KR102059866B1 - Methods for identifying immunobinders of cell-surface antigens - Google Patents

Abstract

The present invention provides methods for identifying immunobinding agents, such as scFvs, that can specifically bind cell surface antigens, and compositions identified in accordance with the methods.

Description

Method for identifying immune binding agents for cell-surface antigens {METHODS FOR IDENTIFYING IMMUNOBINDERS OF CELL-SURFACE ANTIGENS}

The present invention provides a method of identifying an immune binding agent, such as an scFv antibody, capable of specifically binding to a cell surface antigen.

Immunobinding agents, including antibodies, conjugates and derivatives thereof, are of great commercial importance as therapeutic and diagnostic agents. Traditional methods for making or screening antibodies typically use soluble antigens. However, in the case of certain membrane-bound protein antigens, if the antigen is solubilized from the membrane, conformational epitopes on the antigen are modified, resulting in failure of antibody production or selection. In addition, one of the major problems in immunoblotting and affinity chromatography methods is to select an antibody having a moderate affinity for an antigen. This includes numerous cross-reactive or sticky antibodies, which puts a burden on the sequential screening process. Cells expressing membrane-bound antigens are used directly for antibody production, but efficient screening methods capable of detecting and enriching antibodies with high affinity for cell-surface antigens are still lacking.

Summary of the invention

The present invention provides a method of identifying an immune binding agent, such as an scFv antibody, capable of specifically binding to a cell surface antigen. The method of the present invention is generally characterized in that the labeled antigen-expressing cells are contacted with the labeled immune-binding agent-expressing cells and the immuno-binding agent-expressing cells that bind to the antigen-expressing cells are separated using a cell sorter. It is done. These methods are particularly useful for rapid and efficient identification of immunoconjugates for conformational epitopes present in integral membrane proteins, such as GPCRs. The invention also provides isolated immuno-binding agents and immuno-binding agents-encoding nucleic acids, which are identified using the methods of the invention.

In one aspect, the present invention provides a method of identifying an immunobinding agent that specifically binds to the cell surface antigen. The method provides a plurality of immunobinding agent-expressing cells functionally bound to a first classifiable label; Providing a plurality of antigen-expressing cells functionally bound to a second classifiable label, wherein the antigen is displayed on the surface of the antigen-expressing cell; Contacting the antigen-expressing cell with the immune binding agent-expressing cell; A cell sorter (e.g., a fluorescence activated cell sorter) is used to isolate one or more immune-binding agent-expressing cells capable of specifically binding to an antigen-expressing cell from a plurality of immune-binding agent-expressing cells, wherein a single cell complex (E.g., a complex formed between an antigen and a B-cell receptor), the presence of the first and second classifiable markers is an indicator of binding of an immune binding agent-expressing cell to an antigen-expressing cell, thereby binding immunity to the antigen. Characterized by the identification of the binder.

In an embodiment, the isolated immunobinding agent-expressing cells are clonal isolated. In an embodiment, the immunobinding agent-expressing cells are clonal expansion. In another embodiment, the immunobinding agent-encoding nucleic acid sequence is isolated from the immunobinding agent-expressing cell. A suitable method of isolating an immunobinder-encoding nucleic acid sequence is PCR, eg, single-cell PCR. The immunobinder-encoding nucleic acid sequence can be isolated after clonal separation of the cells and/or after clonal expansion.

In an embodiment, immune-binding agent-expressing cells isolated using the methods of the invention are functionally characterized by cell-based assays. A suitable cell-based assay is CELISA.

In an embodiment, the immunobinding agent is an antibody. Such antibodies include murine, rabbit, rabbitized, chicken, camel, camelized, human, humanized and chimeric antibodies. Suitable antibody formats include, without limitation, Fab, Dab, Nanobody and scFv.

In an embodiment, the antigen is expressed from a heterologous gene. In another embodiment, the antigen is a genetically engineered antigen. In another embodiment, the antigen is an intrinsic membrane protein. Suitable intrinsic membrane proteins include, but are not limited to, GPCRs (eg CXCR2) or ion channels.

In an embodiment, the first or second classifiable label is a fluorescent label. Suitable fluorescent labels include, but are not limited to, fluorescent proteins, antibody/fluorine conjugates and fluorescent cell labels.

In an embodiment, the antigen-expressing cell is a yeast or mammalian cell (eg, a human cell). In an embodiment, the antigen-expressing cell expresses an exogenous antigen. In an embodiment, antigen-expressing cells are transfected with an expression vector.

In an embodiment, the immunobinding agent-expressing cell is a yeast or mammalian cell. Suitable mammalian cells include, but are not limited to, B-cells, such as rabbit B-cells. In an embodiment, the B-cells are isolated from an immunized animal, eg, an immunized animal by DNA vaccination. In an embodiment, the immunobinding agent-expressing cell comprises an immunobinding agent expressed from an expression vector.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding an immunobinding agent identified by the method of the present invention.

In another aspect, the present invention is characterized in that the immuno-binding agent-encoding nucleic acid sequence identified by the present invention is introduced into an expression environment in which the encoded immune-binding agent is produced, thereby producing an immuno-binding agent capable of binding to the antigen. Provides a way.

In another aspect, the present invention provides an immunobinding agent produced by the method of the present invention.

In another aspect, the invention also immunizes animals with DNA encoding cell surface antigens; Isolating B-cells from immunized animals; Labeling the B-cells with a first classifiable label; Providing a plurality of antigen-expressing cells functionally bound to a second classifiable label, wherein the antigen is displayed on the surface of the antigen-expressing cell; One or more B-cells capable of specifically binding to the antigen-expressing cells using a cell sorter are separated from the plurality of B-cells (herein, the first and second classifiable labels in a single cell complex The presence of B-cells is an indicator of binding of B-cells to antigen-expressing cells, whereby B-cell clones that bind to the antigens can be identified), which specifically binds to the cell surface antigens. Methods for identifying B-cell clones are provided.

1 schematically shows B-cells labeled with fluorescent antibody 2 that interact with target-expressing cells stained with intracellular dye 3. (4: selected target/antigen; 5: B cell receptor (BCR)).
Figure 2: FACS selection process of rabbit B cells binding to ESBA903 soluble target. Figure 2A: Lymphocytes pass along the anterior chamber and are scattered laterally. 2B: Among these, IgG+ IgM- cells (probably memory B cells) are selected (represented by circles). Figure 2C: Cells double-stained with ESBA903-PE and ESBA903-PerCP (indicated by circles) are expected to encode high affinity IgGs for ESBA903. Cells with the brightest fluorescence (not circled) are sorted in 96-well plates.
Figure 3: Beads coated with anti-TNFalpha antibody (PE label) bind to TNFalpha-transfected CHO cells (upper panel). Control beads coated with anti-CD19 antibody (APC label) do not bind to TNFalpha-transfected CHO cells (middle panel). Beads coated with anti-TNFalpha antibody (PE label) do not bind to wild-type (wt) CHO cells (lower panel). The dot plot on the left represents the anterior and lateral scattering, representing the size and granularity of the event, respectively. A population of single beads (approximately 3 μm) passes at P2. Ultimately, CHO cells bound to beads (approximately 3 μm) pass through at P1. The middle dot plots represent P1 events (CHO cells) for their PE or APC staining. Thus, cells that interact with anti-TNFalpha beads appear at the P3 gate, and cells that react with anti-CD19 beads appear at the P4 gate. On the right, the statistics for each sample are presented in detail.
Figure 4: Anti-TNFalpha-PE coated beads and anti-CD19-APC coated beads were mixed with TNFalpha-transfected CHO cells. CHO cells pass through (P1) and among them, cells that bind to anti-TNFalphaPE coated beads or anti-CD19-APC coated beads appear at P3 and P4, respectively. Unbound beads can be seen at gate P2.
Figure 5a: FACS analysis of three CHO-TNFalpha-memory B cell suspensions. Top left: Dot plot showing cell suspension scattering in the anterior and lateral directions. Live cells containing a large population of transgenic CHO cells and a small population of memory B cells are passed through. Bottom left: Dot plot showing APC and FITC fluorescence. Here, memory B cells (IgG+/IgM-) are passed. These two dot plots are the same for all three samples; Thus, they appear only once.
5B: Histogram and population lineage of three samples: Top: CHO-TNFalpha cells from ESBA105 immunized rabbits + ESBA105 + memory B cells; Middle: CHO-TNFalpha cells from non-immunized rabbits + ESBA105 + memory B cells; Bottom: ESBA105 immunized rabbit CHO-TNFalpha cells + memory B cells. In the histogram, memory B cells that bind to CHO cells are passed.
Figure 6: FACS analysis of a suspension consisting of immunized lymphocytes mixed with TNFalpha transgenic CHO cells "coated" with ESBA105. Figure 6a: Dot plot showing scattering in the front and side of the suspension. Live cells containing a large population of transgenic CHO cells and a small population of lymphocytes are passed through. Figure 6b: Dot plot showing APC and FITC fluorescence. Here, memory B cells (IgG+/IgM-) are passed. 6C: Histogram showing calcein fluorescence of passed memory B cells. The passed population is sorted (memory B cells binding to the CHO-TNFalpha-ESBA105 complex).
Figure 7: Bright field micrographs of beads coated with anti-TNF alpha IgG interacting with CHO-TNF alpha (B220) transgenic cells.
Figure 8: Bright field microscopy limbs (left column) and fluorescence micrographs (right) of CHO-TNFalpha/ESBA105 cells (large cells) bound to B cells (which have anti-EABA105 antibodies (smaller cells) on the surface). column).

details

The present invention provides a method of identifying an immune binding agent, such as an scFv antibody, capable of specifically binding to a cell surface antigen. The method of the present invention is generally characterized in that the labeled antigen-expressing cells are contacted with the labeled immune-binding agent-expressing cells and the immuno-binding agent-expressing cells that bind to the antigen-expressing cells are separated using a cell sorter. It is done. These methods are particularly useful for rapid and efficient identification of immunoconjugates for conformational epitopes present in integral membrane proteins, such as GPCRs. The present invention also provides isolated immuno-binding agents and immuno-binding agent-encoding nucleic acids, which are identified using the methods of the present invention.

