242-250(2007); both of which are incorporated herein by reference. In some embodiments, the assay utilizes cells that express, or have been engineered to express, the transmembrane protein of interest on the cell surface, and antibody titers can be determined by measuring antibody binding to the cells.

[0112] Once an appropriate immune response has been achieved, a cell population is collected from the immunized animal. The cells may be collected from a number of sources, including, but not limited to, the spleen, lymph nodes, bone marrow, and peripheral blood of the immunized animal. In one embodiment, the cells are a population of cells obtained from the spleen of the immunized animal, i.e., a population of splenocytes. In some embodiments, the cell population is a heterogeneous population of cells, containing cells from different tissues, organs, or regions of the immunized animal. In certain embodiments, the cell population is a homogenous population of cells obtained from one organ, such as the spleen, of the immunized animal. In one embodiment, the population of cells includes tissue-derived cells from one or more of the spleen, lymph nodes, and bone marrow. In other embodiments, the cell population is obtained from the blood of the immunized animal.

[0113] Cells collected from immunized animals include "antibody-producing cells," which refers to cells that express antibodies, either naturally (as a result of B cell activation) or as a result of recombinant technology and genetic engineering (such as hybridoma cells). Thus, the term "antibody-producing cells" encompasses immune cells such as lymphocytes. In certain non-limiting examples, lymphocytes can be antigen-dependent B cell lineages, including memory B cells, plasmablasts, and terminally differentiated plasma cells that express antibodies, as well as recombinant cells such as hybridomas and non-lymphoid cells engineered to express antibodies. Additionally, "antibody-producing cells" encompass cells in which expressed antibodies are bound or fixed to the cell membrane, i.e., cell surface antibodies, and cells that secrete antibodies.

[0114] In certain embodiments, the population of antibody-producing cells is derived from the spleen, lymph nodes, bone marrow, or peripheral blood of an immunized animal. In certain embodiments, the population of antibody-producing cells comprises peripheral blood cells, B cells, plasma cells, plasma cell myelomas, or combinations thereof. In some embodiments, the population of antibody-producing cells comprises recombinant cells, e.g., hybridomas. In certain embodiments, the population of antibody-producing cells is a lymphocyte population, e.g., B cells. In one embodiment, the population of antibody-producing cells is composed of memory B cells.

[0115] Also, in some cases, additional antibody-producing cells may be derived from the initial population of antibody-producing cells obtained by the method of the present disclosure. Thus, cell lines, plasma cells, memory B cells, hybridomas, plasma cell myelomas, and recombinant antibody-expressing cells may be derived or obtained from antibody-producing cells. For example, antibody-producing cells may be fused to myeloma cells to generate hybridomas, or may be otherwise immortalized, such as infected with a virus (e.g., EBV), or may be differentiated by cell sorting techniques based on protein markers expressed by specific B cell types.

[0116] In certain embodiments, the term "antibody-producing cells" refers to cells that express an antibody that binds to a transmembrane protein of interest. In some embodiments, the antibody that binds to a transmembrane protein of interest is located on the cell surface. In certain instances, the population of antibody-producing cells is a heterogeneous population of antibody-producing cells, which contains antibody-producing cells that express antibodies against multiple antigens. In one particular embodiment, the population of antibody-producing cells is a heterogeneous population of antibody-producing cells that contains antibody-producing cells that express an antibody that binds to a transmembrane protein of interest and at least one other antigen. In other embodiments, the population of antibody-producing cells may be a homogeneous population of antibody-producing cells that contains antibody-producing cells that express an antibody that binds to only one antigen. In certain embodiments, the population of antibody-producing cells is a homogeneous population of antibody-producing cells that includes antibody-producing cells that express an antibody that binds to a transmembrane protein of interest.

[0117] As shown in Tables 1-5, immunization of animals with a variety of different immunogens and immunogen combinations resulted in the generation of antibody-producing cells, including a population of antibody-producing cells that express antibodies that bind to the transmembrane protein of interest. Tables 1-5 also show that immunization of genetically engineered animals that do not express an endogenous gene encoding the transmembrane protein of interest generated antibody-producing cells that express antibodies that bind to the transmembrane protein of interest.

[0118] Harvesting of antibody-producing cells expressing antibodies to the transmembrane protein of interest. As described herein, obstacles to the generation of antibodies against transmembrane proteins include the inability to provide sufficient amounts of conformationally correct transmembrane protein antigens to antibodies or cells that produce antibodies against the transmembrane protein of interest.For example, purified endogenous transmembrane proteins are conformationally compromised when isolated from their native membrane environment, and current lipid-based and cell-based membrane preparations result in high levels of non-specific binding, are difficult to formulate, and often provide a subset of unfolded or improperly folded transmembrane protein antigens.

[0119] The present disclosure overcomes such obstacles by utilizing a lipid bilayer membrane scaffold protein complex to present a transmembrane protein antigen to antibodies produced by antibody-producing cells. More specifically, a complex comprising a transmembrane protein of interest and a lipid and a membrane scaffold protein commonly present in the membrane of a native cell is utilized to present the transmembrane protein in its native conformation to the antibody. Thus, the lipid bilayer membrane scaffold protein complex is used in the method of the present invention to identify and recover a specific subset of antibodies (or cells expressing antibodies) in a population that bind to an epitope on a transmembrane protein that is readily accessible in nature.

[0120] Once the lipid bilayer membrane scaffold protein complex containing the transmembrane protein of interest is formed, the complex can be used as a sorting agent to detect and isolate a population of antibody-producing cells that express an antibody that binds to the transmembrane protein of interest. For example, a heterogeneous population of antibody-producing cells can be obtained from a mammal immunized as described herein, and the population of antibody-producing cells can then be contacted with a lipid bilayer membrane scaffold protein complex that presents the transmembrane protein of the antigen of interest to the antibody produced by the cells. The complex and population of antibody-producing cells can then be incubated to allow binding between the transmembrane protein of interest presented by the complex and the antibody produced by the cells. The binding between the antibody and the transmembrane protein of interest can then be detected, and the bound cells can be collected for further use.

[0121] In some examples, the population of antibody-producing cells obtained from the immunized animal may be a heterogeneous population of antibody-producing cells, which contains antibody-producing cells expressing antibodies against one or more antigens. In certain embodiments, the population of antibody-producing cells is a heterogeneous population of antibody-producing cells, including specific antibody-producing cells expressing an antibody that binds to the transmembrane protein of interest and specific antibody-producing cells expressing an antibody that binds to at least one other antigen. In certain embodiments, the heterogeneous population of antibody-producing cells consists of at least 0.01%, at least 0.1%, at least 0.5%, at least 1.0%, at least 1.5%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80, at least 90% or more of the antibody-producing cells expressing an antibody that binds to the transmembrane protein of interest.

[0122] In other embodiments, the population of antibody-producing cells may be a homogenous population of antibody-producing cells that contains antibody-producing cells that express an antibody that binds to only one antigen. In certain embodiments, the population of antibody-producing cells is a homogenous population of antibody-producing cells that includes antibody-producing cells that express an antibody that binds to a transmembrane protein of interest. In certain embodiments, the homogenous population of antibody-producing cells consists of at least 70%, at least 80, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the antibody-producing cells that express an antibody that binds to the transmembrane protein of interest.

[0123] In certain embodiments, B cells can be detected and collected from the heterogeneous cell population obtained from the immunized animal at the same time that the antibody-producing cells are obtained to provide an antibody-producing B cell population. In some embodiments, B cells can be obtained from the heterogeneous cell population obtained from the immunized animal, and then the antibody-producing cells can be obtained from the heterogeneous cell population. In yet another embodiment, B cells can be detected in the antibody-producing cell population obtained from the immunized animal to provide an antibody-producing B cell population.

[0124] In one particular embodiment, antibody-producing B cells expressing an antibody against a transmembrane protein of interest can be detected in a heterogeneous cell population obtained from an immunized animal and isolated by FACS based on cell surface B cell markers.

[0125] B cell markers are known in the art. For example, applicable B cell markers that can be detected through the use of FACS include, but are not limited to, IgG, IgM, IgE, IgA, IgD, CD1, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD38, CD40, CD78, CD80, CD138, CD319, TLR4, IL-6, PDL-2, CXCR3, CXCR4, CXCR5, CXCR6, IL-10 and TGFβ. In certain embodiments, B cells can be detected using a label specific for IgG, IgM, or a combination thereof. In one embodiment, antibody-producing B cells are detected using an antibody specific for IgG.

[0126] In certain embodiments, after immunization, splenocytes are harvested from the immunized animal. After removal of red blood cells, IgG from the immunized animal is isolated using the methods described herein. +A population of antigen-positive B cells is isolated from a heterogeneous cell population.For example, splenocytes are contacted with anti-IgG antibody and washed to remove excess non-bound cells and antibody.Then, cells are stained with fluorescent antibody (i.e., secondary antibody) that binds to the labeled B cell marker (e.g., anti-IgG antibody).Then, as described herein, the stained cells can be analyzed by flow cytometry and isolated for further use.

[0127] In some embodiments of the methods, peripheral blood mononuclear cells (PBMCs) are obtained from a mammal known to have humoral immunity to an antigen of interest. IgG1 expressing antibodies that recognize the antigen of interest are then isolated according to the methods of the present disclosure. + Then, antigen positive B cells can be isolated for further processing.

[0128] In some embodiments of the method, the population of antibody-producing cells comprises antibody-producing B cells. In one such embodiment, the antibody-producing population comprises antibody-producing B cells expressing an antibody against a transmembrane protein of interest, which can be detected in a heterogeneous cell population obtained from the immunized animal and isolated by contacting the heterogeneous cell population with a lipid bilayer membrane scaffold protein complex that presents the transmembrane protein of interest, and a molecule that binds to a B cell marker, such as an antibody that binds to a B cell surface marker. For example, after immunization, splenocytes are harvested from the immunized animal. After removal of red blood cells, the cell populations are identified as expressing anti-IgG and IgG antibodies in the heterogeneous cell population. +To allow binding between positive B cells, the cell population is incubated with a labeled anti-IgG antibody. At the same time as, or after or before, the heterogeneous cell population is incubated with a labeled lipid bilayer membrane scaffold protein complex that displays the transmembrane protein of interest, so that the membrane scaffold protein can bind to the antibody produced by the cells. The population of antibody-producing B cells that expresses an antibody against the transmembrane protein of interest can then be obtained by detecting and isolating (harvesting) the cells that are bound to both the labeled anti-IgG antibody (B cells) and the labeled lipid bilayer membrane scaffold protein complex, for example, by flow cytometry.

[0129] In some examples, the methods include immunizing an animal as described herein.

[0130] For example, in examples involving contacting or incubating a population of cells, such as a population of antibody producing cells, with a lipid bilayer membrane scaffold protein complex, the lipid bilayer membrane scaffold protein complex is provided to the cell culture medium at a concentration of at least 0.1 mg / mL, at least 0.2 mg / mL, at least 0.3 mg / mL, at least 0.4 mg / mL, at least 0.5 mg / mL, at least 0.6 mg / mL, at least 0.7 mg / mL, at least 0.8 mg / mL, at least 0.9 mg / mL, at least 1.0 mg / mL, at least 2.0 mg / mL, at least 3.0 mg / mL, at least 4.0 mg / mL, at least 5.0 mg / mL, at least 10.0 mg / mL or more. In certain embodiments, the complex is supplied to the cells at a concentration of 0.2 mg / mL to 20.0 mg / mL, 0.2 mg / mL to 15.0 mg / mL, 0.2 mg / mL to 10 mg / mL, 0.2 mg / mL to 7.0 mg / mL, 0.2 mg / mL to 5.0 mg / mL, 0.5 mg / mL to 10.0 mg / mL, 0.5 mg / mL to 7.0 mg / mL, 0.5 mg / mL to 5.0 mg / mL, 1.0 mg / mL to 10.0 mg / mL, 1.0 mg / mL to 7.0 mg / mL or 1.0 mg / mL to 5.0 mg / mL, inclusive. In certain embodiments, the conjugate is at 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3.0 mg / mL, 3.1 mg / mL, 3.2 mg / mL, 3.3 mg / mL, 3.4 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3 ... Provided to cells at concentrations of 0.5mg / mL, 3.0mg / mL, 3.5mg / mL, 4.0mg / mL, 4.5mg / mL, 5.0mg / mL, 5.5mg / mL, 6.0mg / mL, 6.5mg / mL, 7.0mg / mL, 7.5mg / mL, 8.0mg / mL, 8.5mg / mL, 9.0mg / mL, 9.5mg / mL, 10.0mg / mL, 12.0mg / mL, 15.0mg / mL, 20.0mg / mL or greater.

[0131] In one embodiment of the method, the immunized animal is a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, that is immunized with a nucleic acid sequence encoding a human transmembrane protein of interest or a portion thereof, or a combination thereof.

[0132] In a particular embodiment of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., rat or mouse) and incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a full-length human transmembrane protein, allowing the antibody produced by the cells to bind to the human transmembrane protein of interest (antigen) presented by the complex. The cells are then incubated with PE-streptavidin, allowing the binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing antibodies that bind to an epitope on the full-length human transmembrane protein of interest.

[0133] In some embodiments of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., rat or mouse) and incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a portion of a human transmembrane protein, such as a truncated human transmembrane protein, to allow binding of the antibody produced by the cells to an epitope on the portion of the human transmembrane protein of interest (antigen) presented by the complex. The cells are then incubated with PE-streptavidin to allow binding of the streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from bound B cells can be detected by FACS to identify and recover a homogenous population of antibody-producing B cells expressing antibodies that bind epitopes on the antigen presented by the complex.

