Anti-rabbit CD19 antibodies and methods of use

Abstract

We report an antibody that binds to rabbit CD19, comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 33, or 34, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40, as well as methods for using the antibody, particularly methods used in identifying and selecting rabbit B cells that produce the antibody. TIFF2022515543000025.tif82140

Description

[Technical field]

[0001] The present invention relates to antibodies against rabbit CD19 (anti-rabbit CD19 antibodies), methods for their production and uses. [Background technology]

[0002] B cells develop from pro-B and pre-B cells, mature in the bone marrow to express antigen-specific cell surface antibody molecules, and then progress through the transitional T1 and T2 stages of immature B cell development in lymphoid tissues. In the spleen, the majority of B cells are distributed to the mature follicular compartment, or in smaller numbers to the marginal zone, B1a, and regulatory B10 cell subsets. Antigen-specific mature B cells activated by foreign antigens undergo clonally expanding and differentiating into short-lived plasma cells. Follicular B cells can also enter germinal centers where antigen-selective B cells proliferate, and their antigen receptors undergo affinity maturation and class switch recombination (CSR). B cells either leave the germinal center as memory B cells or differentiate into long-lived plasmablasts that migrate to the bone marrow. B cells are also found in the peritoneal cavity, including the regulatory B10 cell subset, which can produce interleukin-10, the B1a subset that produces the majority of natural Abs, and the B1b subset that generates adaptive antibody responses to TI antigens. Whereas most mature B cells express CD20, CD19 is expressed by mature B cells, as well as some pre-B cells, plasmablasts, and short- and long-lived plasma cells. CD19 is expressed at higher density by peritoneal B1a and B1b cells (Tedder, TF, Nat Rev. Rheumatol. 5 (2009) 572-577 (Non-Patent Document 1); LeBien, TW and Tedder, TF, Blood 112 (2008) 1570-1580 (Non-Patent Document 2)).

[0003] CD19 is a transmembrane protein (B cell coreceptor). The human CD19 structural gene encodes a cell surface molecule that assembles with the B cell receptor to lower the threshold for antigen receptor-dependent stimulation. CD19 acts as a B cell coreceptor, primarily together with CD21 and CD81. CD19 and CD21 are required for normal B cell differentiation (Carter, RH et al., Immunol. Res. 26 (2002) 45-54 (Non-Patent Document 3)). Upon activation, the cytoplasmic tail of CD19 is phosphorylated, leading to binding by Src family kinases and recruitment of PI-3 kinase.