In one aspect, the present invention provides a method of identifying an immunobinding agent that specifically binds to the cell surface antigen. The method provides a plurality of immunobinding agent-expressing cells functionally bound to a first classifiable label; Providing a plurality of antigen-expressing cells functionally bound to a second classifiable label, wherein the antigen is displayed on the surface of the antigen-expressing cell; Contacting the antigen-expressing cell with the immune binding agent-expressing cell; Isolation of one or more immune-binding agent-expressing cells capable of specifically binding to antigen-expressing cells using a cell sorter (e.g., a fluorescence activated cell sorter) from a plurality of immune-binding agent-expressing cells (wherein a single cell complex ( For example, the presence of first and second classifiable markers, which are complexes formed between an antigen and a B-cell receptor), is an indicator of binding of an immunobinder-expressing cell to an antigen-expressing cell, thereby binding an immunobinder to the antigen. It is characterized by being able to sympathize with).

In an embodiment, the isolated immune binding agent-expressing cells are clonal isolated.

In certain embodiments, the clonal isolated immunobinder-expressing cells are cloned using methods well known to those skilled in the art.

In another embodiment, the immunobinding agent-encoding nucleic acid sequence is isolated from the immunobinding agent-expressing cell. Isolation of the nucleic acid sequence may occur after clonal separation or clonal expansion. A suitable method of isolating an immunobinder-encoding nucleic acid sequence is PCR, eg, single-cell PCR.

In an embodiment, immune-binding agent-expressing cells isolated using the methods of the invention are functionally characterized by cell-based assays. A suitable cell-based assay is CELISA.

In an embodiment, the immunobinding agent is an antibody. Such antibodies include murine, rabbit, rabbitized, chicken, camel, camelized, human, humanized and chimeric antibodies. Suitable antibody formats include, without limitation, Fab, Dab, Nanobody and scFv.

In an embodiment, the antigen is expressed from a heterologous gene. In another embodiment, the antigen is a genetically engineered antigen. In another embodiment, the antigen is an intrinsic membrane protein. Suitable intrinsic membrane proteins include, but are not limited to, G-protein coupled receptors (GPCRs (eg CXCR2)) or ion channels.

In an embodiment, the first or second classifiable label is a fluorescent label. Suitable fluorescent labels include, but are not limited to, fluorescent proteins, antibody/fluorine conjugates and fluorescent cell labels.

In an embodiment, the antigen-expressing cell is a yeast or mammalian cell (eg, a human cell). In an embodiment, the antigen-expressing cell expresses an exogenous antigen. In an embodiment, antigen-expressing cells are transfected with an expression vector.

In an embodiment, the immunobinding agent-expressing cell is a yeast or mammalian cell. Suitable mammalian cells include, but are not limited to, B-cells, such as rabbit B-cells. In an embodiment, the B-cells are isolated from an immunized animal, eg, an immunized animal by DNA vaccination. In an embodiment, the immunobinding agent-expressing cell comprises an immunobinding agent expressed from an expression vector.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding an immunobinding agent identified by the method of the present invention.

In another aspect, the present invention is characterized in that the immuno-binding agent-encoding nucleic acid sequence identified by the present invention is introduced into an expression environment in which the encoded immune-binding agent is produced, thereby producing an immuno-binding agent capable of binding to the antigen. Provides a way.

In another aspect, the present invention provides an immunobinding agent produced by the method of the present invention.

In another aspect, the invention also immunizes animals with DNA encoding cell surface antigens; Isolating B-cells from immunized animals; Labeling the B-cells with a first classifiable label; Providing a plurality of antigen-expressing cells functionally bound to a second classifiable label, wherein the antigen is displayed on the surface of the antigen-expressing cell; One or more B-cells capable of specifically binding to the antigen-expressing cells using a cell sorter are separated from the plurality of B-cells (herein, the first and second classifiable labels in a single cell complex The presence of B-cells is an indicator of binding of B-cells to antigen-expressing cells, whereby B-cell clones that bind to the antigens can be identified), which specifically binds to the cell surface antigens. Methods for identifying B-cell clones are provided.

Justice

In order to further facilitate understanding of the present invention, specific terms are defined as follows. Additional definitions are described throughout the detailed description.

The term “antibody” refers to the entire antibody and antigen binding fragment (ie, “antigen-binding portion”, “antigen binding polypeptide”, or “immune binding agent”) or a single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy chains (heavy: H) and two light chains (light: L) linked between chains by disulfide bonds, or antigen-binding portions thereof. Each heavy chain consists of a heavy chain variable region (here abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (here abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions, termed framework regions (FR), interspersed with better conserved regions, termed complementarity determining regions (CDRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody allows immunoglobulins to mediate binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (Clq).

The term “chimeric antibody” refers to (a) a constant region, or a portion thereof, of a class in which the antigen binding site (variable region) is different or modified by modification, replacement, or exchange, effector function and/or species constant region, or Are bound to completely different molecules, such as enzymes, toxins, hormones, growth factors, drugs, etc. that impart new properties to the chimeric antibody; (b) The variable region, or a portion thereof, refers to an antibody molecule in which the variable region has been modified, replaced or exchanged with a variable region having different or modified antigen specificity.

The term “antigen-binding portion” (or simply “antibody portion”) of an antibody refers to one or more fragments of an antibody (eg, TNF) that retain the ability to specifically bind to an antigen. It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single domain, such as a dAb fragment (Ward et al., (1989) Nature 342:544-546), consisting of the VH domain; And (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs that may be optionally linked by a synthetic linker. In addition, even if the two domains of the Fv fragment, VL and VH are encoded by separate genes, these are known as monovalent molecules (single-chain Fv (scFv) by pairing the VL and VH regions using a recombinant method. ; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) It can be linked by a linker. Such single chain antibodies are also included within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are selected for utility in the same manner as the original antibody. Antigen-binding moieties can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of native immunoglobulins. Antibodies may be of different isotypes and may be, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

The term “immune binding agent” refers to a molecule containing all or part of the antigen binding site of an antibody, eg, heavy and/or light chain variable domains, such that the immunobinding agent specifically recognizes the target antigen. Non-limiting examples of immunobinding agents include full-length immunoglobulin molecules and scFvs, as well as, but not limited to (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fab' fragment, essentially a Fab having a portion of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3. sup.rd ed. 1993); (iv) Fd fragment consisting of VH and CH1 domains; ( v) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vi) a Dab fragment consisting of the VH or VL domain (Ward et al., (1989) Nature 342:544-546), Camelid ( See: Hamers-Casterman, et al., Nature 363:446-448 (1993), and Dumoulin et al., Protein Science 1:500-515 (2002)) or Shark antibodies (e.g., Ig-NARs Nanobodies® in sharks). ); and (vii) an antibody fragment, including a nanobody, which is a heavy chain variable region containing a single variable domain and two constant domains.

The term “functional property” as used herein refers to an improvement (eg, relative to a conventional polypeptide) and/or in the art, for example, in order to improve the manufacturing property or therapeutic effect of the polypeptide. It is a property of a polypeptide (eg, immunobinding agent) that is advantageous to the skilled person. In one aspect, the functional properties are improved stability (eg, thermal stability). In another aspect, the functional properties are improved solubility (eg, under cellular conditions). In another aspect, the functional property is non-aggregating. In another aspect, the functional property is an improvement in expression (eg, in prokaryotic cells). In another aspect, the functional property is an improvement in the refolding yield following the purification process after introduction into the body. In certain embodiments, the functional property is not an improvement in antigen binding affinity.

The term “frameworks” refers to a technology-recognized portion of an antibody variable region that exists between the more branching CDR regions. Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and the 3 found in heavy or light antibody variable regions, such as allowing CDRs to form antigen-binding surfaces. It provides a scaffold to maintain the canine CDRs in a three-dimensional space. Such frameworks can also be referred to as scaffolds as they provide support for the expression of more branched CDRs. Other CDRs and frameworks of the immunoglobulin superfamily, such as ankyrin repeats and fibronectin, can be used as antigen binding molecules (e.g., U.S. Patent Nos. 6,300,064, 6,815,540 and U.S. Publication Nos. 20040132028).