[0134] In embodiments of the method comprising a lipid bilayer membrane scaffold protein complex comprising a portion of a transmembrane protein of interest, the transmembrane protein presented by the complex does not comprise the full-length transmembrane protein of interest. In some embodiments, the transmembrane protein of interest is a truncated transmembrane protein as described herein. In certain embodiments, the truncated transmembrane protein of interest comprises at least the entire transmembrane portion of the transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not comprise one or more extracellular loop domains, the amino-terminal portion and / or the carboxy-terminal portion of the full-length transmembrane protein of interest. In some embodiments, the truncated protein comprises an extracellular domain of the transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of the transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not comprise the amino-terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not comprise the carboxy-terminal portion of the full-length transmembrane protein of interest. In some embodiments, the truncated transmembrane protein does not include the extracellular loop domains of the full-length transmembrane protein of interest.

[0135] In another embodiment of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof. Splenocytes are isolated from the immunized non-human animal (e.g., rat or mouse) and incubated with an antibody labeled against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a chimeric transmembrane protein comprising a portion of the human transmembrane protein of interest and a portion of a non-human homologue of the transmembrane protein of interest to allow binding of the chimeric transmembrane protein of interest (antigen) presented by the complex to the antibody produced by the cells. The cells can then be washed. PE fluorescence and signals (e.g., fluorochrome-labeled anti-IgG signals) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells that express an antibody that binds to an epitope on the human portion of the chimeric transmembrane protein of interest.

[0136] In embodiments of the method involving the use of a chimeric transmembrane protein of interest, the chimeric protein may comprise portions of a transmembrane protein of interest from two different species as described herein. For example, the chimeric transmembrane protein may comprise one or more portions of a human transmembrane protein operably linked to one or more portions of a non-human homologue of the transmembrane protein of interest. In some examples, the chimeric transmembrane protein comprises an extracellular domain of the transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of the transmembrane protein of interest. The non-human homologue may be any non-human animal, such as, for example, a mouse, a rat, a goat, a hamster, a pig, a chimpanzee, a horse, a sheep, a monkey, and a guinea pig. In certain embodiments, the non-human mammal is a mouse, a rat, or a goat. In certain embodiments, the non-human mammal is a mouse.

[0137] In an exemplary embodiment of the method of the present invention, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein of interest and an amino-terminal domain and / or a carboxy-terminal domain from a non-human homologue of the transmembrane protein. In one embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein and a carboxy-terminal domain from a non-human homologue of the transmembrane protein. In another embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein and a carboxy-terminal domain from a non-human homologue of the transmembrane protein. In another embodiment, the chimeric transmembrane protein of interest comprises an amino-terminal domain and a carboxy-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a non-human homologue of the transmembrane protein. In one embodiment, the chimeric transmembrane protein of interest comprises an amino-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a non-human homologue of the transmembrane protein. In yet another embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains derived from a non-human homolog and an amino-terminal domain derived from a non-human homolog of the transmembrane protein.

[0138] In another embodiment of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or a rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof. Splenocytes are isolated from the immunized non-human animal and incubated with an antibody labeled against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a non-human homolog of the transmembrane protein to allow binding of the non-human transmembrane protein (antigen) presented by the complex with the cross-reactive antibody produced by the cells. The cells can then be washed. The PE fluorescence and signal (e.g., a fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and recover a homogeneous population of antibody-producing B cells that express cross-reactive antibodies that bind to epitopes presented on the non-human transmembrane protein of interest and the human transmembrane protein of interest.

[0139] In embodiments of the method of the present invention that include the use of a non-human homolog of a transmembrane protein of interest or a portion thereof, the non-human homolog may be any non-human organism. Specific examples of non-human organisms include, but are not limited to, non-human mammals, reptiles, fish, bacteria, insects, and viruses. In some embodiments, the non-human protein may be from mouse, rat, goat, hamster, pig, chimpanzee, horse, sheep, monkey, and guinea pig. In certain embodiments, the non-human protein homolog is from mouse, rat, or goat. In certain embodiments, the non-human homolog is a mouse homolog of the transmembrane protein of interest.

[0140] In some embodiments of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a full-length human transmembrane protein of interest, a nucleic acid sequence encoding the protein, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., rat or mouse) and incubated with a portion of the full-length human transmembrane protein of interest (a peptide blocking agent) to allow binding of the peptide blocking agent to antibodies produced by the cells that specifically bind to an epitope located on the blocking agent. The cells are also incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population, and incubated with a biotinylated lipid bilayer membrane scaffold protein complex encompassing the full-length transmembrane protein to allow binding of the full-length transmembrane protein of interest (antibody) presented by the complex to antibodies produced by the remaining unbound cells in the population. The cells are then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin (e.g., Bir-A)-labeled membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed to remove all cells bound to the peptide blocking agent, unbound and excess PE-streptavidin, and labeled antibodies against B cell markers can be removed from sorting. PE fluorescence and signals (e.g., fluorochrome-labeled anti-IgG signals) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells that express antibodies that bind to epitopes on the full-length human transmembrane protein of interest that are not present in the portion corresponding to the peptide blocking agent.

[0141] In embodiments of the method that include the use of a blocking agent, the blocking agent may be any molecule, such as, for example, a peptide, a polypeptide, a chemical compound, etc. In such embodiments, the blocking agent may be a polypeptide or peptide corresponding to a portion of a transmembrane protein of interest, a polypeptide or polypeptide corresponding to a portion of an MSP protein, or a detectable marker, such as a His tag or a Flag tag. In some embodiments, the blocking agent comprises a detectable label. In other embodiments, the blocking agent does not comprise a detectable label. In one embodiment, the blocking agent is a peptide or polypeptide portion of a transmembrane protein of interest and is incubated with a population of antibody-producing cells to allow binding of the blocking agent to antibodies produced by the antibody-producing cells that specifically bind to an epitope located on the blocking agent. In one embodiment, the blocking agent is a truncated transmembrane protein of interest. In certain embodiments, the blocking agent is a polypeptide corresponding to one or more extracellular loop domains, the amino-terminal portion and the carboxy-terminal portion of a full-length transmembrane protein of interest. In certain embodiments, the blocking agent may be a polypeptide that comprises the amino-terminal portion of a full-length transmembrane protein of interest. In another embodiment, the blocking agent may be a polypeptide that comprises the carboxy-terminal portion of a full-length transmembrane protein of interest. In one embodiment, the blocking agent may be a polypeptide comprising an extracellular loop domain of a full-length transmembrane protein of interest. In some embodiments, the blocking agent comprises an extracellular domain of a transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of a full-length transmembrane protein of interest. In another embodiment, the blocking agent is a peptide or polypeptide portion of an MSP protein, which is incubated with a population of antibody-producing cells to allow binding of the blocking agent to antibodies produced by the antibody-producing cells that specifically bind to an epitope located on the blocking agent.In yet another embodiment, the blocking agent is a peptide or polypeptide detectable marker located in the MSP or transmembrane protein of interest and is incubated with a population of antibody-producing cells to allow binding of the blocking agent to antibodies produced by the antibody-producing cells that specifically bind to an epitope located on the blocking agent.

[0142] In other embodiments of the method, non-human animals or genetically engineered non-human animals, such as mice or rats, are immunized with a nucleic acid sequence (immunogen) encoding a full-length human transmembrane protein of interest, or a combination thereof.

[0143] In some embodiments, splenocytes are isolated from immunized non-human animals (e.g., rats or mice) and incubated with antibodies labeled against B cell markers (e.g., fluorochrome-labeled anti-IgG antibodies) to identify B cell populations. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that encompasses a full-length human transmembrane protein to allow binding of the antibody produced by the cells to the full-length transmembrane protein of interest (antigen) presented by the complex. The cells can then be washed. PE fluorescence and signals (e.g., fluorochrome-labeled anti-IgG signals) from the bound B cells can be detected by FACS to identify and recover a homogenous population of antibody-producing B cells that express antibodies that bind to epitopes located in portions of the full-length human transmembrane protein of the antigen of interest that also correspond to amino acid sequences or domains present in or encoded by the immunogen.

[0144] In another embodiment of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., rat or mouse) and incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a portion of the human transmembrane protein used as an immunogen, allowing binding of the epitope (antigen) of the portion of the human transmembrane protein of interest presented by the complex to the antibody produced by the cells. The cells are then incubated with PE-streptavidin, allowing binding of the streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from bound B cells can be detected by FACS to identify and recover a homogenous population of antibody-producing B cells expressing antibodies that bind epitopes on the antigen presented by the complex.

[0145] In embodiments of the methods of the invention involving the use of a portion of a transmembrane protein of interest, the portion of the transmembrane protein is a peptide or polypeptide and does not include the full-length transmembrane protein of interest. In one embodiment, the truncated transmembrane protein of interest may be a modified transmembrane protein that does not include one or more extracellular loop domains, the amino terminal portion and / or the carboxy terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the modified transmembrane protein of interest may be a truncated transmembrane protein. In some embodiments, the portion of the transmembrane protein of interest includes a detectable label. In other embodiments, the portion of the transmembrane protein of interest does not include a detectable label. In certain embodiments, the truncated transmembrane protein of interest includes at least the entire transmembrane portion of the transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not include the amino terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not include the carboxy terminal portion of the full-length transmembrane protein of interest. In some embodiments, the truncated protein comprises an extracellular domain of a transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of a transmembrane protein of interest. In one embodiment, the truncated transmembrane protein does not include an extracellular loop domain of a full-length transmembrane protein of interest.

[0146] In another aspect of the method, the genetically engineered animal does not express the transmembrane protein of interest from its endogenous gene and is immunized with a nucleic acid sequence encoding a non-human transmembrane protein of interest or a portion thereof, or a combination thereof.

[0147] The non-human transmembrane protein of interest and the genetically engineered animal may be from the same animal or different animals. The non-human transmembrane protein of interest may be from any non-human organism. Non-limiting examples of non-human organisms include non-human mammals, reptiles, fish, bacteria, insects, and viruses. In some embodiments, the non-human transmembrane protein is from an animal, such as, for example, a mouse, a rat, a goat, a hamster, a pig, a chimpanzee, a horse, a sheep, a monkey, and a guinea pig. In certain embodiments, the non-human animal is a mammal. In other embodiments, the non-human transmembrane protein is from a mouse, a rat, or a goat. In certain embodiments, the non-human mammal is a mouse.

[0148] In a particular embodiment of the method, the genetically engineered animal is a non-human animal, such as a mouse or a rat, and is immunized with a nucleic acid sequence (immunogen) encoding a full-length non-human transmembrane protein of interest, or a combination thereof. Splenocytes are isolated from the immunized genetically engineered animal (e.g., a rat or a mouse) and incubated with an antibody labeled against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that encompasses the full-length non-human transmembrane protein, allowing the binding of the transmembrane protein of interest (antigen) presented by the complex to the antibody produced by the cells. The cells are then incubated with PE-streptavidin, allowing the binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. PE fluorescence and signals (e.g., fluorochrome-labeled anti-IgG signals) from bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing antibodies that bind to epitopes on the full-length non-human transmembrane protein of interest.

[0149] In another embodiment of the method, a genetically engineered non-human animal, for example, a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (for example, a rat or mouse) and incubated with a labeled antibody against a B cell marker (for example, a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that encompasses the full-length human transmembrane protein of interest, allowing the antibody produced by the cells to bind to the human transmembrane protein of interest (antigen) presented by the complex. The cells are then incubated with PE-streptavidin, allowing the binding of streptavidin to each of the biotin-labeled (for example, Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. The PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing cross-reactive antibodies that bind to the full-length non-human transmembrane protein of interest and epitopes on the non-human transmembrane protein of interest.

[0150] In some embodiments of the method, a genetically engineered non-human animal, e.g., a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with a labeled antibody (e.g., a fluorochrome-labeled anti-IgG antibody) against a B cell marker to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a portion of a full-length non-human transmembrane protein, such as a truncated transmembrane protein, to allow binding of the antibody produced by the cells to an epitope on the portion of the transmembrane protein of interest (antigen) presented by the complex. The cells are then incubated with PE-streptavidin to allow binding of the streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from bound B cells can be detected by FACS to identify and recover a homogenous population of antibody-producing B cells expressing antibodies that bind epitopes on the antigen presented by the complex.

[0151] In embodiments of the method comprising a lipid bilayer membrane scaffold protein complex comprising a portion of a transmembrane protein of interest, the transmembrane protein presented by the complex does not comprise the full-length transmembrane protein of interest. In one embodiment, the transmembrane protein of interest is a truncated transmembrane protein that does not comprise one or more extracellular loop domains, the amino-terminal portion and / or the carboxy-terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein of interest comprises at least the entire transmembrane portion of the transmembrane protein of interest. In some embodiments, the truncated protein comprises an extracellular domain of the transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of the transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not comprise the amino-terminal portion of the full-length transmembrane protein of interest. In certain embodiments, the truncated transmembrane protein does not comprise the carboxy-terminal portion of the full-length transmembrane protein of interest. In one embodiment, the truncated transmembrane protein does not comprise the extracellular loop domain of the full-length transmembrane protein of interest.

[0152] In a particular embodiment of the method, a genetically engineered non-human animal, such as a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length non-human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized animal and incubated with a labeled antibody against a B cell marker (e.g., a population of fluorochrome-labeled anti-IgG antibodies) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex encompassing a chimeric transmembrane protein comprising a portion of a non-human homolog of the transmembrane protein of interest and a portion of a human transmembrane protein of interest to allow binding of the non-human portion of the chimeric transmembrane protein of interest (antigen) presented by the complex to the non-human portion of the chimeric transmembrane protein of interest (antigen) presented by the complex and the antibody produced by the cells. The cells can then be washed. PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing antibodies that bind to epitopes on the non-human portion of the chimeric transmembrane protein of interest.