[0004] Antibodies against human CD19 are described, for example, in WO 2004 / 106381 (Patent Document 1), WO 2005 / 012493 (Patent Document 2), WO 2006 / 089133 (Patent Document 3), WO 2007 / 002223 (Patent Document 4), WO 2006 / 133450 (Patent Document 5), WO 2006 / 121852 (Patent Document 6), WO 2003 / 048209 (Patent Document 7), U.S. Patent No. 7,109,304 (Patent Document 8), U.S. Patent Application Publication No. 2006 / 0233791 (Patent Document 9), U.S. Patent Application Publication No. 2006 / 0280738 (Patent Document 10), and the like. Reference 10), U.S. Patent Application Publication No. 2006 / 0263357 (Patent Document 11), U.S. Patent Application Publication No. 2006 / 0257398 (Patent Document 12), European Patent Application Publication No. 1648512 (Patent Document 13), European Patent Application Publication No. 1629012 (Patent Document 14), U.S. Patent Application Publication No. 2008 / 0138336 (Patent Document 15), International Publication No. 2008 / 022152 (Patent Document 16), International Publication No. 2011 / 147834 (Patent Document 17), and Bruenke, J. et al., Br. J. Hematol. 130 (2005) 218-228 (Non-Patent Document 4), Vallera, DA et al., Cancer Biother.Radiopharm.19(2004)11-23(Non-Patent Document 5); Ghetie,MA et al., Blood 104(2004)178-183(Non-Patent Document 6); Lang,P. et al., Blood 103(2004)3982-3985(Non-Patent Document 7); Loeffler,A. et al., Blood 95(2000)2098-2103(Non-Patent Document 8); Le Gall,F. et al., FEBS Lett.453(1999)164-168(Non-Patent Document 9); Li,Q. et al., Cancer Immunol. Immunother. 47 (1998) 121-130 (Non-Patent Document 10), Eberl, G. et al., Clin. Exp. Immunol. 114 (1998) 173-178 (Non-Patent Document 11), Pietersz, GA et al., Cancer Immunol. Immunother. 41 (1995) 53-60 (Non-Patent Document 12), Myers, DE et al., Leuk. Lymphoma. 18 (1995) 93-102 (Non-Patent Document 13), Bejcek, B.E. et al., Cancer Res. 55 (1995) 2346-2351 (Non-Patent Document 14); Hagen, IA et al., Blood 85 (1995) 3208-3212 (Non-Patent Document 15); Vlasfeld, LT et al., Cancer Immunol. Immunother. 40 (1995) 37-47 (Non-Patent Document 16); Rhodes, EG et al., Bone Marrow Transplant. 10 (1992) 485-489 (Non-Patent Document 17); Zola, H. et al., Immunol. Cell Biol. 69 (1991) 411-422 (Non-Patent Document 18); Watanabe, M. et al., Cancer Res. 50 (1990) 3245-3248 (Non-Patent Document 19); Uckun, FM et al., Blood 71 (1988) 13-29 (Non-Patent Document 20), and Pezzutto, A. et al., J Immunol. 138 (1987) 2793-2799 (Non-Patent Document 21). Monoclonal antibody SJ25-C1 is commercially available (product number 4737, Sigma-Aldrich Co. USA, SEQ ID NOs: 21-24). Antibodies with increased affinity for FcγRIIIA are described in WO 2008 / 022152 (Patent Document 16).

[0005] The rabbit genome has been sequenced and assembled in 2009 with a redundancy of 6.51 (see http: / / www.broadinstitute.org / science / projects / mammals-models / rabbit / rabbit-genome-sequencing-project (Non-Patent Document 22)).

[0006] No monoclonal anti-rabbit CD19 antibodies have been reported so far, and although claimed by several polyclonal antibodies, cross-reactivity of anti-human or anti-mouse CD19 antibodies with rabbit CD19 could not be demonstrated.

[0007] Therefore, there is a need to provide a monoclonal anti-rabbit CD19 antibody. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2004 / 106381 [Patent Document 2] International Publication No. 2005 / 012493 [Patent Document 3] International Publication No. 2006 / 089133 [Patent Document 4] International Publication No. 2007 / 002223 [Patent Document 5] International Publication No. 2006 / 133450 [Patent Document 6] International Publication No. 2006 / 121852 [Patent Document 7] International Publication No. 2003 / 048209 [Patent Document 8] U.S. Patent No. 7,109,304 [Patent Document 9] US Patent Application Publication No. 2006 / 0233791 [Patent Document 10] US Patent Application Publication No. 2006 / 0280738 [Patent Document 11] US Patent Application Publication No. 2006 / 0263357 [Patent Document 12] US Patent Application Publication No. 2006 / 0257398 [Patent Document 13] European Patent Application Publication No. 1648512 [Patent Document 14] European Patent Application Publication No. 1629012 [Patent Document 15] US Patent Application Publication No. 2008 / 0138336 [Patent Document 16] International Publication No. 2008 / 022152 [Patent Document 17] International Publication No. 2011 / 147834 [Non-patent literature]