The term “epitope” or “antigen determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial arrangement. See, for example, the following literature: Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

The terms "specific binding", "selective binding", "selectively binding", and "specifically binding" refer to an antibody that binds an epitope on a predetermined antigen. Typically antibodies bind with an affinity (KD) of less than ^{about 10 -7} M, such as less than ^{about 10 -8} M, 10 ^-9 M or 10 ^{-10 M or even lower.}

The term “K _D ”refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. ^{Typically, the antibodies of the invention are approximately 10 -8} M, 10 ^-9 M or ^{less than 10 -10} M or even lower, as measured using surface plasmon resonance (SPR) technology, e.g. with a BIACORE device. As such, it binds to the antigen with a dissociation equilibrium constant of less than ^{approximately 10 -7 M.}

“Identity” as used herein refers to a sequence match between two polypeptides, molecules or between two nucleic acids. When the positions of both compared sequences are occupied by the same base or amino acid monomer subunit (e.g., adenine occupies the position in each of the two DNA molecules), then each molecule is identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of x100 compared positions. For example, if 6 out of 10 positions in two sequences match, then the identity of the two sequences is 60%. In one example, the DNA sequences CTGACT and CAGGTTT share 50% identity (three of a total of six positions match). In general, two sequences are compared when aligned for maximum identity. Such alignment is conveniently performed with a computer program such as the Align program (DNAstar, Inc.), for example, Needleman et al. (1970) J. Mol. Biol. It can be provided using the method of 48:443-453. The percent identity between the two amino acid sequences is also included in the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, E. Meyers and W. Miller's algorithm (Comput.Appl). Biosci., 4:11-17 (1988)). In addition, the percent identity between the two amino acid sequences is the Blossum 62 matrix or the PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6, or 4 and the length weight of 1, 2, 3, 4, 5, or 6. Measurements can be made using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm included in the GAP program in the GCG software package (available at www.gcg.com) used.

“Similar” sequences are those that, when aligned, share the same and similar amino acid residues, wherein similar residues are conservative substitutions for the corresponding amino acid residues in the aligned reference sequence. In contrast, "conservative substitution" of a residue in a reference sequence refers to substitution with a residue that is physically or functionally similar to a corresponding reference residue, including, for example, the ability to form a covalent or chemical bond. , Have similar size, shape, electrical charge, and chemical properties. Thus, a "conservatively substituted modified" sequence is different from a reference sequence or wild-type sequence in which one or more conservative substitutions are present. The "percent similarity" between two sequences is a function of the number of positions containing conservative substitutions or matching residues shared by the two sequences divided by the number of x100 compared positions. For example, if 6 out of 10 positions in two sequences match and 2 out of 10 contain conservative substitutions, then the positive similarity of the two sequences is 80%.

The term “conservative sequence modification” as used herein is intended to refer to an amino acid modification that does not negatively affect or change the binding characteristics of an antibody containing an amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as, for example, site-directed mutations and PCR-mediated mutations. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art. These groups include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine, asparagine, glutamine, Serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g. threonine, valine, isoleucine) ) And amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, predicted non-essential amino acid residues can be replaced with other amino acid residues from the same side chain family. Methods for the identification of nucleotide and amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (eg, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

"Amino acid consensus" as used herein can be generated by using a matrix of at least two, preferably more, aligned amino acid sequences, allowing gaps during alignment to determine the most common amino acid residues at each position. Refers to the amino acid sequence that is present. The consensus sequence is a sequence containing the amino acids that appear most frequently at each position. When two or more amino acids are represented equally at a single position, the consensus includes both or both of these amino acids.

The amino acid sequence of a protein can be analyzed at several levels. For example, conservation or variability can be indicated at a single residue level, multiple residue levels, multiple residues with gaps, and the like. Residues may represent conservation of the same residue or may be conserved at the class level. Examples of amino acid classes include polar but uncharged R groups (serine, threonine, asparagine and glutamine); Positively charged R groups (lysine, arginine, and histidine); Negatively charged R groups (glutamic acid and aspartic acid); Hydrophobic R groups (alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, valine and tyrosine); And special amino acids (cysteine, glycine and proline). Other classes are known to those skilled in the art and can be defined using structure determination methods or other data to evaluate substitutionality. In that sense, a substitutable amino acid may refer to an amino acid capable of being substituted and maintaining functional conservation at that position.

However, amino acids of the same class can be varied to the extent due to their physiological properties. For example, it is recognized that certain hydrophobic R groups (eg, alanine) are more hydrophilic (ie, higher hydrophilic or less hydrophobic) than other hydrophobic R groups (eg, valine or leucine). Relative hydrophilicity or hydrophobicity is determined by art-recognized methods (see, e.g. Rose et al., Science, 229:834-838 (1985) and Cornette et al., J. Mol. Biol., 195:659-685 ( 1987)).

As used herein, when aligning one amino acid sequence (e.g., a first VH or VL sequence) with another one or more additional amino acid sequences (e.g., one or more VH or VL sequences in a database), one sequence (e.g. , A first VH or VL sequence) can be compared to a “corresponding position” in one or more additional amino acid sequences. As used herein, "corresponding position" refers to an equivalent position in a compared sequence when the sequences are optimally aligned, ie when the sequences are aligned to achieve the highest percent identity or percent similarity.

The term “nucleic acid molecule” refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-threaded or double-threaded, but is preferably double-threaded DNA. A nucleic acid is "functionally linked" when it is in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is functionally linked to the coding sequence when it affects the transcription of the sequence.

The term “vector” refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One of the types of vectors is "plasmid", which refers to a circular, double-threaded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of self-replicating within the host cell into which they have been introduced (eg, bacterial vectors have replication of bacterial origin and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and replicate with the host genome.

The term “host cell” refers to a cell into which an expression vector has been introduced. The host cell can be a bacterial, microbial, plant or animal cell. Bacteria susceptible to transformation include enterobacteriaceae such as Escherichia coli or Salmonella; Bacilllaceae such as Bacillus subtilis; Pneumococcus; It is a member of Streptococcus, and Haemophilus influenza. Suitable microorganisms are Saccharomyces erevisiae and Pichia pastoris. Suitable animal host cell lines are CHO (Chinise Hamster Ovary lines) and NS0 cells.

The terms “treat”, “treating” and “treatment” refer to the therapeutic or prophylactic methods described herein. The method of “treatment” is to prevent one or more symptoms or severity of the disease or recurring disease in a patient in need of such treatment, for example, in a patient with a GPCR-mediated disease or ultimately a patient who may develop such a disease. To treat, delay, reduce, or prolong the patient's survival than would be expected in the absence of such treatment, the antibody of the present invention is administered.

The term “effective dose” or “effective dosage” refers to an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to treat or at least partially arrest the disease and complications in a patient already suffering from the disease. The amount effective for this use depends on the severity of the disease being treated and the general condition of the patient's own immune system.

The term “patient” refers to a human or non-human animal. For example, the methods and compositions of the present invention can be used to treat patients with GPCR-mediated diseases.

The term "rabbit" as used herein refers to an animal belonging to the rabbit family.

The term “cell sorter” refers to a means of cell separation based on the presence of a detectable “sortable” label. Such means include, but are not limited to, fluorescence activated cell sorters. Without limitation, fluorescent proteins, such as Green fluorescent protein, antibody/fluorine conjugates, and cell labels, including fluorescent cell labels, such as fluorescent calcium ionophores, can be used as classifiable labels.

The term “cell complex” refers to one or more antigen-expressing cells that are bound to one or more immune-binding agent-expressing cells, wherein binding is mediated by an antigen on the surface of the antigen-expressing cell. In some embodiments, the binding of an antigen-expressing cell to an immune-binding agent-expressing cell consists of a direct interaction between an antigen on the surface of the antigen-expressing cell and an immune-binding agent on the surface of the immune-binding agent-expressing cell.

The term “clonal isolation” refers to a means for the isolation of individual cell clones from a population of cells. A suitable means is, without limitation, limiting dilution and transferring the cells to a multiwell plate so that each well no longer contains a single cell.

The term "obtaining an immunobinding agent-encoding nucleic acid sequence" refers to a means for obtaining the nucleic acid sequence of an immunobinding agent expressed by an immunobinding agent-expressing cell. Suitable means include, without limitation, nucleic acid isolation, PCR amplification, and DNA sequencing of an immunobinder-encoding nucleic acid sequence from an immunobinder-expressing cell. In some embodiments, the immunobinding agent-encoding nucleic acid sequence is one that is amplified by PCR from a single cell, ie, is “single cell PCR”.

The term “exogenous antigen” refers to an antigen that is not normally expressed in a particular host cell. For example, the exogenous antigen can be from a different line, phylum, class, order, genus or species from the host cell, such as human antigens expressed in yeast cells. Additionally or alternatively, exogenous antigens may be from the same species, but may be improperly expressed in host cells, for example lung-specific antigens expressed in brain cells. “Exogenous antigens” also include antigenic mutants that are not normally found in normal cells, such as cancer-specific mutant antigens expressed in lung cells.

The term "genetically engineered antigen" refers to an antigen produced by recombinant DNA technology and contains chimeric antigens or contains point mutations, deletions and/or insertions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are for illustrative purposes only and are not limiting.

Various aspects of the invention are described in more detail below. Various aspects, preferences, and ranges can be combined as desired. Also, depending on the particular aspect, the selected definition, aspect, or range cannot be applied.

The present invention provides a selection method using FACS in order to identify and isolate immune-binding agent-expressing cells attached together with cells expressing an antigen. In certain embodiments, the immunobinding agent is an antibody.

Antigen expression

Target antigens for preparation of anti-drugs may be soluble or may be proteins, peptides, nucleotides, carbohydrates, lipids, and other molecules that are expressed on the cell surface or incorporated into the plasma membrane. Antigens can be natural or synthetic. Preferably, the target antigen is a protein or peptide. Non-limiting examples of target antigens include CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, Bestrophins, TMEM16A, GABA receptor, glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1 .7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P1R), NMDA channels, and the like. In one embodiment, the target antigen is a transmembrane protein. In another embodiment, the target antigen is a multispan transmembrane protein, such as G protein coupled receptors (GPCRs), ion channels, and the like.