[0153] In embodiments of the method involving the use of a chimeric transmembrane protein of interest, the chimeric protein may comprise portions of a transmembrane protein of interest from two different species as described herein. For example, the chimeric transmembrane protein may comprise one or more portions of a human transmembrane protein operably linked to one or more portions of a non-human homolog of the transmembrane protein. In some examples, the chimeric transmembrane protein comprises an extracellular domain of the transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain between one or more membrane spanning domains of the transmembrane protein of interest. The non-human homolog may be any non-human animal, such as, for example, a mouse, a rat, a goat, a hamster, a pig, a chimpanzee, a horse, a sheep, a monkey, and a guinea pig. In certain embodiments, the non-human mammal is a mouse, a rat, or a goat. In certain embodiments, the non-human mammal is a mouse.

[0154] In an exemplary embodiment of the method, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein and an amino-terminal domain and / or a carboxy-terminal domain from a non-human homolog of the transmembrane protein. In one embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains and an amino-terminal domain and / or a carboxy-terminal domain from a non-human homolog of the transmembrane protein. In another embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains and an amino-terminal domain from a human transmembrane protein and a carboxy-terminal domain from a non-human homolog of the transmembrane protein. In another embodiment, the chimeric transmembrane protein of interest comprises an amino-terminal domain and a carboxy-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a non-human homolog of the transmembrane protein. In one embodiment, the chimeric transmembrane protein of interest comprises an amino-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a non-human homolog of the transmembrane protein. In yet another embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains derived from a non-human homolog and an amino-terminal domain derived from a non-human homolog of the transmembrane protein.

[0155] In other embodiments of the method, genetically engineered non-human animals, such as mice or rats, are immunized with a portion of a full-length non-human transmembrane protein of interest, the nucleic acid sequences encoding them, or a combination thereof (immunogen).

[0156] In one such exemplary embodiment, splenocytes are isolated from the immunized genetically engineered animal and incubated with antibodies labeled against B cell markers (e.g., fluorochrome-labeled anti-IgG antibodies) to identify the B cell population. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that encompasses a full-length non-human transmembrane protein to allow binding of the antibody produced by the cells to the full-length transmembrane protein of interest (antigen) presented by the complex. The cells can then be washed. PE fluorescence and signals (e.g., fluorochrome-labeled anti-IgG signals) from the bound B cells can be detected by FACS to identify and recover a homogenous population of antibody-producing B cells that express antibodies that bind to epitopes located in portions of the full-length non-human transmembrane protein of the antigen of interest that also correspond to amino acid sequences or domains present in or encoded by the immunogen.

[0157] In another embodiment of the method, a genetically engineered non-human animal, for example, a mouse or rat, is immunized with a nucleic acid sequence encoding a full-length non-human transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (for example, a rat or mouse) and incubated with a labeled antibody against a B cell marker (for example, a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a portion of the non-human transmembrane protein used as an immunogen, allowing the binding of the epitope (antigen) of the portion of the human transmembrane protein of interest presented by the complex to the antibody produced by the cells. The cells are then incubated with PE-streptavidin, allowing the binding of streptavidin to each of the biotin (for example, Bir-A) labeled membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed. PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from bound B cells can be detected by FACS to identify and recover a homogenous population of antibody-producing B cells expressing antibodies that bind epitopes on the antigen presented by the complex.

[0158] In another embodiment of the method, a genetically engineered animal that does not express the transmembrane protein of interest from an endogenous gene is immunized with the chimeric transmembrane protein of interest, or a nucleic acid encoding same.

[0159] In examples involving the use of chimeric transmembrane proteins of interest, the chimeric protein may comprise portions of transmembrane proteins of interest from two different species as described herein. For example, the chimeric transmembrane protein may comprise one or more portions of a human transmembrane protein operably linked to one or more portions of a non-human homologue of the transmembrane protein. In some examples, the chimeric transmembrane protein comprises an extracellular domain of a human transmembrane protein of interest, e.g., an N-terminal extracellular domain, a C-terminal extracellular domain, and / or an extracellular loop domain operably linked to one or more membrane spanning domains of the transmembrane protein of interest. The non-human homologue may be any non-human animal, such as, for example, a mouse, a rat, a goat, a hamster, a pig, a chimpanzee, a horse, a sheep, a monkey, and a guinea pig. In certain embodiments, the non-human mammal is a mouse, a rat, or a goat. In certain embodiments, the non-human mammal is a mouse.

[0160] In an exemplary embodiment of the method of the present invention, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein of interest and an amino-terminal domain and / or a carboxy-terminal domain from a non-human homologue of the transmembrane protein. In one embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein and a carboxy-terminal domain from a non-human homologue of the transmembrane protein. In another embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a human transmembrane protein and a carboxy-terminal domain from a non-human homologue of the transmembrane protein. In another embodiment, the chimeric transmembrane protein of interest comprises an amino-terminal domain and a carboxy-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a non-human homologue of the transmembrane protein. In one embodiment, the chimeric transmembrane protein of interest comprises an amino-terminal domain from a human transmembrane protein and one or more extracellular loop domains from a non-human homologue of the transmembrane protein. In yet another embodiment, the chimeric transmembrane protein of interest comprises one or more extracellular loop domains from a non-human homolog operably linked to a transmembrane domain, and an amino-terminal domain from a non-human homolog of the transmembrane protein.

[0161] In one particular embodiment of the method, a genetically engineered non-human animal, e.g., a mouse or rat, is immunized with a nucleic acid sequence encoding a chimeric transmembrane protein comprising a portion of a human transmembrane protein and a portion of a non-human homologue of the transmembrane protein, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with a labeled antibody (e.g., a fluorochrome-labeled anti-IgG antibody) against a B cell marker to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex encompassing the full-length human transmembrane protein of interest (antigen), allowing binding of the human antigen presented by the complex to the antibody produced by the cells specific for the human portion of the chimeric transmembrane protein of interest. The cells can then be washed. PE fluorescence and signal (e.g., a fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing an antibody that binds to an epitope on the human portion of the chimeric transmembrane protein of interest.

[0162] In another embodiment of the method, a genetically engineered non-human animal, e.g., a mouse or rat, is immunized with a nucleic acid sequence encoding a chimeric transmembrane protein comprising a portion of a human transmembrane protein of interest and a portion of a non-human homologue of the transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex (antigen) encompassing a portion of the full-length human transmembrane protein of interest, allowing binding of the human antigen presented by the complex to the antibody produced by the cells specific for the human portion of the chimeric transmembrane protein of interest. The cells can then be washed. PE fluorescence and signal (e.g., a fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing an antibody that binds to an epitope on the human portion of the chimeric transmembrane protein of interest.

[0163] In yet another embodiment, a genetically engineered non-human animal, e.g., a mouse or rat, is immunized with a nucleic acid sequence encoding a chimeric transmembrane protein comprising a portion of a human transmembrane protein of interest and a portion of a non-human homologue of the transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex (antigen) encompassing the full-length non-human transmembrane protein of interest, allowing binding of the non-human antigen presented by the complex to the antibody produced by the cells specific for the non-human portion of the chimeric transmembrane protein of interest. The cells can then be washed. PE fluorescence and signal (e.g., a fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing an antibody that binds to an epitope on the non-human portion of the chimeric transmembrane protein of interest.

[0164] In another embodiment of the method, a genetically engineered non-human animal, e.g., a mouse or rat, is immunized with a nucleic acid sequence encoding a chimeric transmembrane protein comprising a portion of a human transmembrane protein of interest and a portion of a non-human homologue of the transmembrane protein of interest, or a combination thereof, and splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify a population of B cells. The cells are also incubated with a biotinylated lipid bilayer membrane scaffold protein complex (antigen) encompassing a portion of the full-length non-human transmembrane protein of interest, allowing binding of the non-human antigen presented by the complex to the antibody produced by the cells specific for the non-human portion of the chimeric transmembrane protein of interest. The cells can then be washed. PE fluorescence and signal (e.g., a fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells expressing an antibody that binds to an epitope on the non-human portion of the chimeric transmembrane protein of interest.

[0165] In another embodiment of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a lipid bilayer membrane scaffold protein complex that includes a full-length transmembrane protein of interest or a portion thereof.

[0166] In some embodiments of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a lipid bilayer membrane scaffold protein complex that includes a full-length human transmembrane protein of interest. Splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with one or more blocking agents that correspond to each MSP protein included in the complex and each detectable marker included in the complex, or are attached to the transmembrane protein of interest, such as a His tag or a Flag tag, to allow binding of the blocking agent to antibodies produced by the cells that specifically bind to the epitope located on the blocking agent. The cells are also incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are then incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a full-length human transmembrane protein to allow binding of the full-length transmembrane protein of interest (antigen) presented by the complex to antibodies produced by the remaining unbound cells in the population. The cells are then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin (e.g., Bir-A)-labeled membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed to remove all cells bound to the blocking agent, as well as unbound and excess PE-streptavidin, and the labeled antibodies against the B cell markers can be removed from sorting. The PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells that express antibodies that bind to an epitope on the full-length human transmembrane protein of interest.

[0167] In one embodiment of the method, a non-human animal or a genetically engineered non-human animal, such as a mouse or rat, is immunized with a lipid bilayer membrane scaffold protein complex that includes a full-length human transmembrane protein of interest. Splenocytes are isolated from the immunized non-human animal (e.g., a rat or mouse) and incubated with one or more blocking agents that correspond to each MSP protein included in the complex and each detectable marker included in the complex, or are attached to the transmembrane protein of interest, such as a His tag or a Flag tag, allowing the binding of the blocking agent to antibodies produced by the cells that specifically bind to the epitope located on the blocking agent. The cells are also incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are then incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a portion of the human transmembrane protein, allowing the binding of the portion of the transmembrane protein of interest (antigen) presented by the complex to antibodies produced by the remaining unbound cells in the population. The cells are then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin (e.g., Bir-A)-labeled membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed to remove all cells bound to the blocking agent, as well as unbound and excess PE-streptavidin, and the labeled antibodies against the B cell markers can be removed from sorting. The PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells that express antibodies that bind to an epitope of the portion of the full-length human transmembrane protein of interest presented by the complex.

[0168] In another embodiment of the method, a genetically engineered non-human animal, such as a mouse or rat, that does not express the transmembrane protein of interest from an endogenous gene is immunized with a lipid bilayer membrane scaffold protein complex that includes the full-length non-human transmembrane protein of interest. Splenocytes are isolated from the immunized non-human animal (e.g., rat or mouse) and incubated with one or more blocking agents, which correspond to each MSP protein included in the complex and each detectable marker included in the complex, or are attached to the transmembrane protein of interest, such as a His tag or a FLAG tag, to allow binding of the blocking agent to antibodies produced by the cells that specifically bind to the epitope located on the blocking agent. The cells are also incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are then incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes the full-length non-human transmembrane protein to allow binding of the full-length transmembrane protein of interest (antigen) presented by the complex to antibodies produced by the remaining unbound cells in the population. The cells are then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin (e.g., Bir-A)-labeled membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed to remove all cells bound to the blocking agent, as well as unbound and excess PE-streptavidin, and the labeled antibodies against the B cell markers can be removed from sorting. The PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells that express antibodies that bind to an epitope on the full-length non-human transmembrane protein of interest.

[0169] In some embodiments of the method, a genetically engineered non-human animal, such as a mouse or rat, that does not express a transmembrane protein of interest from an endogenous gene is immunized with a lipid bilayer membrane scaffold protein complex that includes a portion of the full-length non-human transmembrane protein of interest. Splenocytes are isolated from the immunized animal and incubated with one or more blocking agents, which correspond to each MSP protein included in the complex and each detectable marker included in the complex, or are attached to a portion of the non-human transmembrane protein of interest, such as a His tag or a FLAG tag, to allow binding of the blocking agent to antibodies produced by the cells that specifically bind to the epitope located on the blocking agent. The cells are also incubated with a labeled antibody against a B cell marker (e.g., a fluorochrome-labeled anti-IgG antibody) to identify the B cell population. The cells are then incubated with a biotinylated lipid bilayer membrane scaffold protein complex that includes a portion of the full-length non-human transmembrane protein or the full-length non-human transmembrane protein of interest (antigen), to allow binding of the transmembrane protein of interest presented by the complex to antibodies produced by the remaining unbound cells in the population. The cells are then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin (e.g., Bir-A)-labeled membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells can then be washed to remove all cells bound to the blocking agent, as well as unbound and excess PE-streptavidin, and the labeled antibodies against the B cell markers can be removed from sorting. The PE fluorescence and signal (e.g., fluorochrome-labeled anti-IgG signal) from the bound B cells can be detected by FACS to identify and collect a homogenous population of antibody-producing B cells that express antibodies that bind to an epitope on a portion of the full-length non-human transmembrane protein of interest.