[0009] [Non-Patent Document 1] Tedder, TF, Nat Rev. Rheumatol. 5 (2009) 572~577 [Non-Patent Document 2] LeBien, TW and Tedder, TF, Blood 112 (2008) 1570-1580 [Non-Patent Document 3] Carter, RH et al., Immunol. Res. 26 (2002) 45-54 [Non-Patent Document 4] Bruenke, J. et al., Br.J.Hematol.130(2005)218~228 [Non-Patent Document 5] Vallera, DA et al. Cancer Biother.Radiopharm.19(2004)11~23 [Non-Patent Document 6] Ghetie, MA et al., Blood 104 (2004) 178-183 [Non-Patent Document 7] Lang, P. et al., Blood 103(2004)3982~3985 [Non-Patent Document 8] Loeffler, A. et al., Blood 95(2000)2098~2103 [Non-Patent Document 9] Le Gall, F. et al., FEBS Lett. 453 (1999) 164~168 [Non-Patent Document 10] Li, Q. et al. Cancer Immunol. Immunother. 47 (1998) 121-130 [Non-Patent Document 11] Eberl, G. et al., Clin. Exp. Immunol. 114 (1998) 173-178 [Non-Patent Document 12] Pietersz, GA et al. Cancer Immunol. Immunother. 41 (1995) 53-60 [Non-Patent Document 13] Myers, DE et al., Leuk. Lymphoma. 18 (1995) 93-102 [Non-Patent Document 14] Bejcek, BE et al. Cancer Res. 55 (1995) 2346~2351 [Non-Patent Document 15] Hagen, I. A. et al., Blood 85 (1995) 3208-3212 [Non-Patent Document 16] Vlasfeld, LT et al. Cancer Immunol. Immunother. 40 (1995) 37~47 [Non-Patent Document 17] Rhodes, E. G. et al., Bone Marrow Transplant. 10 (1992) 485-489 [Non-Patent Document 18] Zola, H. et al., Immunol. Cell Biol. 69 (1991) 411~422 [Non-Patent Document 19] Watanabe, M. et al., Cancer Res. 50 (1990) 3245-3248 [Non-Patent Document 20] Uckun, FM et al., Blood 71 (1988) 13-29 [Non-Patent Document 21] Pezzutto, A. et al., J Immunol. 138 (1987) 2793~2799 [Non-Patent Document 22] http: / / www.broadinstitute.org / science / projects / mammals-models / rabbit / rabbit-genome-sequencing-project Summary of the Invention

[0010] The present invention relates to an anti-rabbit CD19 antibody and its use in labeling rabbit B cells.

[0011] The antibody according to the invention is, inter alia: - characterizing B cells in rabbit PBMCs or splenocytes; - Enriching antigen-specific B cells after macrophage depletion, either before or after antigen panning; - selective staining of B cells after co-culture with feeder cells, which expand B cells by orders of magnitude; and - Sorting and selecting B cells with improved yield and quality (optionally in combination with one or more additional markers). At least make it possible.

[0012] One embodiment of the present invention is an isolated antibody that specifically binds to rabbit CD19, comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32 or 33 or 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0013] In one embodiment, the antibody is a monoclonal antibody.

[0014] In one embodiment, the antibody is a chimeric or humanized antibody.

[0015] In one embodiment, the antibody comprises a heavy chain variable domain of SEQ ID NO:30 and a light chain variable domain of SEQ ID NO:26.

[0016] In one aspect, a) obtaining B cells from rabbit blood; b) incubating the B cells with an antibody according to the invention, and c) selecting one or more B cells to which an antibody according to the invention is bound A method for selecting B cells comprising:

[0017] In one embodiment, the method comprises: After step b) and before step c), incubating the B cells in co-culture medium at 37° C. for 1 hour; c) placing in an individual container (single) one or more B cells to which an antibody according to the invention is bound; d) co-culturing the deposited cells (single) with feeder cells in a co-culture medium; e) selecting the B cells proliferating in step d), thereby selecting the B cells. The method further includes one or more of:

[0018] One aspect described herein is a) co-culturing each of the B cells of the population of B cells placed as single cells by FACS based on the binding of a labeled antibody according to the present invention with mouse EL-4 B5 cells as feeder cells; b) selecting B cell clones which proliferate and secrete antibodies in step a); A method for selecting B cells comprising:

[0019] In one embodiment, the co-culture is carried out in the presence of a synthetic feeder mixture comprising IL-1β, TNFα, IL-10, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4, and IL-6.

[0020] In one embodiment, the B cells are rabbit B cells.