There are at least 250 species in the GPCRs family (Strader et al. FASEB J., 9:745-754, 1995; Strader et al. Annu. Rev. Biochem., 63:101-32, 1994). It was estimated that 1% of human genes could encode GPCRs. GPCRs bind to a wide variety of ligands, from photons, small bioamines (i.e. epinephrine and histamine), peptides (i.e. IL-8) to large glycoprotein hormones (i.e. parathyroid hormone). Upon ligand binding, GPCRs regulate the intracellular signaling pathway by activating the guanine nucleotide-binding protein (G protein). Interestingly, GPCRs have functional homologues in human cytomegalovirus and herpesvirus, suggesting that GPCRs may be required during evolution for viral pathogenogenesis (Strader et al., FASEB J., 9:745). -754, 1995; Arvanitakis et al. Nature, 385:347-350, 1997; Murphy, Annu. Rev. Immunol. 12:593-633, 1994).

A characteristic feature of most GPCRs known to date is that seven clusters of hydrophobic amino acid residues are located in the primary structure and cross the cell membrane (span) in each of these regions. This domain is thought to represent a transmembrane alpha-helix linked by three intracellular loops, three extracellular loops, and amino- and carboxyl-terminal domains (K. Palczewski et al., Science 289, 739-45 ( 2000)). Most GPCRs have a single conserved cysteine residue in each of the first two extracellular loops that form disulfide bonds that are thought to stabilize the functional protein structure. The seven transmembrane regions are denoted by TM1, TM2, TM3, TM4, TM5, TM6, and TM7. These structures described in detail above are common among G protein-coupled receptor proteins, and the amino acid sequence corresponding to the region (membrane-spanning region or transmembrane region) through which the protein passes and the amino acid sequence near the membrane-spanning region. Is usually known to be highly conserved among these receptors. Thus, due to the high level of homology in GPCRs, identification of novel GPCRs, as well as identification of both the intracellular and extracellular portions of such novel substances, are readily performed by those skilled in the art. By way of example, the sequence of over 50 GPCRs is provided in Watson and Arkinstall (1994), which is incorporated herein by reference. The document also describes the exact residues comprising each transmembrane domain, for each sequence.

The binding site for the small ligand of the G-protein coupled receptor is located near the extracellular surface and is thought to contain a hydrophilic socket formed by several G-protein coupled receptor transmembrane domains, the socket being It is surrounded by hydrophobic residues of the G-protein coupled receptor. The hydrophilic side of each G-protein coupled receptor transmembrane helix is assumed to face inward to form a polar ligand binding site. TM3 is involved in several G-protein coupled receptors with ligand binding sites, such as those containing TM3 aspartate residues. In addition, TM5 serine, TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine are also involved in ligand binding. Receptors with other larger ligands, such as peptide hormone receptors and glycoproteins (LH, FSH, hCG, TSH), and ligand binding sites for receptors of the Ca2+/glutamate/GABA family likewise have extracellular domains and residues in the loop. .

Important in switching the receptor from inactive to active is the ligand-induced structural change of transmembrane helix 3 (TM3) and 6 (TM6) of GPCRs with 7 transmembrane spanning helixes (U. Gether, and BK Kolbilka, J. Biol. Chem. 273, 17979-17982 (1998)). These helix movements in turn change the structure of the receptor's intracellular loop, promoting the activation of the related heterotrimeric G protein. Mutagenesis studies (S. Cotecchia, J. Ostrowski, MA Kjelsberg, MG Caron and RJ Lefkowitz, J. Biol. Chem. 267, 1633-1639 (1992); E. Kostenis, BR Conklin and J. Wess, Biochemistry 36, 1487-1495 (1997); MA Kjelsberg, S. Coteechia, J. Ostrowski, MG Caron, and RJ Lefkowitz, J. Biol. Chem. 267, 1430-1433 (1992)) as a third intracellular loop (i3) Has been demonstrated to mediate a large part of the coupling between the receptor and the G protein. The I3 loop expressed as a minigene has also been shown to compete directly with adrenergic receptors for Gq binding (LM Luttrell, J. Ostrowski, S. Cotecchia, H. Kendal and RJ Lefkowitz, Science 259, 1453-1457 (1993). )), it was found to be capable of activating G protein as a soluble peptide in cell-free conditions (T. Okamoto et al., Cell 67, 723-730 (1991)).

The antigen may be of an endogenous source of target cells (sometimes referred to as antigen-expressing cells). Alternatively, exogenous molecules can be introduced into cells to express the antigen. Introducing the antigen into cells can be carried out by methods known to those skilled in the art. In one embodiment, a polynucleotide encoding an antigen as a polypeptide can be inserted into a vector in vitro, which vector can be further introduced into a target cell for expression. The polynucleotide may contain the cDNA sequence, DNA sequence, or other sequence known in the art of the target antigen. The vector may be a plasmid, cozmid, liposome, or natural or artificial vector known in the art. Introduction can be transfection, transformation, infection, direct micro-injection of a substance, biolistic particle delivery, electroporation, or other methods known in the art. Target cells expressing the antigen are, for example, cells obtained directly from animals, such as cancer cells, non-cancer cells, early cells, etc., or molecularly engineered cells, such as cultured cells (e.g. Chinese Hamster Ovary (CHO ) Cells, HEK293 cells, etc.), immortalized cells, transfected/infected cells, T cells, and the like. Alternatively, the target cells can be derived from non-animal origin, such as bacteria, insects, and the like. In one embodiment, the cells expressing the target antigen are yeast cells, preferably yeast spheroblasts. Alternatively, the target “cell” can be an artificial cell-analog or structure, such as a liposome, a single-layer membrane body, and the like. Expression of the antigen in the target cell or cell-analog may be transient, i.e. expression may be weakened or stopped after a relatively short period of time (e.g., minutes to days), or stable, i.e. for a relatively long period of time (e.g., several days or It can be maintained at a relatively stable level) after several generations of cell development. In a preferred embodiment, the antigen is expressed on the extracellular surface of the plasma membrane of the target cell. In another preferred embodiment, the antigen is an intrinsic membrane antigen or a multispan membrane antigen. To be placed on the plasma membrane, the antigen can be expressed directly at these sites, or it can be expressed in the cytoplasm of the target cell and then translocated to these sites. The translocation can be performed before or after the natural process of the target cell or antigen expression, for example, the attachment of a signal/tag molecule (eg, Golgi classification signal, an antibody to a specific membrane-binding molecule, etc.), anchoring graft; For example, glycosylphosphatidylinositol (GPI) anchor), or chemical crosslinking to the antigen, mutating the antigen, or translocating by other methods known in the art.

Immune binding agents -Expressing cells and immunization

In one embodiment, the immunobinding agent-expressing cells to be selected by the methods described herein are mammalian B cells, preferably rabbit B-cells.

In a preferred embodiment, the B cells originate from an animal immunized with the target. Immunization of animals can be carried out by methods known to those skilled in the art. Typically, B-cells are isolated from lymphoid organs (eg, spleen or lymph nodes) of immunized animals.

In a preferred embodiment, immunization of the antigen is carried out by DNA immunization/vaccination. Alternatively, cells expressing the target antigen are injected into animals (eg, rabbit, rat, mouse, hamster, sheep, goat, chicken, etc.) for immunization. A preferred animal for the immunization step is a rabbit. DNA immunization/vaccination triggers a rapid immune response and allows only native expression of the target antigen, usually native expression, to occur. Since there is no involvement in the expression and manipulation of recombinant proteins, this process is more efficient and cost-effective than traditional immunization methods using recombinant proteins. In addition, more importantly, antigens expressed in vivo have the same secondary structure and may even have the same post-transcriptional modification as the target protein in nature, which improves the accuracy of recognition by antibodies prepared against the target antigen. Improve. An example of DNA immunization is described in Canadian patent applications CA2350078 and WO04/087216. In the above description, a DNA encoding a polypeptide as a target antigen is directly introduced into an animal through a gene gun method to express the polypeptide in the animal, and an antibody against the polypeptide is formed by this expression. To achieve more potent antibody formation, so-called gene adjuvants are applied simultaneously with the polypeptide-encoding DNA. These gene adjuvants are plasmids that express cytokines (eg GM-CSF, IL-4 and IL-10) and stimulate humoral immune responses in laboratory animals. In a preferred embodiment, the antibody against the target antigen is expressed on the surface of B cells as a B cell receptor (BCR). In another preferred embodiment, the antibody-expressing cells are memory B cells, characterized by the lack of IgM on the surface and distinguishable from conventional B cells.

In a preferred embodiment, the immuno-binding agent-expressing cell is a yeast cell and the immuno-binding agent is preferably an antibody fragment, more preferably a scFv.

Fluorescence-activated cell sorting ( FACS Screening using)

After the immunization step, it is necessary to separate membrane-bound antibodies on B cells that specifically bind to the target antigen from other cells expressing non-specific antibodies. In a preferred embodiment, through antigen-antibody binding, B cells expressing specific antibodies in the plasma membrane are attached to target cells expressing the antigen. In another preferred embodiment, one or more interactions (eg, identical or different antigen-antibody interactions, chemical cross-links, ligand-receptor interactions, etc.) may occur between B cells and target cells. B cells may exist as a pool of B cells expressing different antibodies, or from an immunized animal, from a pool of immunized/non-immunized animals, or from an in vitro engineering process, e.g., V( D)J can exist with other immune cells, collected directly from a library of B cells expressing different antibodies by gene recombination technology.

Isolation of B cells expressing an antibody specific for a target antigen can be performed by a method known in the art. These methods include, but are not limited to, panning on antigen, limited dilution, affinity purification, or other methods that utilize the characteristics of the expressed antibody or antibody-producing B cells.