[0170] In embodiments of the method that include the use of a blocking agent, the blocking agent may include any molecule, such as, for example, a peptide, a polypeptide, a chemical compound, etc. In such embodiments, the blocking agent may be a polypeptide or peptide corresponding to a portion of a transmembrane protein of interest, a polypeptide or polypeptide corresponding to a portion of an MSP protein, or a detectable marker, such as a His tag or a FLAG tag. In some embodiments, the blocking agent includes a detectable label. In other embodiments, the blocking agent includes a detectable label. In one embodiment, the blocking agent is a peptide or polypeptide portion of a transmembrane protein of interest and is incubated with a population of antibody-producing cells to allow binding of the blocking agent to antibodies produced by the antibody-producing cells that specifically bind to an epitope located on the blocking agent. In one embodiment, the blocking agent is a truncated transmembrane protein of interest. In certain embodiments, the blocking agent is a polypeptide corresponding to one or more extracellular loop domains, the amino-terminal portion and the carboxy-terminal portion of a full-length transmembrane protein of interest. In certain embodiments, the blocking agent may be a polypeptide that includes the amino-terminal portion of a full-length transmembrane protein of interest. In another embodiment, the blocking agent may be a polypeptide that includes the carboxy-terminal portion of a full-length transmembrane protein of interest. In one embodiment, the blocking agent may be a polypeptide comprising an extracellular loop domain of a full-length transmembrane protein of interest. In some embodiments, the blocking agent is a truncated protein comprising the extracellular domain of the transmembrane protein of interest, e.g., the N-terminal extracellular domain, the C-terminal extracellular domain, and / or the extracellular loop domain between one or more membrane spanning domains of the transmembrane protein of interest. In another embodiment, the blocking agent is a peptide or polypeptide portion of an MSP protein, which is incubated with a population of antibody-producing cells to allow binding of the blocking agent to antibodies produced by the antibody-producing cells that specifically bind to an epitope located on the blocking agent.In yet another embodiment, the blocking agent is a peptide or polypeptide detectable marker located in the MSP or transmembrane protein of interest and is incubated with a population of antibody-producing cells to allow binding of the blocking agent to antibodies produced by the antibody-producing cells that specifically bind to an epitope located on the blocking agent.

[0171] The disclosed method includes detecting, sorting, and collecting antibody-producing cells that are bound to an antigen presented by a lipid bilayer membrane scaffold protein complex that includes a transmembrane protein of interest. Detection, sorting, and collection may be combined or performed as separate steps. Detection, sorting, and / or collection may include identifying cells that are bound by the transmembrane protein of interest presented by the complex by detection of one or more detectable labels on the complex. The bound cells can then be separated (i.e., sorted) from unbound cells and selected for further use.

[0172] In some embodiments, the interaction between antibody-producing cells and the lipid bilayer membrane scaffold protein complex that displays the transmembrane protein of interest is detected by the conformational change of the transmembrane protein of interest, the activation or inactivation of the transmembrane protein of interest in cells, or by using detectable labels (e.g., fluorescent molecules, FLAG tags, His-tags) to identify and capture the cells that bind to the lipid bilayer membrane scaffold protein complex that contains the transmembrane protein of interest. Detection can be performed by immunostaining with an antibody specific to the label, or by direct staining with a reagent that binds to the label. Numerous detection kits and techniques are well known in the art.

[0173] In certain embodiments of the method, the cells are washed with buffer for about 5 minutes to about 60 minutes to remove unbound antigens, and then washed with buffer for a total of 10 to 120 minutes, e.g., 3 times for 10 minutes, or 1 time for 30 minutes, 2 to 4 times for 10 to 15 minutes each. In one embodiment, the cells can be washed for a time period including one wash of about 10 minutes, or about 15 minutes or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 55 minutes, or a total of about 60 minutes. In some embodiments, the cells can be washed for a time period including two washes of about 5 minutes, or about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes per wash. Additional wash intervals are contemplated and are essentially equivalent to those described herein.

[0174] In some embodiments, detection, sorting, and collection can be performed using fluorescence-activated cell sorting (FACS) to detect, sort, and select single antibody-producing cells that express transmembrane protein-specific antibodies. Protocols for single cell isolation by flow cytometry are well known (Huang, J. et al., 2013, supra). For example, cells that bind to antigens presented by labeled (e.g., fluorescently labeled) lipid bilayer membrane scaffold protein complexes (or fluorescently labeled streptavidin / biotinylated antigens) can be detected and identified as cells that express antibodies that specifically bind to the antigen (i.e., the transmembrane protein of interest), and then collected into individual wells on a 96-well plate or 384-well plate.

[0175] Once collected, single antibody-producing cells can be grown by common cell culture techniques for subsequent DNA preparation. Alternatively, antibody genes can be directly amplified from single antibody-producing cells and then cloned into DNA vectors.

[0176] Single antibody-producing cells may be selected and collected by alternative methods known in the art, including but not limited to manual single-cell picking, limiting dilution, and B-cell panning of adsorbed antigens, all of which are well known in the art. See, e.g., Rolink et al., J Exp Med (1996) 183:187-194; Lightwood, D. et al., J. Immunol. Methods (2006) 316(1-2):133-43.

[0177] Generation of antibodies from nucleic acid obtained from an antibody-producing cell expressing an antibody against a transmembrane protein of interest. Nucleic acid encoding an antibody or fragment thereof can be isolated from antibody-producing cells generated and obtained using the methods described herein.

[0178] In some embodiments, the nucleic acid encodes a fragment of an antibody, such as a variable domain, a constant domain, or a combination thereof. In certain embodiments, the nucleic acid isolated from the antibody-producing cell encodes a variable domain of an antibody. In some embodiments, the nucleic acid encodes an antibody heavy chain or a fragment thereof. In other embodiments, the nucleic acid encodes an antibody light chain or a fragment thereof.

[0179] In certain instances, the nucleic acid isolated from the antibody-producing cell is expressed in a host cell. For example, the nucleic acid isolated from the antibody-producing cell can be expressed (e.g., cloned and recombinantly replicated) in a host cell, such as a mammalian cell, a bacterial cell, or an insect cell. In some embodiments, the host cell is cultured under conditions to express the nucleic acid, and an antibody or a portion thereof can then be produced and isolated for further use. Methods for making antibodies from isolated nucleic acids are well known in the art, and any such method can be used in conjunction with the present disclosure.

[0180] In some embodiments, host cells containing one or more of the above nucleic acids are cultured under conditions to express the full-length antibody, which can then be produced and isolated for further use. In certain embodiments, the host cells contain nucleic acids encoding the variable domains of an antibody, and the cells are cultured under conditions to express the variable domains. In other embodiments, the host cells contain nucleic acids encoding the variable heavy chain (V H ) domain, and the cell comprises a nucleic acid encoding the V H In another embodiment, the host cell is cultured under conditions to express the variable light chain (V L ) domain, and the cell comprises a nucleic acid encoding the V L In certain embodiments, the host cell is cultured under conditions to express the V domain of the antibody. H Nucleic acids encoding domains and V of antibodies H and a nucleic acid encoding a V domain. H Domains and V L The cells are cultured under conditions that allow expression of the domain.

[0181] In some embodiments, DNA may be isolated from the host in order to recombinantly produce the antibody. Generally, immunoglobulin variable heavy and variable light chains (i.e., V H , V L Genes or nucleic acids encoding Vkappa, Vkappa and Vlambda) can be recovered using RT-PCR protocols with nucleic acids isolated from antibody-producing cells. These RT-PCR protocols are well known and routine in the art, as described, for example, in Wang et al., J. Immunol. Methods (2000) 244:217-225, and as described herein.

[0182] Once recovered, the antibody-encoding gene or nucleic acid can be cloned into IgG heavy and light chain expression vectors and expressed via transfection of host cells. For example, the antibody-encoding gene or nucleic acid can be inserted into a replicable vector for further cloning (amplification of the DNA) or for expression in cells (stable or transient). Many vectors, particularly expression vectors, are available or can be engineered to contain the appropriate regulatory elements required to regulate the expression of the antibody-encoding gene or nucleic acid.

[0183] An expression vector in the context of the present disclosure may be any suitable vector, including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors (nucleic acid sequences that include an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.

[0184] In some embodiments, the nucleic acid molecule is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech (1997) 12:355-59), a compressed nucleic acid vector (e.g., as described in U.S. Pat. No. 6,077,835), or a plasmid vector such as pBR322 or pUC 19 / 18. Such nucleic acid vectors and their uses are well known in the art. See, for example, U.S. Pat. Nos. 5,589,466 and 5,973,972.

[0185] In certain embodiments, the expression vector may be a vector suitable for expression in yeast system. Any vector suitable for expression in yeast system may be adopted. Suitable vectors include, for example, vectors that contain constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase, and PGH. See F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987); and Grant et al., Methods in Enzymol 153,516-544 (1987).

[0186] In certain embodiments, the vector comprises a nucleic acid molecule (or gene) encoding a heavy chain of the antibody and a nucleic acid molecule (or gene) encoding a light chain of the antibody, and the antibody is produced by the antibody-producing cell obtained by the method of the present disclosure. Generally, the vector used comprises an expression vector comprising the described nucleic acid molecule (or gene), which is operably linked to an expression control sequence suitable for expression in the host cell.

[0187] The choice of vector will depend in part on the host cell to be used, which include, but are not limited to, cells of prokaryotic or eukaryotic (generally mammalian) origin.

[0188] It is understood that the full-length antibody nucleic acid sequence or gene may then be cloned into a suitable vector. Alternatively, the Fab region of the isolated antibody may be cloned into a vector along with the constant region of any isotype. Thus, any constant region may be utilized in the construction of the isolated antibody, including IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE heavy chain constant regions, or chimeric heavy chain constant regions. Such constant regions may be obtained from any human or animal species, depending on the intended use of the antibody. Also, the antibody variable region or Fab region may be cloned into a suitable vector for the expression of the protein in other formats, such as ScFv, diabody, etc.

[0189] Thus, the present disclosure also provides a mammalian host cell encoding a nucleic acid molecule comprising a full-length antibody specific to a transmembrane protein of interest, wherein the nucleic acid or gene encoding the antibody was isolated from an antibody-producing cell obtained according to the method of the invention. In one embodiment, the primary antibody-producing cell was a B cell isolated from a heterogeneous population of cells obtained from an immunized mammal.

[0190] In some embodiments, the host cell is a bacterial cell or a yeast cell. In some embodiments, the host cell is a mammalian cell. In other embodiments, the host cell may be, for example, Chinese Hamster Ovary Cells (CHO), such as CHO K1, DXB-11 CHO, Veggie-CHO cells; COS (e.g., COS-7); stem cells; retinal cells; Vero cells; CV1 cells; kidney cells, such as HEK293, 293 EBNA, MSR 293, MDCK, aHaK, BHK21 cells; HeLa cells; HepG2 cells; WI38, MRC 5; Colo25; HB 8065; HL-60; Jurkat or Daudi cells; A431 (epidermal) cells; CV-1, U937, 3T3 or L cells; C127 cells, SP2 / 0, NS-0 or MMT cells, tumor cells, and cell lines derived from any of the aforementioned cells. In certain embodiments, the host cell is a CHO cell. In a particular embodiment, the host cell is a CHO K1 cell.

[0191] The term antibody is known in the art and refers to an antigen-binding protein of the immune system. The term "antibody" as referred to herein includes full-length antibodies having an antigen-binding region, and any fragment thereof, in which the "antigen-binding portion" or "antigen-binding region" is retained, or includes a single chain, e.g., a single-chain variable fragment (scFv) thereof. A "full-length antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant (CH) region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (V L ) and the light chain constant CL region. The light chain constant region is composed of one domain, CL. H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0192] As shown herein, the present disclosure can be used to obtain antibodies that bind to transmembrane proteins of interest.Antibodies obtained from the antibody-producing cells described herein can be characterized using methods known in the art.For example, the binding affinity, specific epitope recognition ability or functional ability of any of the antibodies generated herein can be determined.

[0193] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule for its binding partner is generally measured by the dissociation equilibrium constant (KD or K D ) can be expressed as: K D There is an inverse relationship between the molar (M) value and binding affinity, K D The smaller the value (M), the higher the binding affinity of the molecule for its binding epitope.

[0194] The term "higher affinity" or "high affinity" generally refers to an antibody that binds to an antigen more strongly and / or faster and / or remains bound longer. Generally, a high affinity antibody requires a lower concentration (M) of antigen to achieve the desired effect due to the strong binding interaction. Conversely, the terms "low affinity" and "lower affinity" are terms used to reflect weaker binding, such as a reduced ability to form an interaction between a molecule and its binding partner, compared to other binding molecules (e.g., antibodies). Thus, a low affinity binding molecule has a larger K D These antibodies have a low affinity for the antigen and / or tend to bind antigen slowly and dissociate easily.

[0195] The term "kd" (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody, Ig, antibody binding fragment or molecular interaction. This value is also called the k-off value.

[0196] The term "ka" (M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody, Ig, antibody binding fragment or molecule interaction.

[0197] "KD" or "K DThe term "(M)" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, or the equilibrium dissociation constant of an antibody, Ig, antibody binding fragment, or molecule interaction. The equilibrium dissociation constant is obtained by dividing ka by kd.

[0198] Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure. For example, the binding affinity of an antibody to an antigen can be measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA.

[0199] In some embodiments, binding of the antibody to a transmembrane protein of interest confers functional activity in cells in which the transmembrane protein is expressed (in vitro, ex vivo, or in vivo), or binding of the antibody to the transmembrane protein of interest modulates the normal functional activity of the transmembrane protein of interest in the presence or absence of an endogenous ligand for the transmembrane protein of interest.