[0021] One aspect described herein is a) co-culturing one or more B cells of the population of B cells deposited in individual containers by FACS based on binding of a labeled antibody according to the invention, optionally in the presence of mouse EL-4 B5 cells as feeder cells and IL-1β, TNFα, IL-10 as a feeder mix, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4, and IL-6; b) selecting a B cell clone that produces an antibody that specifically binds to the target antigen; b1) determining the nucleic acid sequences encoding the light and heavy chain variable domains of the antibody by reverse transcription PCR; b2) transfecting a cell with a nucleic acid comprising a nucleic acid sequence encoding the antibody light chain variable domain and the heavy chain variable domain; and c) producing the antibody by culturing cells containing a nucleic acid encoding the antibody or a humanized variant thereof produced by the B cell clone selected in step b) and recovering the antibody from the cells or the culture supernatant. A method for producing an antibody that binds to a target antigen, comprising:

[0022] In one embodiment, the B cells are rabbit B cells.

[0023] One aspect described herein is - incubating the plurality of rabbit B cells with an antibody according to the invention conjugated to a detectable label / labeling individual B cells of the plurality of rabbit B cells with an antibody according to the invention conjugated to a detectable label, - selecting / depositing one or more rabbit B cells having antibodies according to the invention bound on their surface / labeled either as individual B cells (B cells deposited as single cells) or as a pool of B cells (in individual containers); and - co-culturing said single cell-placed rabbit B cells or said pool of rabbit B cells with feeder cells, - optionally, after co-culture, incubating the resulting cell mixture with an antibody according to the invention conjugated to a detectable label and selecting / locating / counting rabbit B cells which have on their surface bound / labeled with an antibody according to the invention. A method for co-culturing one or more rabbit B cells, comprising:

[0024] One aspect described herein is a) co-culturing B cells, deposited either as single cells or as a pool of cells, with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to the invention, and c) removing non-B cells by selecting one or more B cells to which an antibody according to the invention is bound. A method for selecting B cells / removing non-B cells for culture comprising:

[0025] In one embodiment, the B cells are rabbit B cells.

[0026] One aspect described herein is a) co-culturing B cells deposited as single cells with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to the invention, and c) determining the number of B cells in the culture by counting the number of cells to which the antibody according to the invention is bound. A method for determining the number of B cells after culture of B cells deposited as single cells, comprising:

[0027] In one embodiment, the B cells are rabbit B cells.

[0028] One aspect of the present invention is a) co-culturing B cells deposited as single cells or a pool of B cells with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to the invention, and c) selecting one or more cells to which an antibody according to the invention is bound, thereby selecting B cells and removing non-B cells from the mixture of cells; A method for removing non-B cells from a mixture of cells (e.g., a culture), comprising:

[0029] In one embodiment, the B cells are rabbit B cells.

[0030] One aspect of the present invention is a) co-culturing B cells deposited as single cells with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to the invention, and c) determining the number of B cells in the culture by counting the number of cells to which the antibody according to the invention is bound. A method for determining the number of B cells in a co-culture of B cells and feeder cells deposited as single cells, comprising:

[0031] In one embodiment, the B cells are rabbit B cells. [The present invention 1001] An antibody that binds to rabbit CD19, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 33 or 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40 An antibody comprising: [The present invention 1002] The antibody of the present invention which is a monoclonal antibody. [The present invention 1003] The antibody of the present invention 1001 or 1002, which is a chimeric antibody or a humanized antibody. [The present invention 1004] The antibody of the present invention 1001 or 1002, comprising a heavy chain variable domain of SEQ ID NO: 30 and a light chain variable domain of SEQ ID NO: 26. [The present invention 1005] The antibody of any of claims 1001 to 1004, which is a full length antibody or an antibody fragment. [The present invention 1006] The antibody of any of claims 1001 to 1005, which is conjugated to a detectable label. [The present invention 1007] The antibody of the present invention, wherein said detectable label is a fluorescent dye. [The present invention 1008] 1. A method for selecting rabbit B cells, comprising: a) incubating a large number of rabbit B cells with any of the antibodies 1001 to 1007 of the present invention; and b) selecting one or more B cells to which any of the antibodies 1001 to 1007 of the invention is bound, thereby selecting rabbit B cells. A method comprising: [The present invention 1009] After step b) and before step c), incubating the rabbit B cells in co-culture medium at 37° C. for 1 hour; and / or c) depositing as single cells one or more rabbit B cells to which any of the antibodies according to the invention 1001 to 1007 is bound, and / or d) co-culturing the single-cell-placed rabbit B cells with feeder cells in a co-culture medium; and / or e) selecting the rabbit B cells proliferating in step d), thereby selecting the rabbit B cells. The method of the present invention 1008 further comprising one or more of: [The present invention 1010] 1. A method for depleting non-B cells in a culture, comprising: a) co-culturing rabbit B cells, deposited either as single cells or as a pool of cells, with feeder cells; b) incubating the cells from the co-culture obtained in step a) with any of the antibodies of the invention 1001 to 1007, and c) selecting one or more rabbit B cells to which any of the antibodies of the invention 1001 to 1007 is bound, thereby removing non-B cells; A method comprising: [The present invention 1011] 1. A method for determining the number of B cells in a co-culture of B cells deposited as single cells and feeder cells, comprising: a) co-culturing rabbit B cells deposited as single cells with feeder cells; b) incubating the cells from the co-culture obtained in step a) with any of the antibodies of the invention 1001 to 1007, and c) determining the number of B cells in said culture by counting the number of cells to which any of the antibodies of the invention 1001 to 1007 is bound. A method comprising: [The present invention 1012] 1. A method for co-culturing one or more rabbit B cells, comprising: - incubating a large number of rabbit B cells with an antibody according to any one of claims 1001 to 1007 of the invention / labeling individual B cells of the large number of rabbit B cells with an antibody according to any one of claims 1001 to 1007 of the invention; - selecting / depositing one or more rabbit B cells having any of the antibodies of the invention 1001 to 1007 bound to their surface / labeled either as individual B cells (B cells deposited as single cells) or as a pool of B cells; and - co-culturing said single cell-placed rabbit B cells or said pool of rabbit B cells with feeder cells A method comprising: [The present invention 1013] The method of any of claims 1009 to 1012, wherein the co-culture is carried out in the presence of a synthetic feeder mixture comprising IL-1β, TNFα, IL-10, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4, and IL-6. [The present invention 1014] a) obtaining B cells from rabbit blood; b) incubating said B cells with any of the antibodies 1001 to 1005 of the invention bound to beads; c) removing unbound B cells; and d) recovering bound B cells from said beads, thereby selecting one or more B cells. 2. A method for selecting B cells, comprising: [The present invention 1015] e) optionally incubating the recovered B cells in co-culture medium at 37° C. for 1 hour; f) placing one or more recovered B cells into individual containers; g) co-culturing the plated cells with feeder cells in a co-culture medium; and h) selecting the B cells proliferating in step g), thereby selecting the B cells. The method of the present invention 1014 further comprises: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] DETAILED DESCRIPTION OF EMBODIMENTS OF THE PRESENTINVENTION CD19 is the pan-B cell marker of choice as it is expressed at almost all stages of B cell development up to terminal differentiation into plasma cells (see, eg, Tedder, supra).

[0033] I. Definition General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is provided in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acid positions of all constant regions and domains of the heavy and light chains can be numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "Kabat numbering." Specifically, the Kabat numbering system (see pages 647-660) of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CH1, hinge, CH2, and CH3, which is further clarified herein by referring to this case as "Kabat EU index numbering").

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0035] It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0036] The skilled artisan is well aware of procedures and methods for converting, for example, the amino acid sequence of a peptide linker or fusion polypeptide into the corresponding coding nucleic acid sequence. Thus, a nucleic acid is characterized by its nucleic acid sequence, consisting of individual nucleotides, as well as by the amino acid sequence of the peptide linker or fusion polypeptide that is encoded by said nucleic acid sequence.

[0037] The use of recombinant DNA technology allows the production of derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion, or insertion, at individual or several nucleotide positions. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be easily performed by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD and Higgins, SG, Nucleic acid hybridization-a practical approach (1985) IRL Press, Oxford, England).