In a preferred embodiment, the antigen-expressing cells are labeled with a tag useful for subsequent detection and/or isolation of attached B cells. The tag may be a crosslinking agent, an antigen/antibody, a small molecule (eg, glutathione (GSH), biotin/avidin, etc.), a magnetic particle, a fluorescent tag, or the like. In a preferred embodiment, the antigen-expressing cells are labeled with a fluorescent protein/peptide. In another embodiment, antibody-producing B cells are also labeled with a tag, preferably with a different fluorescent protein/peptide. In another embodiment, B cells attached to antigen-expressing cells can be detected by emitting fluorescence from fluorescent proteins/peptides labeled on B cells. In another embodiment, attached B cells and antigen-expressing cells can be detected by emitting fluorescence from two different fluorescent proteins/peptides labeled on them. In a preferred embodiment, B cells expressing an antibody specific for the target antigen can be detected and further separated from other antibody-producing B cells by releasing fluorescence from both these and the labeled fluorescent protein/peptide on the attached APCs. . Preferably, the detection and separation can be performed by fluorescence-activated cell sorting (FACS) technology.

Acronym FACS is a trade name and owned by Becton Dickinson (Franklin Lakes, NJ). The term FACS as used herein refers to a form of flow cytometry based on cell sorting.

Fluorescence-activated cell sorting is a specialized type of flow cytometer. This provides a method of classifying one cell and a heterogeneous mixture of biological cells into two or more containers over time, based on the specific light scattering and fluorescence properties of each cell. It is a useful scientific tool, such as the rapid, objective and quantitative recording of fluorescence signals from individual cells as well as physically isolating specific cells.

In a typical FACS system, the cell suspension is placed in a narrow center through which a liquid stream flows rapidly. The flow is arranged so that cells that are larger than their diameter are separated between cells. A vibrating mechanism causes a stream of cells to break down into individual droplets. The system regulates such that there is a low probability of more than one cell present as droplets. Immediately before the stream is destroyed by droplets, the flow is passed through a fluorescence measurement station that measures the corresponding fluorescence properties of each cell. Place the electric charging ring at the very point where the stream breaks down with droplets. As the ring is charged and broken from the stream based on the previous fluorescence intensity measurement, the opposite charge is trapped on the droplet. The charged droplet then falls through an electrostatic deflection system that classifies the droplet's charge into the container. In some systems, charge is applied directly to the stream and the droplets are destroyed, retaining the same sign of charge as the stream. The stream is then returned to neutral after droplet destruction.

Fluorescent labels for the FACS technique depend on the lamp or laser used to excite the fluorochrome and the detector available. The most commonly available laser for single laser machines is the blue argon laser (488 nm). Fluorescent labels operable for this kind of laser include, but are not limited to, 1) for green fluorescence (usually labeled FL1): FITC, Alexa Fluor 488, GFP, CFSE, CFDA-SE, and DyLight 488; 2) For orange fluorescence (usually FL2): PE, and PI; 3) For red fluorescence (usually FL3): PerCP, PE-Alexa Fluor 700, PE-Cy5 (TRI-COLOR), and PE-Cy5.5; And 4) for infrared fluorescence (typically FL4; in some FACS machines): PE-Alexa Fluor 750, and PE-Cy7. Other lasers and their corresponding fluorescent labels include, but are not limited to, 1) red diode lasers (635 nm): Allophycocyanin (APC), APC-Cy7, Alexa Fluor 700, Cy5, and Draq-5; And 2) purple laser (405 nm): Pacific Orange, Amine Aqua, Pacific Blue, 4',6-diamidino-2-phenylindole (DAPI), and Alexa Fluor 405.

In a preferred embodiment, it is preferentially selected to stain B cells with labeled anti-IgG and anti-IgM antibodies and to positively stain only memory B cells with anti-IgG antibodies except for anti-IgM antibodies. Thereby, IgG generally has a higher affinity than IgM; Positive B-cells (which are characteristic for memory B-cells) that express IgG on the surface but not IgM are selected. For this purpose, multicolor staining is preferably used, wherein antibodies specific for IgG and IgM are labeled differently, for example, with APC and FITC, respectively. Preferably, the target antigen and/or target cells expressing the target antigen are also labeled. In one embodiment, the target antigen is indirectly stained by staining the cells expressing the target antigen with an intracellular fluorescent dye.

The present invention is a method of using FACS to select a pool of B cells, capable of identifying and isolating B cells that produce an antibody that binds to a cell expressing a target antigen and specifically binds to the target antigen. Provides. Preferably, B cells are labeled with a fluorescent label and cells expressing the target antigen are separately labeled with different fluorescent labels. These markers may be intracellular, extracellular, or integrated into the plasma membrane. After immunization to produce antibodies, all B cells are collected together and passed through a FACS system. Only B cells that produce antibodies specific for the target antigen will attach to the antigen-expressing cells. Their adhesion shortens the distance between these two cells in the flow compared to the enormous separation between each other cell, causing a detectable “bi-color event” while passing through the scanning laser beam. . Thus, B cells producing the antibody can be identified and sorted into different collection tubes from other non-specific B cells.

In another preferred embodiment, when the cell characteristics, for example, depolarization, fluorescence resonance energy transfer (FRET), etc. are partially modified by the interaction between B cells and corresponding antigen-expressing cells, a label having a higher fluorescence is used as described above. It can be added to cells that can be modified together. Thus, when a B cell and an antigen-expressing cell come into contact, a “tri-color” event or an event containing three or more colors at the same time will occur.

Alternatively, there is no need for a fluorescent label to be present in the identification and classification of B cells in the attached state. In one embodiment, a cell-cell interaction results in a functional change in one of the cells. In another embodiment, these functional changes can be used to identify and further isolate B cells producing antibodies that specifically bind to the target antigen. For example, cell-cell interactions can functionally block or activate receptor signaling in one of the cells, resulting in cell changes detectable by the FACS system, such as changes in Ca ^{2 +} efflux. Thus, by monitoring these detectable functional changes, the B cells in question can also be identified and isolated. One of the specific embodiments of functionally blocking or activating receptor signaling is to incubate B-cells with cells that functionally express GPCRs (G-protein-coupled receptors). An agonist capable of inducing GPDR by adding a signal to the mixture via GPCR mediates Ca ^{2 +} efflux from the endoplasmic reticulum. If the antibody present in the B- cell functional blocks the agonist screen signal, Ca ^{2 +} efflux consequently the cells is blocked by the cell interaction. Ca ^{2 +} efflux can be measured quantitatively, for example by flow cytometry. Thus, only B-cells/target cell aggregates that show an increase or decrease in ^{Ca 2 + efflux are classified.}

Isolated Affinity analysis for antibodies produced by B cells

In certain embodiments, B-cells are cultured under conditions suitable to allow the antibody to be secreted into the culture medium. The produced antibody is, for example, a monoclonal antibody. A helper cell line such as a thymoma helper cell line (eg, EL4-B5, Zubler et al., 1985, J. Immunol., 134(6):3662-3668) can be used for culture.

Optionally, additional affinity assays can be performed prior to further processing to assess the selectivity and competitive ability of the antibodies produced by the isolated B cells. These assays include, but are not limited to, cell-based assays (eg, cell ELISA (CELISA), which is an improved ELISA method that uses whole cells for coating). As discussed in CA2350078, CELISA can be carried out as described in the Examples below. An antibody generated for specific binding to a target is tested to exclude, for example, an antibody directed against a protein expressed on a cell surface other than the target protein by performing a validation step.

Alternatively, the identified and isolated B cells can be directly probed for antibody affinity and the B cells can be isolated from the attached antigen-expressing cells prior to processing.

For antibody production Isolated Further processing of B cells

B cells that have been identified, isolated, and optionally tested in an affinity assay (eg, CELISA) can be further processed to produce the immunobinder. For example, the traditional hybridoma technique can be used. This includes steps such as purification of the immunobinding agents, description of their amino acid sequence and/or nucleic acid sequence.

Alternatively, the characterization of the binders is carried out in their scFv format. In this case, the CDR sequences of the binding agent expressed in sorted B-cells are restored by RT-PCR directly from cultured sorted cells or single cells. Two pools of partially overlapping oligonucleotides (where one of the oligonucleotide pools encodes for the CDRs and the second pool encodes for the framework region of a suitable scFv scaffold) combines the humanized scFv in a one-step PCR process. To occur. Instead of characterization of secreted IgG in cell culture supernatant by HT sequencing, cloning and production, clone selection based on the performance of purified humanized scFvs is performed. ScFv scaffolds suitable for taking CDRs from rabbit antibodies have been identified and characterized (“rabbitized” human FW or rabbit receptor RabTor; see WO09/155726, which is incorporated herein by reference in its entirety). In some cases even rabbit specific evidence of concept for various CRs containing inter-CDR disulfide bonds has been found.

A general description of rabbitized antibodies is described below.

Of immune binding agents Grafting (grafting)

The antigen binding regions or CDRs of an immunobinding agent identified by the method of the present invention can be grouped into the receptor antibody framework. Such grafting can, for example, reduce the immunogenicity of an immunobinding agent or improve its functional properties, for example improve thermodynamic stability.

A general method for grafting CDRs into the human receptor framework is described in the following literature, which is incorporated herein by reference in its entirety: Winter, US Pat. No. 5,225,539.

Specific strategies for grafting CDRs from rabbit monoclonal antibodies are disclosed in US Provisional Patent Applications 61/075,697, and 61/155,041, which are incorporated herein by reference in their entirety. These strategies are related to Winter's, but differ in that the receptor antibody framework is particularly suitable as universal receptors for all human or non-human donor antibodies. In particular, the human single chain framework FW1.4 (a combination of SEQ ID NO: 1 (designated as a43 in WO03/097697) and SEQ ID NO:2 (designated as K127 in WO03/097697)) is the antigen-binding of rabbit antibodies. It was found to be highly compatible with the site. Thus, FW1.4 is a suitable scaffold for constructing stable humanized scFv antibody fragments derived from grafting of rabbit loops.