[0200] In certain embodiments, the antibody against the transmembrane protein of interest is an antagonist of the transmembrane protein function. An antagonist antibody refers to an antibody that is directed against the active site of the antigen (i.e., the transmembrane protein of interest) and can inhibit the activity of the natural ligand for the transmembrane protein or the transmembrane protein itself. For example, the binding of the antibody to the antigen reduces or inhibits the endogenous function of the transmembrane protein of interest. In one non-limiting example, an antagonist antibody can bind to the transmembrane protein of interest and regulate the transmembrane protein function by interfering with the ligand binding to the transmembrane protein, receptor activation, or the like.

[0201] In a further specific embodiment, the antibody against the transmembrane protein of interest is a transmembrane protein antagonist. An agonist antibody refers to an antibody that can activate the transmembrane protein of interest (i.e., antigen) in the absence of the natural ligand itself, and an agonist antibody can induce the functional activity of the transmembrane protein of interest. For example, the binding of the antibody to the antigen induces or increases the intrinsic function of the transmembrane protein of interest. In one non-limiting example, an agonist antibody can bind to the transmembrane protein of interest and can regulate the transmembrane protein function by activating the protein or receptor, for example, by changing the structure of the protein.

[0202] Assays for measuring antibody binding and transmembrane protein function are well known to those of skill in the art, e.g., assays for measuring transmembrane protein internalization, conformational changes, phosphorylation, ligand binding, etc. are well known in the art.

[0203] The present specification is further illustrated by the following examples, which should not be construed as limiting. All cited references, including literature references, issued patents, and published patent applications cited throughout this application, are expressly incorporated herein by reference in their entirety. EXAMPLES

[0204] Example 1. Generation and harvesting of antibody-producing cells. For comparison, several immunization campaigns were initiated to generate immune responses in mice and generate cells that produced antibodies against the antigen of interest (antibody-producing cells), as shown in Table 1. The antibody-producing cells were then harvested according to the methods described herein.

[0205] Generally, mice are immunized by injecting an immunogen, such as DNA or a part thereof that codes for a transmembrane protein of interest, a purified transmembrane protein of interest or a part thereof, a lipid bilayer membrane scaffold protein complex or a part thereof that contains a transmembrane protein of interest or a part thereof, a virus-like particle (VLP) that contains a transmembrane protein of interest or can express DNA that codes for a transmembrane protein of interest, or a combination thereof.In some cases, the immunogen is a human transmembrane protein or a DNA that codes for it.In other cases, mice are immunized with a chimeric transmembrane protein of interest, a truncated form of a transmembrane protein, a modified transmembrane protein of interest, or a DNA that codes for it.

[0206] The transmembrane proteins used in the methods described herein were produced as follows. In general, the nucleotide sequence encoding the transmembrane protein was modified to include a FLAG tag and a histidine tag (10x-His tag) attached to the carboxy terminus of the transmembrane protein. The modified nucleotide sequence was expressed in Sf9 cells or Expi293 cells (Thermo Fisher Scientific). The cells were then solubilized using N-dodecyl-β-D-maltoside (DDM) detergent and centrifuged to obtain a detergent-soluble fraction containing the modified transmembrane protein. The modified protein was then isolated from the detergent-soluble fraction by affinity purification using anti-FLAG affinity beads, which bound to the modified transmembrane protein. The affinity beads and protein were then washed, and the bound transmembrane protein was eluted therefrom and collected. The isolation of the purified transmembrane protein was verified by SDS-PAGE gel electrophoresis and Western blot.

[0207] A lipid bilayer membrane scaffold protein complex was formed incorporating the purified transmembrane protein. The purified transmembrane protein was included in a detergent mixture consisting of DDM and cholesteryl hemisuccinate tris salt (CHS). This transmembrane protein and detergent mixture was combined with 1 to 130 membrane scaffold protein to lipid mixture containing modified MSP1E3D1 membrane scaffold protein, which contains a Bir-A tag at the carboxy terminus for biotinylation, and phosphatidylcholine (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) lipid dissolved in sodium cholate detergent buffer, to create a final mixture consisting of transmembrane protein, detergent, membrane scaffold protein lipid and buffer containing a ratio of 1 transmembrane protein to 20 membrane scaffold protein. The detergent was then extracted from the final mixture using beads, facilitating the construction of a disc-shaped lipid bilayer membrane scaffold protein complex integrating a single transmembrane protein of interest. The disc-shaped lipid bilayer membrane scaffold protein complexes incorporating the transmembrane protein of interest were separated from the disc-shaped lipid bilayer membrane scaffold protein complexes not containing the transmembrane protein of interest using 10x-His tag affinity purification and / or molecular sieve chromatography to obtain the disc-shaped lipid bilayer membrane scaffold protein complexes incorporating the transmembrane protein of interest for further use.

[0208] The mice immunized were varied. In certain cohorts, the mice were genetically engineered so that the transmembrane protein of interest was not expressed from the endogenous gene. In other cohorts, the mice were wild-type mice and expressed the mouse homologue of the transmembrane protein of interest. In some cohorts, the mice were genetically engineered as described in U.S. Patent Nos. 8,502,018, 8,642,835, 8,697,940, 8,791,323, 9,226,484, and WO2019 / 113065, all of which are incorporated herein by reference in their entirety. In some cohorts, the genetically engineered mice immunized were VELOCIMMUNE® mice (Regeneron Pharmaceuticals, Inc., Tarrytown, NY), as described, for example, in U.S. Pat. Nos. 8,502,018 and / or 8,642,835, the entire contents of each of which are incorporated herein by reference. Generally, the VELOCIMMUNE® mice used for immunization contain DNA encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain variable region, and may lack an endogenous mouse gene encoding a transmembrane protein of interest. In certain cohorts, VELOCIMMUNE® mice containing a humanized IgH locus and / or a humanized Igκ locus were immunized. In some cohorts, the immunogen was injected into genetically engineered mice containing DNA encoding a human immunoglobulin heavy chain and an immunoglobulin lambda light chain (Igλ) variable region that lack an endogenous mouse gene encoding a transmembrane protein of interest.

[0209] Antibody immune responses were monitored by cell binding assays using cells engineered to express the transmembrane protein of interest or a portion thereof. Once the desired immune response was identified in the immunized mice, their spleens were harvested. Splenocytes were obtained and red blood cells were removed by lysis.

[0210] In the examples where hybridomas were generated and used, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines, which were then screened and selected to identify cell lines that produced antibodies against the transmembrane protein of interest.

[0211] Antibody-producing cells were also isolated from antigen-positive B cells. Once mice were immunized, the spleens were harvested. Splenocytes were obtained and red blood cells were removed by lysis. Splenocytes were then stained with fluorochrome-labeled antibodies specific for B cell surface markers, such as IgG, to identify B cells.

[0212] In general, a population of antibody-producing B cells is identified by contacting the B cells with a known sorting agent, such as a synthesized transmembrane protein or portion thereof, a chimeric transmembrane protein of interest, a segmented transmembrane protein of interest, a transmembrane protein of interest in a VLP or exosome, or a lipid bilayer membrane scaffold protein complex incorporating a transmembrane protein of interest or portion thereof, allowing the transmembrane protein of interest to bind to an antibody present on the surface of the B cells. The bound B cells are then collected using FACS to obtain a population of antibody-producing B cells expressing an antibody against the transmembrane protein of interest. In samples without complexes (controls), sorting was performed with peptides or virus-like particles (VLPs) via FACS to obtain antibody-producing B cells from a heterogeneous B cell population. In samples with complexes, sorting was performed with fluorescently labeled complexes via FACS to obtain antibody-producing B cells. Here, the B cells were incubated with a biotinylated lipid bilayer membrane scaffold protein complex containing the transmembrane protein of interest. The cells were then washed to remove unbound complexes. The cells were then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins of the lipid bilayer membrane scaffold protein complex. The cells were then washed with phosphate-buffered saline (PBS) to remove excess PE-streptavidin. PE fluorescence of the B cell samples was analyzed by FACS to identify cell populations expressing antibodies that bind to the transmembrane protein of interest.

[0213] Example 2. Generation of antibodies from a population of antibody-producing cells. For comparison, antibodies were isolated for screening by either conventional hybridoma technology or cell sorting with or without lipid bilayer membrane scaffold protein complex. Single cells from the antibody-producing B cell population were isolated in individual wells on a 384-well plate. RT-PCR of antibody genes from these isolated antibody-producing B cells was performed according to the method described in Wang and Stollar Journal of Immunological Methods (2000) 244:217-225, the entire contents of which are incorporated herein by reference. Briefly, cDNA of each single antibody-producing B cell was synthesized via reverse transcriptase (RT) reaction (Superscript™ III, Invitrogen). Then, each resulting RT product was divided and transferred to two corresponding wells on two 384-well plates. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the human IgG heavy chain variable region leader sequence and a 3' primer specific to the mouse heavy chain constant region to form an amplicon. The amplicons were then amplified again by PCR using a 5' degenerate primer set specific to framework 1 of the human IgG heavy chain variable region sequence and a 3' degenerate primer set specific to framework 4 of the human IgG heavy chain variable region sequence. The other set of resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the human kappa or lambda light chain variable region leader sequence and a 3' primer specific to the mouse kappa or lambda light chain constant region to form an amplicon. The amplicons were then amplified again by PCR using a 5' degenerate primer set specific to framework 1 of the human kappa or lambda light chain variable region sequence and a 3' degenerate primer set specific to framework 4 of the human kappa or lambda light chain variable region sequence. The heavy and light chain PCR products were cloned into antibody vectors containing the IgG1 heavy chain constant region and the kappa light chain constant region, respectively. Recombinant human IgG1 antibodies were produced by transient transfection of CHO cells.

[0214] The primary screening of antibodies obtained from antibody-producing cells was tested for cell binding. The dissociation constant of binding between the transmembrane protein of interest and each antibody was determined on a Biacore™ T200 (GE Healthcare). The results of several campaigns under various conditions showed that cell sorting using lipid bilayer membrane scaffold protein complexes that display the transmembrane protein of interest was the most successful in identifying antibodies that bind to the transmembrane protein of interest. See Table 1. [Table 1] TIFF2024501796000002.tif156168

[0215] Example 3. Detection of binding between an antibody and a transmembrane protein of interest encompassed by a lipid bilayer membrane scaffold protein complex. In this example, a nucleotide sequence encoding an exemplary human transmembrane protein (GPCR1) was modified to attach a FLAG-tag and a His-tag (10x-His tag) to the carboxy-terminus of the GPCR1 protein sequence, and the modified nucleotide sequence was expressed intracellularly and purified as described in Example 1. The exemplary GPCR1 transmembrane protein of interest was then incorporated into a lipid bilayer membrane scaffold protein complex, and the lipid bilayer membrane scaffold protein complex encompassing the GPCR1 transmembrane protein of interest was purified as described in Example 1.

[0216] The binding ability of known anti-GPCR1 antibodies to lipid bilayer membrane scaffold protein complexes encompassing the GPCR1 transmembrane protein of interest was confirmed by the octet assay shown in Figures 1A and 1B. In detail, 38 μg / mL of disk-shaped lipid bilayer membrane scaffold protein complexes containing GPCR1 transmembrane protein or 10 μg / mL of control disk-shaped lipid bilayer membrane scaffold protein complexes without GPCR1 (empty complex) were provided to plates with immobilized anti-GPCR1 antibody (positive control AB) or isotype control antibody (negative control AB).

[0217] Binding was detected between the positive control (anti-GPCR1) antibody and a discoid lipid bilayer membrane scaffold protein complex containing the GPCR1 transmembrane protein (0.6 nm), and no binding was detected between the anti-GPCR1 antibody and the empty complex (-0.05 nm), between the negative control antibody and a discoid lipid bilayer membrane scaffold protein complex containing the GPCR1 transmembrane protein (-0.06 nm), or between the negative control antibody and the empty complex (-0.03 nm). See Figure 1A. This confirms that the transmembrane protein of interest presented by the complex but not by the complex itself binds to the antibody directed against the transmembrane protein of interest.

[0218] To confirm that biotinylated lipid bilayer membrane scaffold protein complexes presenting a transmembrane protein of interest can be detected and isolated from control lipid bilayer membrane scaffold protein complexes, biotinylated control lipid bilayer membrane scaffold protein complexes (empty complexes), biotinylated lipid bilayer membrane scaffold protein complexes presenting GPCR1 transmembrane protein (complexed with GPCR1) or control lipid bilayer membrane scaffold protein complexes, biotinylated lipid bilayer membrane scaffold protein complexes (empty complexes) were fed to a plate with streptavidin linked to allow the streptavidin to bind to the Bir-A tag attached to the membrane scaffold protein of the complex. The plate was then incubated with either 50 μg / mL of anti-GPCR1 antibody (positive control AB) or 50 μg / mL of isotype control antibody (negative control AB). See Figure 1B.

[0219] First, Figure 1B shows that the biotinylated lipid bilayer membrane scaffold protein complexes presenting the GPCR1 transmembrane protein of interest and the biotinylated control lipid bilayer membrane scaffold protein complexes were detected and isolated by streptavidin bound to the Bir-A tag attached to each of the membrane scaffold proteins of the complexes. This is shown by detecting the binding between the linked streptavidin and both the biotinylated disc-shaped lipid bilayer membrane scaffold protein complexes containing the GPCR1 transmembrane protein (3.0 nm) and the biotinylated empty complex (0.69 nm).

[0220] Binding was detected between the positive control (anti-GPCR1) antibody and a biotinylated discoid lipid bilayer membrane scaffold protein complex (0.69 nm) containing the GPCR1 transmembrane protein linked to a streptavidin plate, but no binding was detected between the anti-GPCR1 antibody and a biotinylated empty complex (-0.04 nm), the negative control antibody and a biotinylated discoid lipid bilayer membrane scaffold protein complex containing the transmembrane protein (-0.05 nm), or the negative control antibody and an empty biotinylated complex (-0.06 nm). Referring to Figure 1B, the biotinylated lipid bilayer membrane scaffold protein complex linked to a streptavidin plate presents the transmembrane protein of interest and specifically binds to the anti-GPCR1 antibody but not to the control antibody.

[0221] Example 4. Obtaining antibody-producing cells expressing antibodies that specifically bind to an exemplary transmembrane protein of interest. Nucleotide sequences encoding the first, second and third exemplary human transmembrane proteins were modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of the transmembrane proteins, and the modified nucleotide sequences were expressed in cells and purified as described in Example 1. Lipid bilayer membrane scaffold protein complexes encompassing either the first, second or third exemplary transmembrane proteins of interest were generated using Bir-A tagged membrane scaffold protein and purified as described in Examples 1 and 3.

[0222] Splenocytes were harvested from genetically modified VELOCIMMUNE® mice containing a humanized IgH locus and / or a humanized IIgκ locus lacking the endogenous mouse gene encoding the transmembrane protein of interest. Cells were isolated from unimmunized control mice or genetically modified mice immunized by injection of DNA encoding the first transmembrane protein of interest (GPCR1), the second transmembrane protein of interest (GPCR2), or the third transmembrane protein of interest (GPCR3), where each mouse was genetically modified. Splenocytes collected from each mouse were stained with a fluorescent label for a B cell marker (i.e., anti-IgG antibody) and simultaneously incubated with 0.2 mg / mL to 5.0 mg / mL of a biotinylated lipid bilayer membrane scaffold protein complex containing the transmembrane protein incorporated therein. The cells were then washed to remove unbound complex. The cells were then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins of the lipid bilayer membrane scaffold protein complex. The cells were then washed with phosphate-buffered saline (PBS) to remove excess PE-streptavidin. PE fluorescence of cell populations from control and immunized mice was detected by FACS to identify B cell populations expressing antibodies that bind to the transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex. See Figure 2A-F.

[0223] Only two B cells out of one million control cells bound non-specifically to the GPCR1 transmembrane protein presented by the biotinylated lipid bilayer membrane scaffold protein complex (Figure 2A, rectangle), indicating that the lipid bilayer membrane scaffold protein complex containing the transmembrane protein of interest is a highly specific sorting agent for detecting and isolating cells producing antibodies against the transmembrane protein antigen. In comparison, 11 out of one million antibody producing B cells that bound to the first transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex were detected using the method of the present invention (Figure 2B, rectangle).

[0224] As shown in Figure 2C, two B cells out of one million splenocytes obtained from a control mouse were detected as non-specific binders to the biotinylated lipid bilayer membrane scaffold protein complex presenting the second transmembrane protein of interest. Seventy-eight out of one million B cells expressing an antibody specific for a second exemplary GPCR transmembrane protein of interest (GPCR2) were detected using the method of the present invention, as shown in Figure 2D (rectangle).

[0225] In yet another example, only 11 B cells non-specifically bound to a biotinylated lipid bilayer membrane scaffold protein complex presenting a third transmembrane protein of interest were obtained from 1 million splenocytes collected from a control mouse (see Figure 2E), whereas 65 / 1 million B cells expressing a primary antibody specific for a third exemplary GPCR transmembrane protein of interest (GPCR3) were detected and obtained from splenocytes harvested from an immunized genetically modified mouse.

[0226] Example 5. Comparison of known cell sorting strategies with a sorting method involving the use of lipid bilayer membrane scaffold protein complexes. To compare the utility of methods for obtaining antibody-producing cells known in the art with the inventive method disclosed herein, DNA encoding an exemplary human transmembrane protein of interest was purified according to Example 1. For immunization of mouse cohorts with DNA encoding the transmembrane protein of interest, the purified human DNA was injected into genetically engineered VELOCIMMUNE® mice that do or do not express the endogenous mouse gene encoding the transmembrane protein of interest. For the cohorts immunized with the transmembrane protein of interest, a nucleotide sequence encoding a first exemplary human transmembrane protein was expressed in cells, purified as described in Example 1, and administered by injection directly into genetically engineered VELOCIMMUNE® mice that do or do not express the endogenous mouse gene encoding the transmembrane protein of interest.

[0227] In certain cohorts, mice were immunized with VLPs. Plasmids containing DNA encoding the human transmembrane protein of interest were generated and expressed in the viral cells. The lipoparticles self-assembled within the viral cells and were isolated and purified from the viral cells upon budding by pegylation (PEG) precipitation, followed by isopycnic centrifugation and fractionation of the viral cell media. The purified VLPs were injected into mice for immunization using known techniques.

[0228] For comparison, genetically engineered VELOCIMMUNE® mice containing DNA encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain variable region were immunized by injection as described in Table 2 with (VI) or without (VI-KO) an endogenous mouse gene encoding a transmembrane protein of interest.

[0229] A population of antibody-producing B cells was then isolated using the sorting strategy described in Table 2. In detail, splenocytes were collected from the immunized genetically modified mice. The collected splenocytes were stained with a fluorescent label to a B cell marker (i.e., anti-IgG antibody) and simultaneously incubated with 0.2 mg / mL to 5.0 mg / mL of the sorting agent biotinylated transmembrane protein (protein) of interest, or 0.2 mg / mL to 5.0 mg / mL of a biotinylated lipid bilayer membrane scaffold protein complex (complex with TMB) encompassing the transmembrane protein of interest, or 2.0 mg / mL to 20 mg / mL of a biotin-labeled VLP sorting agent (VLP) described in Table 2. The cells were then washed to remove unbound sorting agent. Subsequently, the cells were incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin (Bir-A) labeled sorting agents. Cells were washed with phosphate-buffered saline (PBS) to remove excess PE-streptavidin, and the PE fluorescence of a sample of B cells was analyzed by FACS to identify B cells in the heterogeneous cell population that expressed antibodies that bound to the transmembrane protein of interest presented by the sorting agent.

[0230] Single antibody producing B cells that bind to the transmembrane protein of interest were then isolated into individual wells on a 384-well plate as described in Example 2. RT-PCR of antibody genes from these B cells was performed according to the method described in Wang and Stollar Journal of Immunological Methods (2000) 244:217-225. Briefly, cDNA of each single B cell was synthesized via a reverse transcriptase (RT) reaction (Superscript™ III, Invitrogen). Each resulting RT product was then split and transferred to two corresponding wells on two 384-well plates. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the human IgG heavy chain variable region leader sequence and a 3' primer specific to the mouse heavy chain constant region to form an amplicon. The amplicons were then amplified again by PCR using a 5' degenerate primer set specific to framework 1 of the human IgG heavy chain variable region sequence and a 3' degenerate primer set specific to framework 4 of the human IgG heavy chain variable region sequence. The other set of resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the human kappa or lambda light chain variable region leader sequence and a 3' primer specific to the mouse kappa or lambda light chain constant region to form an amplicon. The amplicons were then amplified again by PCR using a 5' degenerate primer set specific to framework 1 of the human kappa or lambda light chain variable region sequence and a 3' degenerate primer set specific to framework 4 of the human kappa or lambda light chain variable region sequence. The heavy and light chain PCR products were cloned into antibody vectors containing the IgG1 heavy chain constant region and the kappa light chain constant region, respectively. Recombinant human IgG1 antibodies were produced by transient transfection of CHO K1 cells and analyzed for their ability to bind to transmembrane proteins of interest. [Table 2]

[0231] Table 2 shows that the method of obtaining antibody-producing cells displaying a transmembrane protein utilizing a lipid bilayer membrane scaffold-protein complex detects more antibody-producing cells expressing antibodies that specifically bind to the transmembrane of interest when compared to other sorting agents, independent of the immunogen used.

[0232] Cells sorted with purified transmembrane protein sorting agents obtained from genetically modified mice expressing an endogenous mouse gene encoding a transmembrane protein of interest (VI) produced more antibodies that bound to the transmembrane protein of interest when compared to those obtained from genetically modified mice that did not express an endogenous mouse gene encoding a transmembrane protein of interest (VI-KO) and were sorted with a lipid bilayer membrane scaffold protein complex that presented the transmembrane protein. However, upon analysis, the antibodies generated from the VI mice were not functional binders. We show that the method of obtaining antibody-producing cells presenting a transmembrane protein using a lipid bilayer membrane scaffold protein complex obtains antibodies that bind to functionally relevant epitopes of the transmembrane protein.

[0233] Example 6. Generation and isolation of antibodies that bind to a second exemplary GPCR transmembrane protein of interest. The nucleotide sequence encoding the second example of the GPCR transmembrane protein of interest (GPCR2) was modified as described in Example 1 to include a FLAG tag and a 10x-His tag attached to the carboxy-terminus of the GPCR2 protein. In addition, the following stabilizing mutations were introduced into the transmembrane protein: amino acid substitutions: A2a-T4L-delta, as described in Veli-Pekka Jaakola et al., Science. 2008-11-21, 322(5905) pp.1211-1217, which is expressly incorporated herein by reference in its entirety. The modified GPCR2 nucleotide sequence was cloned into an expression vector and expressed in Sf9 cells. The cells were then solubilized, and the modified GPCR2 transmembrane protein was obtained and purified as in Example 1.

[0234] Next, disc-shaped lipid bilayer membrane scaffold protein complexes containing the modified human GPCR2 transmembrane protein were generated as described in Example 1 and herein.

[0235] Genetically modified GPCR2 knockout mice, i.e., VELOCIMMUNE® mice that contain a humanized IgH locus and a humanized Igκ locus that also lack the endogenous mouse gene encoding the transmembrane protein of interest, were immunized by injection of exogenous DNA encoding a modified human GPCR2 protein containing stabilizing mutations (DNA immunogen) or by injection of a modified human GPCR2 protein containing stabilizing mutations (protein immunization) as shown in Table 3.

[0236] Splenocytes were collected from each mouse and stained with a fluorescent label for a B cell marker (i.e., anti-IgG antibody) and simultaneously incubated with 0.2 mg / mL to 5.0 mg / mL of a biotinylated lipid bilayer membrane scaffold protein complex containing a transmembrane protein incorporated therein. The cells were then washed to remove unbound complexes. The cells were then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotinylated (e.g., Bir-A) membrane scaffold proteins of the lipid bilayer membrane scaffold protein complex. The cells were then washed with phosphate-buffered saline (PBS) to remove excess PE-streptavidin. PE fluorescence of cell populations from control and immunized mice was detected by FACS to identify B cell populations expressing antibodies that bind to the second exemplary GPCR transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex.

[0237] Single antibody producing B cells that bound to the transmembrane protein of interest were then isolated into individual wells on a 384-well plate, and antibody encoding DNA was isolated from each cell and used to generate the antibody as described in Example 2 for further analysis.

[0238] FIG. 3 shows that the inventive method for obtaining antibody-producing cells detects antibody-producing cells that express an antibody that specifically binds to a transmembrane of interest, regardless of the type of immunogen used. In particular, antibody-producing cells were obtained from genetically engineered mice immunized with either DNA (DNA) encoding a transmembrane protein (TMP) or DNA encoding a purified transmembrane protein (TMP) of interest using a biotinylated lipid bilayer membrane scaffold protein complex presenting the transmembrane protein of interest according to the disclosed method. Antibodies were generated for screening as described in Example 2. Full-length transmembrane proteins of the antigen of interest were expressed in 293 cells (TMB overexpressing) using standard transfection techniques, and the antigen-expressing 293 cells were compared to control 293 cells (parental cells) that were not transfected with DNA encoding the exemplary transmembrane protein of interest. The cells were then incubated with the antibody to allow binding of the antigen on the surface of the cells to the antibody that specifically binds to the exemplary transmembrane protein of interest. The MSD (Meso Scale Diagnostics) immunoassay was performed according to the manufacturer to identify cell-binding antibodies generated from antibody-producing cells obtained from five different immunized mice. Mice 1, 3, 4 and 5 were immunized with purified human GPCR2 transmembrane protein and mouse 2 was immunized with DNA encoding human GPCR2 transmembrane protein. Antibodies above the dashed line were able to bind to the transmembrane protein of interest. In contrast, antibodies below the dashed line were weak binders or were unable to bind to the transmembrane protein of interest, as shown by comparison with positive (squares) and negative (triangles) control antibodies.

[0239] The results showed that the antibody-producing cells obtained using the methods of the present invention were able to produce sufficient amounts of antibodies capable of binding to a transmembrane protein of interest, regardless of whether the mice were immunized with DNA encoding the transmembrane protein of interest or with a purified transmembrane protein of interest.

[0240] As shown in Table 3, B cells were obtained from each immunized mouse using a biotinylated lipid bilayer membrane scaffold protein complex presenting the transmembrane protein (a complex containing TMB), antibodies were generated from a subset of the collected B cells, and the antibodies were tested by immunoassay for their ability to bind to the antigen of interest. Antibody-producing cells expressing antibodies that specifically bind to the transmembrane protein of interest (antigen) were obtained in abundance using the methods of the present invention, regardless of whether the mouse was immunized with DNA encoding the transmembrane protein or with purified transmembrane protein. [Table 3]

[0241] Example 7. Obtaining antibody-producing cells expressing antibodies that bind to specific domains of an exemplary transmembrane protein of interest. The methods of the present invention have also been used to generate antibodies that specifically bind to epitopes located in specific domains of a transmembrane protein of interest.