In addition, it was found that FW1.4 can be optimized by substituting the 5th or 6th residue position in the heavy chain of FW1.4 and/or substituting the 1st position in the light chain of FW1.4. Thereby, it was surprisingly found that the loop arrangement of all kinds of rabbit CDRs in VH can be completely maintained completely independent of the sequence of the donor framework. The 5th or 6th residue of the heavy chain of FW1.4 as well as the 1st position of the light chain are conserved in the rabbit antibody. Co-residues for positions 5 or 6 in the heavy chain, as well as for position 1 in the light chain, are deduced from the rabbit repertoire and introduced into the sequence of the human receptor framework. As a result, the modified framework 1.4 (referred to as rFW1.4 in the literature) is substantially compatible with rabbit CDRs. In contrast to the wild-type single chain of rabbits, rFW1.4 containing different rabbit CDRs is well expressed and almost completely retains the affinity of the original donor rabbit antibody.

Thus, an exemplary immune binding agent receptor framework

(i) a variable heavy chain framework having at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity with SEQ ID No. 1; And/or

(ii) a variable light chain framework having at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity with SEQ ID No.2.

In a preferred embodiment, the variable light chain comprises threonine (T) at position 87 (AHo numbering).

In a preferred embodiment, the immune binding agent receptor framework is

(i) a variable heavy chain framework selected from the group consisting of SEQ ID No.1, SEQ ID No.4 and SEQ ID No.6; And/or

(ii) a variable light chain framework of SEQ ID No.2 or SEQ ID No.9.

In a preferred embodiment, the variable heavy chain framework is linked to the variable light chain framework through a linker. The linker is a suitable linker, for example a linker comprising 1 to 4 repeats of the sequence GGGGS (SEQ ID No. 10), preferably (GGGGS) ₄ peptides (SEQ ID No. 8), or in the literature: Alfthan et al. (1995) Protein Eng. It may be a linker as disclosed in 8:725-731.

In a most preferred embodiment, the immunobinding agent receptor framework is a sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID No.3. More preferably, the immunobinding agent receptor framework comprises or is SEQ ID No.3.

In another preferred embodiment, the immunobinding agent receptor framework is a sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID No.5. More preferably, the immunobinding agent receptor framework comprises or is SEQ ID No.5.

In another preferred embodiment, the immunobinding agent receptor framework is a sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID No. 7. More preferably, the immunobinding agent receptor framework comprises or is SEQ ID No.7.

In addition, the exemplary variable heavy chain framework of SEQ ID No. 1, which further comprises one or more amino acid residues that generally support the arrangement of CDRs derived from rabbit immunobinding agents, can be used. In particular, the residue is present at one or more amino acid positions selected from the group consisting of 24H, 25H, 56H, 82H, 84H, 89H and 108H (AHo numbering). These positions have been demonstrated to affect CDR alignment and are therefore considered for mutations to accommodate donor CDRs. Preferably, at least one of the residues is selected from the group consisting of: threonine at position 24 (T), valine at position 25 (V), glycine or alanine at position 56 (G/ A), lysine at position 82 (K), threonine at position 84 (T), valine at position 89 (V) and arginine at position 108 (R) (AHo numbering).

In a preferred embodiment, the variable heavy chain framework is or comprises SEQ ID No.4 or SEQ ID No.6. The two variable heavy chain frameworks can be mixed with a suitable light chain framework, for example.

The sequence disclosed above is as follows (the X residue is the CDR insertion site):

SEQ ID No.1: variable heavy chain framework of FW1.4(a43)

SEQ ID No.2: variable light chain framework of FW1.4 (K127)

SEQ ID No.3: Framework of FW1.4

SEQ ID No.4: variable heavy chain framework of rFW1.4

SEQ ID No.5: framework of rFW1.4

SEQ ID No.6: variable heavy chain framework of rFW1.4 (V2)

SEQ ID No.7: framework of rFW1.4(V2)

SEQ ID No.8: linker

SEQ ID No. 6: substituted variable light chain framework of FW1.4

Therefore, unlike the general method of Winter, the framework sequence used in the humanization method of the present invention requires a framework sequence that exhibits maximum sequence similarity with the sequence of a non-human (e.g., rabbit) antibody from which donor CDRs are derived. It is not. In addition, framework residues that are grafted from the donor sequence are not required to support the CDR alignment.

The receptor antibody framework may also include one or more of the stability enhancing mutations described in US Provisional Patent Application No. 61/075,692, which is incorporated herein by reference in its entirety. Examples of solubility enhancing substitutions in the heavy chain framework were found at positions 12, 103 and 144 (AHo numbering). More preferably, the heavy chain framework is (a) serine at position 12 (S); (b) serine (S) or threonine (T) at position 103 and/or (c) serine (S) or threonine (T) at position 144. In addition, stability enhancing amino acids may be present at one or more of positions 1, 3, 4, 10, 47, 57, 91 and 103 (AHo numbering) of the variable light chain framework. More preferably, the variable light chain framework includes glutamic acid at position 1 (E), valine at position 3 (V), leucine at position 4 (L), and serine at position 10 (S); Arginine at position 47 (R), serine at position 57 (S), phenylalanine at position 91 (F) and/or valine at position 103 (V).

Example

Throughout the examples, the following materials and methods are used unless otherwise stated.

Substance and method

In general, unless otherwise indicated, conventional chemical techniques, molecular biology, recombinant DNA techniques, immunology (especially, for example antibody techniques), and standard techniques of polypeptide production are used in the practice of the present invention. See, for example, the following documents: Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); And Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Cell ELISA (CELISA)

The following examples illustrate the use of CELISA to analyze cells expressing CXCR2:

CHO cells expressing CXCR2 can be seeded into 96-well half area plates at a density of 50,000 cells/well. After incubation at 37° C. overnight, the cells can be fixed at room temperature for 30 minutes with 1% formaldehyde in PBS. The cell layer can be washed 3 times, and non-specific binding sites can be blocked with cell culture medium for 1 hour at room temperature. Subsequently, after three washing steps, the supernatant may be diluted 1:1 in the culture medium and added to the well. In three control wells, a commercially available rabbit anti-CXCR2 antibody can be spiked into the supernatant. Subsequently, the supernatant may be incubated on the cell layer for 1 hour and 30 minutes at room temperature. Finally, rabbit IgGs are detected with goat anti-rabbit IgG Fc antibody coupled to HRP. Upon addition of the peroxidase substrate (Blue POD substrate from Roche), a colorimetric reaction develops, which can be stopped with 1M HCl after 25 minutes. Absorbance can be measured at 450 nm.

Example One

B cell selection system using soluble antigen

Here, it is described that the FACS (fluorescence-activated cell sorting)-based selection system described in the present invention can select the target, specifically, B cells that bind to the soluble protein through the B cell receptor (BCR). do. In this example, the target is a single chain anti-VEGF antibody ESBA903 labeled with fluorescent dyes (PE and PerCP). A lymphocyte suspension is prepared from the spleen of rabbits immunized with the recombinant target. Cells are then incubated with PE and PerCP labeled scFvs as well as antibodies specific for IgG (APC-labeled) or IgM (FITC labeled). On the surface, ESBA903-positive B-cells expressing IgG but not IgM were sorted and selected in 96-well plates (Fig. 2). As shown by panel A of Figure 2, lymphocytes pass along the anterior chamber and are scattered laterally. Among these, IgG+ IgM- cells (probably memory B cells) are selected (panel B). Cells double-stained with scFv-PE and scFv-PerCP are expected to encode high affinity IgGs for scFv (Panel C). Cells with the brightest fluorescence are sorted in 96-well plates with the sorting statistics listed in panel D. Using a thymoma helper cell line (EL4-B5: Zubler et al, 1985, see J. Immunol. 134(6):3662-3668), selected B cells are amplified, differentiated into plasma cells, and then antibodies are secreted. The affinity of these IgG molecules to the target protein is confirmed by ELISA and Biacore assay. Kinetic indicators for the 7 selected clones are shown in Table 1. These clones from approximately 200 sorted cells exhibit high binding affinity in the low nanomolar to piconol range. Finally, mRNAs for secreted IgG molecules were isolated from these six clones and the CDRs were grafted onto the ESBATech single chain framework RabTor, also named Framework rFW1.4.

Table 1 : Kinetic values for 7 B cell culture supernatant

Table 1a : Classification statistics for Figure 2

Selected literature:

Zuber et al. Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction. J Immunol. 1985; 134(6):3662-3668.

Transmembrane Target

The selection system described above works efficiently when the target is soluble and also when recombinant proteins are available. However, some of these targets are multispan transmembrane proteins (eg GPCRs and ion channels). Traditional immunization with recombinant proteins is not advisable or impossible in these cases. Further, FACS selection of B cells cannot be performed on the basis of binding of purified and labeled proteins when the antigen is an intrinsic membrane protein. In line with this problem, the above-mentioned process was improved as follows.

1) Immunization with DNA instead of recombinant protein:

DNA vaccination elicits a rapid immune response against natural antigens. Since no recombinant protein is required, this technique is very cost effective on the one hand, and more importantly, on the other hand, this method allows the natural expression of the intrinsic membrane complex and/or multispan membrane protein. .

2) FACS selection of B-cells that bind to cells expressing the intrinsic membrane target protein:

Flow cytometers typically measure the fluorescence emitted by single cells when they cross a laser beam. However, some of the researchers already have cell-cell interactions, such as cadhedrins; Panorchan et al, 2006, J. Cell Science, 119, 66-74; Leong et Hibma, 2005, J. Immunol. , 302, 116-124) or integrins (Gawaz et al, 1991, J. Clin. Invest, 88, 1128-1134). However, such studies have not demonstrated whether such cell-cell interactions remain intact during the physical phase of cell sorting. In addition, it has not been suggested that binding of the B cell receptor to its target present on the surface of other cells is strong enough to allow such material classification.

In order to select B-cells that bind to the transmembrane target, cells (e.g., CHO or HEK293 cells) can be transiently or preferentially stably transfected with a selected target, or cells expressing a naturally selected target are selected. Can be used. Such target cells are stained with an intracellular fluorescent dye (eg calcein) and incubated with immunized rabbit memory B lymphocytes. B lymphocytes are stained with fluorescent antibodies that bind to cell surface specific markers. Thus, it is possible to select a bi-color "event" (see Fig. 1) consisting of two cells that adhere to each other through a BCR-target interaction.

Further processing of these B cells is carried out as described above, whereby monoclonal antibodies are produced, for example in IgG or scFv format. To evaluate the affinity of these antibodies for the target, CELISA (ELISA, where the coating step is performed with whole cells) is performed. In this way, the selectivity of the antibody and the ability to compete with the ligand can be evaluated. Finally, the CDRs of this clone are cloned into the rabbitization framework (RabTor) of the present invention by gene synthesis using an oligo extension method.

Although it is not necessary to limit the readout for B-cell sorting to cell-cell interactions, it can also be used to select for the ability to functionally block/activate receptor signaling of these interactions. For example, B-cells can be cultured with cells that functionally express GPCRs. An agonist signaling via GPCR can be added to the mixture to induce GPCR-mediated Ca2+ outflow from the endoplasmic reticulum. When antibodies present in B-cells functionally block agonist signaling, Ca2+ outflow is also consequently blocked by the cell-cell interaction. Ca2+ efflux can be quantitatively measured by flow cytometry. Thus, only B-cell/target cell aggregates that do not show Ca2+ outflow are sorted.

Example 2

Bead coated with anti- TNFα antibody and membrane- bound CHO expressing TNFα Intercellular Detection of interactions

Before initiating the selection of B cells for transmembrane proteins, it must be demonstrated that cell-cell interactions (and in particular those between BCR and transmembrane proteins on target cells) can be positively selected by FACS. To determine if high pressure in the flow cytometer stream breaks the non-covalent bond between the two cells, the following experiment was performed.

Membrane-bound TNFα (B-220 cells) (contains a mutant membrane-bound TNFα containing a point mutation at the TACE cleavage site that prevents cleavage and shedding of the TNFα ligand; e.g. Scallon et al. J Pharmacol Exp Exp. CHO cells stably transfected with Ther 2002; 301:418-26) are incubated with beads coated with PE-labeled anti-TNFα antibody. In the set-up, the beads mimic memory B cells. As a negative control, beads coated with non-transfected CHO cells as well as APC-labeled unrelated antibody (anti-CD19) are used. After incubating for 2 hours while stirring at 4° C., the cell-bead suspension was analyzed by FACS (using a 130 μm nozzle). 3 shows that specific binding between anti-TNFα beads and TNFα-transfected CHO cells can be clearly detected by FACS. In fact, in this sample (top panel) 2/3 of the beads bound to the cells (585 bound vs. 267 unbound). In contrast, in the control samples (middle and lower panels), there were few beads bound to CHO cells. In addition, both bead populations (anti-TNFα-PE and anti-CD19-APC) are mixed with TNFα-transfected CHO cells. 4 shows that about half of the anti-TNFα beads bind to CHO cells, while almost all of the anti-CD19 beads remain unbound. The percentage of beads that bind to cells in each sample is detailed in Table 2. Thus, it is demonstrated that specific selection of single B cells that bind to the intrinsic membrane target protein through the B cell receptor may be possible using a flow cytometer.

Table 2 : Percentage of beads bound to CHO cells in each sample

Table 2a : Classification statistics for the top panel of FIG. 3; Beads coated with anti-TNFα antibody bind to TNFα-transfected CHO cells

Table 2b : Classification statistics for the middle panel of Figure 3; Beads coated with anti-CD19 antibody do not bind TNFα-transfected CHO cells

Table 2c : Classification statistics for the lower panel of Figure 3; Beads coated with anti-TNFα antibody do not bind to CHO wild-type cells

Table 2d : Classification statistics for Figure 4

Example 3

Detection of interaction between B cells isolated from anti- TNFα antibody immunized rabbits and CHO cells expressing membrane-bound TNFα saturated with anti- TNFα antibody

For the experiment shown in Figure 5, lymphocytes are isolated from immunized rabbit spleens or non-immunized rabbit spleens with anti-TNFα antibody (ESBA105, produced internally). They are stained with anti-rabbit IgG-APC and anti-rabbit IgM-FITC (AbD serotec) and then pre-sorted to obtain a pure population of memory B cells (IgG+/IgM-). At the same time, TNFα-expressing CHO cells (donated by Dr. P Scheurich, Univ. of Stuttgart) were loaded with 1 μg/ml of calcein-red (Invitrogen), a cytoplasmic dye for fluorescently staining living cells. These cells are then washed once and incubated with (or without, as a negative control) 100 μg/ml of ESBA105, and finally washed again 3 times with PBS. Finally, memory B cells were mixed with CHO cells at a ratio of about 1:10 and ^{incubated for 2 hours on a 4°C rotating plate (concentration: 3*10 7} cells/ml).

The following samples are prepared:

1) ESBA105 immunized rabbit CHO-TNFα cells + ESBA105 + memory B cells

2) Non-immunized rabbit CHO-TNFα cells + ESBA105 + memory B cells

3) ESBA105 immunized rabbit CHO-TNFα cells + memory B cells

After incubation for 2 hours, three samples were measured by FACS using a 70 μm nozzle. According to the population system shown in Table 3a, 5% of ESBA105 immunized cells bind to “ESBA105-coated” TNFα transgenic CHO cells. By comparison, only 0.5% of non-immunized B cells bind to these "ESBA105-coated" TNFα transgenic CHO cells (Table 3b), and 0.6% of the immunized B cells are on the CHO cell surface in the absence of ESBA105. Combine (Table 3c). These results indicate that specific interactions between BCR (B cell receptor) and transmembrane targets can be detected by FACS.

Table 3a : Classification statistics for the top panel of FIG. 5B

Table 3b : Classification statistics for the middle panel of FIG. 5B

Table 3c: Classification statistics for the lower panel of FIG. 5B

Example 4

ESBA105 saturated with isolated from immunized rabbit B cells and ESBA105 membrane-expressing CHO cells bound TNFα between the detection of the interaction

In further experiments, pre-sorting of memory B cells is not performed. The entire lymphocyte population is incubated with stained CHO-TNFα-ESBA105 cells. Transgenic CHO cells are prepared as described above. However, in order to prevent their proliferation in B cell culture medium after sorting in a 96-well plate, the cell-cycle of the cells was stopped with mitomycin C (M4287-2MG) prior to calcein staining. ESBA105 immunized rabbit lymphocytes are mixed with stained CHO cells in a ratio of 3:1 (concentration of cell suspension: approximately 3-10 ⁷ cells/ml) and incubated for 2 hours on a rotating plate at 4°C. Thereafter, the cell suspension was analyzed by FACS and the memory B cells binding to CHO-TNFα-ESBA105 were sorted according to the gate shown in FIG. 6, which is 1, 10 or 100 cells/ml as shown in Table 3. Sorted cells are 5.5% of the memory B cell population, each 0.2% of the total event.

Sorted cells were collected in a 96-well plate and incubated for 13 days at 37° C. with _{5% CO 2.} The culture supernatant is then collected and subjected to direct ELISA to check for the presence of ESBA105 binding to IgGs. The results of ELISA (Table 3) show that ESBA105 specific antibodies can be detected in numerous wells, and also in wells where single B cells are sorted. Biacore (GE Healthcare) analysis of these supernatants (Table 4) confirms that these antibodies truly bind to the ESBA105 target.

Table 3 : ELISA analysis of B cell culture supernatant collected 13 days after sorting. There were no B cells sorted in column A wells to confirm the specificity of the OD450 signal. In wells A11 and A12, polyclonal rabbit anti-ESBA105 antibody (AK3A; 2 μg/ml) was spiked into the supernatant as a positive control. Wells with an OD450 significantly higher than the baseline were highlighted in dark.

Table 4: Kinetic values and concentrations measured by Biacore for B cell culture supernatant. Only the supernatant from which one cell was sorted per well was measured. BLQ: Below the quantification limit

Example 5

Selection of lymphocytes from rabbits immunized with CXCR2 (27 classifications/29)

Three rabbits are immunized with the CXCR2 expression vector. After several intradermal application of CXCR2-cDNA, serum is collected and tested for the presence of specific antibodies on CXCR2 transfected cells. Subsequently, the lymph node cells are removed ^{, divided into 5, 1.6×10 7} cells each, frozen, and stored in a liquid nitrogen tank.

One aliquot is thawed and stained with antibodies specific for IgG (APC-labeled) or IgM (FITC labeled). At the same time, CXCR2-expressing CHO cells were treated with mitomycin C to prevent further growth without killing the cells, and loaded with 1 μg/ml calcein-red. The two cell preparations are ^{then mixed to a final cell concentration of 10 7} cells, with lymphocytes doubling as abundantly as CXCR2-transfected CHO cells. After incubating for 2 hours at 4° C. with gentle stirring, the cell suspension was filtered through a 50-µm filter and loaded onto the FACS. Gating is performed as described in Figure 6. One “event” per month (memory B cells bound to CXCR2-transfected CHO cells) is sorted (900 events in total) in a total of 10 x 96-well plates. Classified events were 3.1% of the memory B cell population, each representing 0.035% of the total amount of cells in the sample.