[0242] In a first example, a nucleotide sequence encoding a full-length human transmembrane protein (full-length TMB) was modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of the transmembrane protein, and purified and isolated as described in Example 1. Additionally, a nucleotide sequence encoding a truncated human transmembrane protein that is missing the N-terminal extracellular portion was further modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of the transmembrane protein, and the modified nucleotide sequence was expressed in cells and purified as described in Example 1. A lipid bilayer membrane scaffold protein complex that includes truncated TMB was generated using Bir-A-labeled membrane scaffold protein and purified as described in Examples 1 and 3. Here, the truncated TMB protein includes each of the extracellular loop domain, the transmembrane domain, and the intracellular C-terminal domain of the wild-type human transmembrane protein, but does not include the extracellular N-terminal domain. Thus, the lipid bilayer membrane scaffold protein complex that includes truncated TMB presents only the extracellular loop domain of the wild-type human transmembrane protein to cells.

[0243] Genetically engineered VELOCIMMUNE® mice containing DNA encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain variable region were immunized with DNA encoding full-length TMB. Immune responses were monitored and splenocytes were harvested from the immunized mice. Splenocytes collected from each mouse were stained with a fluorescent label for B cell markers (i.e., anti-IgG antibodies) and simultaneously incubated with 0.2 mg / ml to 5.0 mg / mL of a biotinylated lipid bilayer membrane scaffold protein complex that displays cleaved TMB incorporated therein to allow binding of epitopes on the extracellular domain of cleaved TMB to antibodies on the surface of B cells. The cells were then washed to remove unbound complexes. The cells were then incubated with PE-streptavidin to allow binding of streptavidin to each of the biotinylated (e.g., Bir-A) membrane scaffold proteins of the lipid bilayer membrane scaffold protein complex. The cells were then washed with phosphate-buffered saline (PBS) to remove excess PE-streptavidin. PE fluorescence of cell populations collected from mice was detected by FACS to identify B cell populations expressing antibodies specific to the extracellular loop domains of transmembrane proteins of interest.

[0244] Single antibody-producing B cells that bound to the transmembrane protein of interest were then isolated into individual wells on a 384-well plate, antibody encoding DNA was isolated from each cell, and antibodies were generated as described in Example 2 for further analysis.

[0245] As shown in FIG. 4, antibodies that bind to epitopes located in the extracellular loop domains of an exemplary transmembrane protein of interest were obtained using the methods of the invention (boxed).

[0246] In another example, a nucleotide sequence encoding a full-length human transmembrane protein (full-length TMB) is modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy-terminus of the transmembrane protein, purified and isolated as described in Example 1. Additionally, a nucleotide sequence encoding a portion of a human transmembrane protein (a TMB fragment) is generated, and the TMB fragment nucleotide sequence is expressed in a cell and purified as described in Example 1, where the TMB fragment polypeptide TMB corresponds to the N-terminal portion of the full-length transmembrane protein of interest.

[0247] A lipid bilayer membrane scaffold protein complex encompassing full-length TMB was generated using Bir-A tagged membrane scaffold protein and purified as described in Examples 1 and 3.

[0248] In general, genetically engineered VELOCIMMUNE® mice containing DNA encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain variable region are immunized with DNA encoding full-length TMB or purified with full-length TMB. The immune response is monitored and splenocytes are harvested from the immunized mice. Splenocytes harvested from each mouse are incubated with TMB fragments to allow binding between the TMB fragments and antibodies on the cell surface specific for epitopes located at the N-terminus of full-length TMB. This step effectively blocks all antibody-producing cells expressing antibodies specific for epitopes in the N-terminal domain of full-length TMB from binding to full-length TMB presented by the biotinylated lipid bilayer membrane scaffold protein complex containing embedded full-length TMB. The cells are then stained with a fluorescent label for a B cell marker (i.e., anti-IgG antibody) to identify antibody-producing B cells in the splenocyte population, and incubated with 0.2 mg / mL to 5.0 mg / mL of a biotinylated lipid bilayer membrane scaffold protein complex encompassing full-length TMB to allow binding of epitopes on the extracellular loop domain of full-length TMB with antibodies on the surface of the antibody-producing B cells. The cells are then washed to remove unbound complexes. The cells are incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells are then washed in phosphate-buffered saline (PBS) to remove excess PE-streptavidin and cells bound to TMB fragments from sorting. PE fluorescence of the cell population collected from the mouse is detected by FACS to identify a population of antibody-producing B cells expressing antibodies specific to the extracellular loop domain of the transmembrane protein of interest.

[0249] Single antibody-producing B cells that bind to the transmembrane protein of interest are then isolated into individual wells on a 384-well plate, and the antibody encoding DNA is isolated from each cell and used to generate antibodies as described in Example 2 for further analysis.

[0250] In another example, a chimeric transmembrane protein of interest, comprised of a portion of a human transmembrane protein of interest and a portion of a mouse homologue of the transmembrane protein of interest, is used to generate antibodies that specifically bind to epitopes located in specific domains of the transmembrane protein of interest.

[0251] Here, the chimeric transmembrane protein is made by generating a nucleotide sequence encoding a portion of a human transmembrane protein of interest operably linked to a portion of a mouse homolog of the transmembrane protein of interest, and further modifying the nucleotide sequence to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of the chimeric transmembrane protein. The nucleotide sequence is then purified and isolated as described in Example 1. Furthermore, the nucleotide sequence encoding the chimeric transmembrane protein is expressed intracellularly and purified as described in Example 1 to generate a labeled chimeric transmembrane protein of interest (chimeric TMB). In this example, the chimeric transmembrane protein of interest has an N-terminus corresponding to the mouse homolog of the transmembrane protein and an extracellular loop domain, a transmembrane domain, and an intracellular C-terminal domain of the human transmembrane protein of interest.

[0252] Furthermore, a nucleotide sequence encoding a wild-type human transmembrane protein (human TMB) is modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of the transmembrane protein, allowing it to be purified and isolated, and then the nucleotide sequence encoding the human transmembrane protein is expressed in cells and purified as described in Example 1.

[0253] Lipid bilayer membrane scaffold protein complexes including human TMB or chimeric TMB are generated using Bir-A tagged membrane scaffold proteins and purified as described in Examples 1 and 3.

[0254] Genetically modified VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy chain (IgH) and human immunoglobulin light chain variable regions are immunized with human TMB or DNA encoding purified human TMB protein. Immune responses are monitored and splenocytes are harvested from the immunized mice. Splenocytes collected from each mouse are stained for B cell markers (i.e., anti-IgG antibodies) with fluorescent labels and simultaneously incubated with a biotinylated lipid bilayer membrane scaffold protein complex encompassing the chimeric TMB, allowing binding between epitopes on the human portion of the chimeric-TMB presented by the complex and antibodies on the surface of the B cells. The cells are then washed to remove unbound complexes.

[0255] The cells are incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells are then washed in phosphate-buffered saline (PBS) to remove excess PE-streptavidin and cells bound to TMB fragments from sorting. PE fluorescence of the cell population collected from the mouse is detected by FACS to identify a population of antibody-producing B cells that express antibodies specific to the extracellular loop domain of the transmembrane protein of interest.

[0256] This immunization and sorting strategy effectively removes all antibody-producing B cells that express antibodies specific for epitopes of human-TMB located in the mouse portion of the chimeric TMB.

[0257] In another immunization and sorting strategy, genetically modified VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy chain (IgH) and human immunoglobulin light chain variable regions are immunized with DNA encoding chimeric TMB or purified chimeric TMB protein. Immune responses are monitored and splenocytes are harvested from the immunized mice. Splenocytes collected from each mouse are stained with a fluorescent label for a B cell marker (i.e., anti-IgG antibody) and simultaneously incubated with a biotinylated lipid bilayer membrane scaffold protein complex that encompasses human TMB, allowing binding between epitopes located on the extracellular portion of human-TMB presented by the complex and antibodies on the surface of B cells. The cells are then washed to remove unbound complexes.

[0258] The cells are incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells are then washed in phosphate-buffered saline (PBS) to remove excess PE-streptavidin from the sorting. PE fluorescence of the cell population collected from the mouse is detected by FACS to identify a population of antibody-producing B cells expressing antibodies specific to the extracellular loop domain of the human transmembrane protein of interest.

[0259] This particular immunization and sorting strategy also effectively removes all antibody-producing B cells that express antibodies specific for epitopes located in the murine portion of the chimera-TMB.

[0260] Single antibody-producing B cells that bind to the transmembrane protein of interest presented by the complex can then be isolated into individual wells of a 384-well plate, and antibody encoding DNA is isolated from each cell for antibody generation and analysis as in Example 2.

[0261] Example 8. Obtaining antibody-producing cells expressing specific cross-reactive antibodies to a transmembrane protein of interest. The methods of the invention were also used to generate antibodies that recognize both the murine and human forms of an exemplary transmembrane protein of interest.

[0262] Nucleic acids encoding two different exemplary human transmembrane proteins, human TMB1 and human TMB2, were modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of each human transmembrane protein amino acid sequence and purified as described in Example 1. Additionally, nucleic acids encoding the mouse homologs of each of the two human transmembrane proteins, mouse TMB1 and mouse TMB2, were modified to attach a FLAG tag and a His tag (10x-His tag) to the carboxy terminus of each mouse transmembrane protein amino acid sequence and the modified nucleotide sequences were purified as described in Example 1. Next, nucleic acids encoding human TMB1, human TMB2, mouse TMB1, and mouse TMB2, respectively, were each expressed in cells and each protein was isolated and purified as described in Example 1. Lipid bilayer membrane scaffold protein complexes including one of human TMB1 protein, human TMB2 protein, mouse TMB1 protein, and mouse TMB2 protein were generated using Bir-A labeled membrane scaffold protein and purified as described in Example 1.

[0263] Genetically engineered VELOCIMMUNE® mice containing DNA encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain variable region that do not express an endogenous mouse homolog to a transmembrane protein of interest (i.e., mouse TMB1 or mouse TMB2) were immunized by injection with exogenous DNA encoding a mouse or human transmembrane protein, or a combination of exogenous DNA encoding the human TMB1 and mouse TMB2 proteins, as described in Table 4.

[0264] To monitor the immune response, splenocytes were harvested from the immunized mice. Splenocytes harvested from each mouse were stained for B cell markers (i.e., anti-IgG antibodies) with fluorescent labels and simultaneously incubated with biotinylated lipid-bilayer membrane scaffold protein complexes containing either specific human TMB proteins (complexes containing human TMB) or specific mouse TMB proteins (complexes containing mouse TMB) to allow binding between the transmembrane protein of interest presented by the biotinylated lipid-bilayer membrane scaffold protein complexes and antibodies on the surface of the B cells. The cells were then washed to remove unbound complexes and incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complexes. The cells were then washed in phosphate-buffered saline (PBS) to remove excess PE-streptavidin from the sorting. PE fluorescence of cell populations collected from mice was detected by FACS to identify populations of antibody-producing B cells expressing antibodies specific to the transmembrane protein of interest.

[0265] Single antibody-producing B cells that bound to the transmembrane protein of interest were then isolated into individual wells on a 384-well plate, antibody encoding DNA was isolated from each cell, and antibodies were generated as described in Example 2 for further analysis.

[0266] As shown in Table 4, immunization of mice genetically modified with DNA encoding only mouse TMB1 and sorting of antibody-producing B cells bearing lipid bilayer membrane scaffold protein complexes displaying human TMB1 protein identifies antibody-producing B cells expressing cross-reactive antibodies capable of binding mouse TMB1 and human TMB1 proteins.

[0267] Furthermore, by immunization of mice genetically modified with DNA encoding both mouse TMB1 protein and human TMB1 protein, and sorting of antibody-producing B cells with either a lipid bilayer membrane scaffold protein complex presenting human TMB1 protein or a lipid bilayer membrane scaffold protein complex presenting mouse TMB1 protein, antibody-producing B cells expressing cross-reactive antibodies capable of binding mouse TMB1 and human TMB1 proteins were identified.

[0268] Table 4 also shows that immunization of mice genetically modified with DNA encoding only mouse TMB2 and sorting of antibody-producing B cells with lipid bilayer membrane scaffold protein complexes displaying human TMB2 protein or sorting of antibody-producing B cells with lipid bilayer membrane scaffold protein complexes displaying mouse TMB2 protein identifies antibody-producing B cells that express cross-reactive antibodies capable of binding mouse TMB2 and human TMB2 protein. Immunization of mice genetically modified with DNA encoding only mouse TMB2 and sorting of antibody-producing B cells with lipid bilayer membrane scaffold protein complexes displaying human TMB2 protein or sorting of antibody-producing B cells with lipid bilayer membrane scaffold protein complexes displaying mouse TMB2 protein also identifies antibody-producing B cells that express cross-reactive antibodies capable of binding mouse TMB1 and human TMB1 protein. [Table 4]

[0269] Example 9. Obtaining antibody-producing cells expressing antibodies specific to a transmembrane protein of interest from mice immunized with a lipid bilayer membrane scaffold protein complex containing the transmembrane protein. To determine whether a lipid bilayer membrane scaffold protein complex encompassing a transmembrane protein of interest can be used as an immunogen to obtain antibody-producing cells expressing antibodies specific to the transmembrane protein of interest, human TMB2 comprising a nucleotide sequence encoding an exemplary human transmembrane protein and a FLAG tag and a His tag (10x-His tag) attached to its carboxy terminus was generated and purified as described in Example 8. A nucleotide sequence encoding a modified human TMB2 protein was expressed in cells, and the modified TMB2 protein was isolated and purified as described in Example 8. A lipid bilayer membrane scaffold protein complex encompassing human TMB2 protein was generated using Bir-A-tagged membrane scaffold protein and purified as described in Example 8.