Selected lymphocytes are incubated in an incubator at 37° C. for 21 days. Every 2-3 days, 100 μl of the culture supernatant is collected from the wells and replaced with fresh medium. During the culture period, B cells proliferate and differentiate into plasma cells to secrete antibodies. To visualize that the supernatant contains a CXCR2 specific antibody, CELISA is performed. To this end, CHO cells expressing CXCR2 are seeded at a density of 50,000 cells/well in half an area of a 96-well plate. After incubation at 37° C. overnight, the cells are fixed in 1% formaldehyde in PBS for 30 minutes at room temperature. The cell layer is then washed 3 times and the non-specific binding sites are blocked with cell culture medium for 1 hour at room temperature. Next, after three washing steps, the supernatant is diluted 1:1 in the culture medium and added to the wells. In three control wells, a commercially available rabbit anti-CXCR2 antibody is spiked into the supernatant. The supernatant is incubated on the cell layer for 1 hour and 30 minutes at room temperature. Finally, rabbit IgGs are detected with goat anti-rabbit IgG Fc antibody coupled to HRP. Upon addition of the peroxidase substrate (Blue POD substrate from Roch), the colorimetric reaction is predicated, which is stopped with 1M HCl after 25 minutes. The absorbance is measured at 450 nm.

The CELISA revealed that 1.8% (16/900) of the wells showed a positive signal. All positives are confirmed by a second CELISA. The supernatant may also contain other cell lines: CHO-K1 (wild type) for identifying the ultimate nonspecific binding clone, CHO-human CXCR1 and CHO- for demonstrating agitation-activity against closely related receptors or counterparts of species. Test for mouse CXCR2. Finally, the supernatant is tested by direct ELISA for binding to a peptide consisting of 48 CXCR2 N-terminal amino acids. The results are presented in Table 5. All of the selected supernatants produced _{a potent OD 450 signal for human CXCR2 in CELISA.} Some of these were also slightly positive in control experiments on CHO wild-type cells, meaning they could bind in a non-specific manner. None of the clones were cross-reactive with human CXCR1 or mouse CXCR2. Finally, only some of the clones bound to the CXCR2 N-terminal peptide, indicating the possibility of another binding site on CXCR2. Without the possibility of immobilizing whole cells on the Biacore chip, it is currently not possible to quantitatively measure the affinity of the selected antibody for the CXCR2 receptor. However, the data gathered indicate that antibodies specific for human CXCR2 were selected using the cell-cell interaction sorting system described above.

Table 5 : Summary of CELISA results from anti-CXCR2 clones isolated during classification 27 and 29

Equivalent

Numerous improvements and alternative embodiments of the present invention will be apparent to those skilled in the art in view of the foregoing description. Accordingly, the present disclosure is to be considered for illustrative purposes and is for the purpose of teaching those skilled in the art the best way to practice the present invention. Structural details may be changed without substantially departing from the spirit of the invention, and the exclusive use of all improvements within the scope of the appended claims is preserved. The invention is limited only to the extent required by the appended claims and applicable laws.

All documents cited herein and similar, including patents, patent applications, papers, books, academic papers, research reports, web pages, drawings and/or appendices, are expressly full text, regardless of the format of such documents and similar. It is used as a reference. If one or more of the cited literature and similar, including defined terms, term usage, described technology, etc., differs from or contradicts this document, this document will control.

SEQUENCE LISTING <110> ESBATECH, AN ALCON BIOMEDICAL RESEARCH UNIT <120> mETHODS FOR IDENTIFYING IMMUNOBINDERS OF CELL-SURFACE ANTIGENS <130> CELL-CELL INTERACTION <150> CH00832/09 <151> 2009-06-02 <150> US61/155,105 <151> 2009-02-24 <150> US61/155,041 <151> 2009-02-24 <150> PCT/CH2009/000222 <151> 2009-06-25 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain framework of FW1.4 (a43) <220> <221> CDR <222> (26)..(75) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (90)..(139) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (172)..(221) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser 130 135 140 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 145 150 155 160 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln 210 215 220 Gly Thr Leu Val Thr Val Ser Ser 225 230 <210> 2 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> variable light chain framework of FW1.4 (KI27) <220> <221> CDR <222> (24)..(73) <223> CDR; at least 1 and up to 50amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (89)..(138) <223> CDR; at least 1 and up to 50amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (171)..(220) <223> CDR; at least 1 and up to 50amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 2 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly 225 230 <210> 3 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> framework of FW1.4 <220> <221> CDR <222> (24)..(73) <223> CDR; at least one and up to 50 amino acids can be present of absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (89)..(138) <223> CDR; at least one and up to 50 amino acids can be present of absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (171)..(220) <223> CDR; at least one and up to 50 amino acids can be present of absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (277)..(326) <223> CDR; at least one and up to 50 amino acids can be present of absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (341)..(390) <223> CDR; at least one and up to 50 amino acids can be present of absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (423)..(472) <223> CDR; at least one and up to 50 amino acids can be present of absent. Xaa can be any naturally occurring amino acid. <400> 3 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Ala Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 325 330 335 Glu Trp Val Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 385 390 395 400 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410 415 Val Tyr Tyr Cys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly Thr Leu Val Thr 465 470 475 480 Val Ser Ser <210> 4 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain framework of rFW1.4 <220> <221> CDR <222> (26)..(75) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (90)..(139) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (172)..(221) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser 130 135 140 Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg 145 150 155 160 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln 210 215 220 Gly Thr Leu Val Thr Val Ser Ser 225 230 <210> 5 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> framework of rFW1.4 <220> <221> CDR <222> (24)..(73) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (89)..(138) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (171)..(220) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (277)..(326) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (341)..(390) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (423)..(472) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 5 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Thr Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 325 330 335 Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys 385 390 395 400 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410 415 Val Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly Thr Leu Val Thr 465 470 475 480 Val Ser Ser <210> 6 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> variable heavy chain framework of rFW1.4(V2) <220> <221> CDR <222> (26)..(75) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (90)..(138) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (171)..(220) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser Lys 130 135 140 Asp Thr Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala 145 150 155 160 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly 210 215 220 Thr Leu Val Thr Val Ser Ser 225 230 <210> 7 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> framework of rFW1.4(V2) <220> <221> CDR <222> (24)..(73) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (89)..(138) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (171)..(220) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (277)..(326) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (341)..(390) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (423)..(472) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 7 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Thr Val Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 325 330 335 Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys 385 390 395 400 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410 415 Val Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly Thr Leu Val Thr 465 470 475 480 Val Ser Ser <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 9 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> substituted variable light chain framework of FW1.4 <220> <221> CDR <222> (24)..(73) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (89)..(138) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <220> <221> CDR <222> (171)..(220) <223> CDR; at least 1 and up to 50 amino acids can be present or absent. Xaa can be any naturally occurring amino acid. <400> 9 Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly 225 230 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 10 Gly Gly Gly Gly Ser 1 5

Claims (20)

(a) providing a plurality of B-cells;
(b) contacting the antigen with a plurality of B-cells, wherein the antigen comprises a first sortable label, and wherein the plurality of B-cells comprises a second sortable label; Staining with an anti-IgM antibody comprising a classifiable label of 3;
(c) using a cell sorter to separate one or more B-cells capable of specifically binding the antigen from a plurality of B-cells, wherein the first and second non-third classifiable labels in the complex The presence of the classifiable label of 2 is indicative of the binding of B-cells to the antigen, thereby identifying B-cell clones that bind to the antigen; And
(d) isolating heavy and light chain CDRs from the immunobinding agents expressed by the B-cell clones identified in step (c) and grafting them into the receptor antibody framework; Including,
A method of making an immunobinding agent that binds to an antigen of interest. The method of claim 1, wherein the plurality of B-cells are provided in a plurality of lymphocytes. The method of claim 1, wherein the plurality of B-cells are stained before contacting the antigen in step (b). The method of claim 1, wherein the plurality of B-cells are stained after contact with the antigen in step (b). The method of claim 1, wherein the identification of the B-cell clone comprises the isolation of the B-cells obtained in step (c) by replication. The method of claim 5, wherein clonal proliferation of the cells isolated by replication is subsequently performed. The method of claim 5, further comprising isolating nucleic acid molecules encoding immunobinding agents from B-cell clones that bind to the antigen. The method of claim 1, wherein the immunobinding agent is an antibody. The method of claim 8, wherein the antibody is full length immunoglobulin, Fab, Dab, scFv or nanobody. The method of claim 1, wherein the one or more isolated B-cells are cultured under appropriate conditions to secrete the antibody into the culture medium. The method of claim 10, wherein the antibody is tested for specific binding to the antigen. The method of claim 10, wherein the antibody is a mouse, rabbit, rat, hamster, sheep, chicken, camel, goat, or human antibody. The method of claim 1, wherein the classifiable label is a fluorescent label. The method of claim 1, wherein the antigen is a soluble antigen. The method of claim 1, wherein the B-cells are B-cells from rabbits. The method of claim 15, wherein the rabbit is immunized with the antigen. The method of claim 1, comprising immunizing the non-human animal with the antigen and isolating a plurality of B-cells from the immunized animal prior to step (a). The method of claim 17, wherein the classifiable label is a fluorescent label. The method of claim 17, wherein the antigen is a soluble antigen. The method of claim 17, wherein the animal is a rabbit.

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