[0270] Furthermore, a nucleotide sequence encoding a His tag and a nucleotide sequence encoding a FLAG tag affixed to the modified human TMB2 protein were generated, expressed separately in cells, and purified as described in Example 1 to obtain FLAG-peptides and HIS-peptides, respectively. Next, a nucleotide sequence encoding a membrane scaffold protein included in a lipid bilayer membrane scaffold protein complex was generated, expressed separately in cells, and purified as described in Example 1 to obtain MSP-peptides.

[0271] As shown in Table 5, genetically engineered VELOCIMMUNE® mice containing DNA encoding a human immunoglobulin heavy chain (IgH) and a human immunoglobulin light chain variable region that do not express an endogenous mouse homolog to a transmembrane protein of interest (i.e., mouse TMB2) were immunized by injecting 0.54 mg / mL of a lipid bilayer membrane scaffold protein complex (complex containing human TMB2) that encompasses the human TMB2 protein. The immune response was monitored and splenocytes were harvested from the immunized mice. Splenocytes collected from each immunized mouse were stained with a fluorescent label for a B cell marker (i.e., anti-IgG antibody) and simultaneously incubated with MSP-peptide, FLAG-peptide, and HIS-peptide to allow binding of the MSP-peptide, FLAG-peptide, and HIS-peptide to antibodies on the surface of B cells specific for epitopes located on the MSP-peptide, FLAG-peptide, and HIS-peptide, respectively. This step effectively blocks all antibody-producing B cells that express antibodies specific for components of the complex other than the human TMB2 protein presented by the complex.

[0272] The remaining B cells were subsequently incubated with a biotinylated lipid bilayer membrane scaffold protein complex containing either human TMB2 protein (complex containing human TMB2) or mouse TMB2 protein (complex containing mouse TMB2) to allow binding of the transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complex to the antibody on the surface of the B cells. The cells were then washed to remove unbound complexes and incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complex. The cells were then washed in phosphate-buffered saline (PBS) to remove excess PE-streptavidin from the sorting. PE fluorescence of the cell populations collected from the mice was detected by FACS to identify the population of antibody-producing B cells expressing antibodies specific to the transmembrane protein of interest.

[0273] Single antibody-producing B cells that bound to the transmembrane protein of interest were then isolated into individual wells on a 384-well plate, antibody encoding DNA was isolated from each cell, and antibodies were generated as described in Example 2 for further analysis.

[0274] As shown in Table 5, immunization of genetically modified mice with a lipid bilayer membrane scaffold protein complex including human TMB2 protein and sorting of antibody-producing B cells having the lipid bilayer membrane scaffold protein complex presenting human TMB2 protein identified antibody-producing B cells expressing cross-reactive antibodies that enable binding of antibodies specific to mouse TMB2 and human TMB2 protein homologs as well as human TMB2 protein. Furthermore, immunization of genetically modified mice with a lipid bilayer membrane scaffold protein complex including mouse TMB2 protein and sorting of antibody-producing B cells having the lipid bilayer membrane scaffold protein complex presenting mouse TMB2 protein identified antibody-producing B cells expressing only cross-reactive antibodies that can bind to mouse TMB2 protein and human TMB2 protein. [Table 5]

[0275] Example 10. Generation of antibody-producing cells expressing antibodies that bind to an exemplary SLC transmembrane protein of interest and obtaining SLC protein-specific antibodies from the cells. The nucleotide sequence encoding the first example of the SLC transmembrane protein of interest is modified as described in Example 1 to include a FLAG tag and a 10x-His tag attached to the N-terminus of the exemplary SLC protein. The nucleotide sequence encompassing the modified SLC transmembrane protein is cloned into an expression vector and expressed in Sf9 cells. The cells are then lysed and the modified SLC transmembrane protein is obtained and purified as in Example 1.

[0276] Next, disc-shaped lipid bilayer membrane scaffold protein complexes containing the modified human SLC transmembrane protein are generated as described in Example 1 and herein.

[0277] Genetically modified SLC knockout mice, i.e., VELOCIMMUNE® mice that contain a humanized IgH locus and a humanized Igκ locus that also lack the endogenous mouse gene encoding the SLC transmembrane protein of interest, are immunized by injection of exogenous DNA encoding the modified human SLC protein and by injection of the modified human SLC protein, as described in Example 1.

[0278] Splenocytes are collected from each immunized mouse, stained for B cell markers (i.e., anti-IgG antibodies) with a fluorescent label, and simultaneously incubated with biotinylated lipid bilayer membrane scaffold protein complexes containing 0.2 mg / mL to 5.0 mg / mL of SLC transmembrane protein embedded therein. The cells are then washed to remove unbound complexes. Subsequently, the cells are incubated with PE-streptavidin to allow binding of streptavidin to each of the biotin-labeled (e.g., Bir-A) membrane scaffold proteins in the lipid bilayer membrane scaffold protein complexes. The cells are then washed with phosphate-buffered saline (PBS) to remove excess PE-streptavidin. PE fluorescence of cell populations from control and immunized mice was detected by FACS to identify B cell populations expressing antibodies that bind to the exemplary SLC transmembrane protein of interest presented by the biotinylated lipid bilayer membrane scaffold protein complexes.

[0279] Single antibody-producing B cells that bound to the SLC transmembrane protein of interest were isolated into individual wells on a 384-well plate, antibody encoding DNA was isolated from each cell, and antibodies were generated as described in Example 2 for further analysis.

[0280] Although several aspects of the present disclosure have been described and illustrated herein, alternative aspects can be effected by those skilled in the art to accomplish the same objectives, and it is therefore intended by the appended claims to cover all such alternative aspects which fall within the true spirit and scope of the present disclosure.

Claims

1. The method is to obtain antibody-producing cells expressing an antibody that binds to a transmembrane protein of interest. hand, (a) contacting a population of antibody-producing cells from a mammal with a lipid bilayer membrane scaffold protein complex that includes the transmembrane protein of interest to allow the transmembrane protein of interest to bind to an antibody on the cell surface; (b) harvesting the cells bound to the transmembrane protein of interest. 。

2. The complex comprises a disc-shaped phospholipid surrounded by at least one membrane scaffold protein. The method of claim 1 , comprising a plurality of lipids that form a bilayer.

3. The population of antibody-producing cells is located in the spleen, lymph nodes, peripheral blood, bone marrow, or a combination thereof. The method of claim 1 , wherein the nucleic acid sequence is obtained from a nucleic acid sequence ...

4. The method of claim 1 , wherein the population of antibody-producing cells comprises peripheral blood cells, B cells, plasma cells, or a combination thereof.

5. The method of claim 4, wherein the population of antibody-producing cells comprises B cells.

6. The method of claim 1 , wherein the mammal is a mammal that has previously been immunized with the transmembrane protein of interest.

7. The mammal is provided with a nucleic acid encoding at least a portion of the transmembrane protein of interest, or or a non-human mammal that has been immunized with at least a portion of said transmembrane protein of interest. The method according to claim 6, wherein

8. The method of claim 7, wherein the non-human mammal is genetically engineered.

9. The genetically engineered non-human mammal expresses the transmembrane protein of interest from an endogenous gene. The method of claim 8, wherein the gene does not express a quality factor.

10. The genetically engineered non-human mammal comprises a nucleic acid sequence encoding a human heavy chain variable region, and and a nucleic acid sequence encoding a human light chain variable region.

11. The method of claim 8, wherein the genetically engineered non-human mammal is a mouse.

12. 10. The method of claim 9, wherein the transmembrane protein of interest is a human transmembrane protein.

13. The genetically engineered non-human mammal is a mammal that is a mammalian animal that is a mammalian animal that is a mammalian animal. or a nucleic acid encoding said non-human homologue of said transmembrane protein, The method of claim 12.

14. The cells harvested in step (b) contain the human transmembrane protein and the transmembrane 14. The method of claim 13, wherein the human expresses an antibody that binds to the non-human homolog of the common protein. 。

15. The genetically engineered non-human mammal is capable of expressing a chimeric transmembrane protein of interest or a The subject is immunized with a nucleic acid encoding a chimeric transmembrane protein of interest, The protein comprises a portion of a non-human homolog of the transmembrane protein of interest and a portion of the human transmembrane protein of interest. The method of claim 12, comprising a portion of a common protein.

16. The cells harvested in step (b) contain an epitope located on the human transmembrane protein. The method of claim 15, wherein an antibody that binds to the top is expressed.

17. The transmembrane protein of interest is a chimeric transmembrane protein, a portion of a human transmembrane protein operably linked to a portion of a non-human transmembrane protein The method of claim 8 , comprising:

18. The genetically engineered non-human mammal is 18. The method of claim 17, wherein the subject is immunized with a nucleic acid encoding a transmembrane protein.

19. The cells collected in step (b) contain the human portion of the chimeric transmembrane protein. The method of claim 18, wherein the antibody binds to

20. The genetically engineered non-human mammal is immunized with a truncated form of the transmembrane protein. The method of claim 8 ,

21. The genetically engineered non-human mammal is 21. The method of claim 20, wherein the subject is immunized with a full-length form of a nucleic acid encoding the protein.

22. The truncated form of the transmembrane protein is an N-amino acid of the full-length form of the transmembrane protein. The method of claim 20, which does not contain a terminal domain or a C-terminal domain.

23. The truncated form of the transmembrane protein comprises an extracellular loop and is collected in step (b). The cell is located on the extracellular loop of the truncated form of the transmembrane protein. The method of claim 22, wherein the antibody that binds to the epitope is expressed.

24. Step (a) contacts the population of antibody-producing cells with at least one blocking agent. The method of claim 1 , further comprising:

25. The at least one blocking agent binds to a portion of the transmembrane protein of interest.

25. The method of claim 24, wherein the polypeptide is

26. The polypeptide is an N-terminal domain of the transmembrane protein of interest or The method of claim 25, which binds to the C-terminal domain of a transmembrane protein.

27. the transmembrane protein comprises an extracellular loop, and the cells harvested in step (b) an antibody that binds to an epitope located on the extracellular loop of the transmembrane protein of interest; The method of claim 26, wherein the body is expressed.

28. The population of antibody-producing cells is then cultured in a lipid bilayer scaffold containing the transmembrane protein of interest. from a mammal immunized with the protein complex, said complex being a first detectable and wherein the transmembrane protein of interest further comprises a second detectable label. The method according to claim 24.

29. The at least one blocking agent (i) binds to the first detectable label; (ii) a first blocking agent, and (ii) a second blocking agent that binds to said second detectable label.

30. The method of claim 28, further comprising a blocking agent.

30. The at least one blocking agent may comprise a third blocking agent that binds to the membrane scaffold protein.

30. The method of claim 29, further comprising a blocking agent.

31. the transmembrane protein of interest is a G protein-coupled receptor (GPCR) protein; tetraspanin proteins, and ion channel proteins. The method according to claim 1 .

32. 32. The method of claim 31 , wherein the transmembrane protein of interest is a GPCR protein.

33. 32. The method of claim 31 , wherein the transmembrane protein of interest is an ion channel protein. method.

34. 32. The method of claim 31 , wherein the transmembrane protein of interest is a tetraspanin protein. method.

35. The complex comprises at least one membrane scaffold protein conjugated to a detectable label. The method of claim 1 further comprising a protein.

36. harvesting said cells bound to said transmembrane protein of interest; detecting binding of said cell to said transmembrane protein of interest; The cells bound to the transmembrane protein of interest are then subjected to separating the cells from unbound cells; and and collecting the bound cells.

37. 36. The method of claim 35, wherein the detectable label is biotin, and optionally the at least one membrane scaffold protein is conjugated to biotin by an Avi tag, or a Bir-A tag.

38. The detection comprises: The cells of step (b) are treated with streptavidin-phycoerythrin (PE-Streptavidin). and incubating with 100 μg / ml of 100 μl ... identifying the bound cells using fluorescence activated cell sorting (FACS); 38. The method of claim 37, comprising:

39. 37. The method of claim 36, wherein the detectable label is a fluorescent molecule.

40. 40. The method of claim 39, wherein the detecting comprises identifying the bound cells using FACS. Method of posting.

41. (i) isolating from the cells collected in step (b) the antibody expressed by the cells; A nucleic acid comprising a nucleotide sequence encoding the heavy chain variable region, and a nucleic acid expressed by said cell. A nucleic acid comprising a nucleotide sequence encoding the light chain variable region of the antibody to be isolated. The method of claim 1 , further comprising:

42. The process further comprises, after step (i), (ii) the nucleic acid encoding the heavy chain variable region isolated in step (i); The nucleic acid comprising a nucleotide sequence and the nucleotide sequence encoding the light chain variable region. transfecting a host cell to introduce said nucleic acid comprising the sequence; and (iii) under conditions that express an antibody comprising the heavy chain variable region and the light chain variable region; and culturing the host cell from step (ii). Law.

43. The host cell in step (ii) is a first expression vector comprising a sequence, and said nucleotide sequence encoding said light chain variable region; 43. The method of claim 42, wherein the nucleic acid sequence is transfected with a second expression vector comprising the sequence.

44. 43. The method of claim 42, wherein the host cell is a mammalian cell.

45. 45. The method of claim 44, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. The method described.

46. The lipid bilayer membrane scaffold protein complex containing the transmembrane protein of interest is one or more 46. ​​The method of any one of claims 1 to 45, wherein the complex is formed by mixing the lipids provided, at least one membrane scaffold protein, and the transmembrane protein of interest in the presence of the detergent, and removing the one or more detergents to induce formation of the complex comprising the transmembrane protein of interest.

47. The method of any one of claims 1 to 46, wherein at least one membrane scaffold protein comprises an Avi tag.

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