[0038] Methods and techniques useful for carrying out the invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997), Glover, ND and Hames, BD (eds.), DNA Cloning: A Practical Approach, Vols. I-II (1985), Oxford University Press, Freshney, RI (ed.), Animal Cell Culture-a practical approach, IRL Press Limited (1986), Watson, JD et al., Recombinant DNA, 2nd Edition, CHSL Press (1992), Winnacker, EL, From Genes to Clones, NY, VCH Publishers (1987), Celis, J. (ed.), Cell Biology, 2nd Edition, Academic Press (1998), and Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition, Alan R. Liss, Inc., NY (1987).

[0039] The term "about" refers to a range of ±20% of the numerical value that follows it. In one embodiment, the term about refers to a range of ±10% of the numerical value that follows it. In one embodiment, the term about refers to a range of ±5% of the numerical value that follows it.

[0040] An "acceptor human framework" for purposes herein is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise that same amino acid sequence or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0041] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally measured using the dissociation constant (k d Affinity can be measured by common methods known in the art, including those described herein.

[0042] The terms "anti-rabbit CD19 antibody" and "antibody that specifically binds to rabbit CD19" refer to an antibody that is capable of binding to rabbit CD19 with sufficient affinity and is useful as a diagnostic agent when the antibody targets rabbit CD19.

[0043] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired rabbit CD19 binding activity.

[0044] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to rabbit CD 19. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0045] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0046] The "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. The five major classes of antibodies include IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0047] The term "Fc region" is used herein to define a C-terminal region fragment of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine dipeptide (Gly446-Lys447), respectively, of the Fc region may or may not be present. Numbering according to the Kabat EU index.

[0048] The term "constant region (of an antibody)" is used herein to define that portion of an immunoglobulin heavy chain excluding the variable domain. This term includes native sequence constant regions and variant constant regions. In one embodiment, the human IgG heavy chain constant region extends from Ala114 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or the glycine-lysine dipeptide (Gly446-Lys447), respectively, of the Fc region may or may not be present. Numbering according to the Kabat EU index.

[0049] The antibody according to the present invention comprises as Fc region, in one embodiment, an Fc region derived from human origin. In one embodiment, the Fc region comprises all parts of human constant region. The Fc region of the antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. The effect of the antibody on the complement system depends on certain conditions, and the binding to C1q is caused by a defined binding site in the Fc region. Such binding sites are known in the prior art and are described, for example, in Lukas, TJ et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virol. 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324, and European Patent Publication No. 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat). Antibodies of human subclasses IgG1, IgG2, and IgG3 usually exhibit complement activation, C1q binding, and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q, and does not activate C3. "Antibody Fc region" is a term well known to those skilled in the art and is defined based on papain cleavage of an antibody. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of human IgG1 or IgG4 subclass. In one embodiment, the Fc region is of the human IgG4 subclass, comprising the mutations S228P and / or L235E (numbering according to EU index of Kabat). In one embodiment, the Fc region is of the human IgG1 subclass, comprising the mutations L234A, L235A, and optionally P329G (numbering according to EU index of Kabat).

[0050] As used herein, the term "detectable label" encompasses chromogens (fluorescent or luminescent groups, and dyes), enzymes, NMR-active groups, metal particles, or haptens such as digoxigenin. In one embodiment, the detectable label is a fluorescent dye. Metal chelates that can be detected by electrochemiluminescence are also signal-emitting groups in one embodiment, e.g., ruthenium(bispyridyl)3 2+ Ruthenium chelates, such as chelates, are particularly preferred. Suitable ruthenium labelling groups are described, for example, in EP 0 580 979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138.

[0051] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I or III as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup kappa III as in Kabat et al., supra.

[0052] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences are generally represented in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0053] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0054] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the original transformed cell and the progeny derived from the original transformed cell, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the invention.

[0055] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0056] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0057] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that comprises stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0058] HVR is (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and / or (c), including amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Includes.

[0059] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0060] An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chromatogr. B848 (2007) 79-87.

[0061] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes nucleic acid molecules that are contained in a cell that originally contained the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0062] "Isolated nucleic acid encoding an anti-rabbit CD19 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) are present in one or more locations in a host cell.

[0063] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, excluding possible variant antibodies (such variants are generally present in minor amounts), e.g., containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which generally include various antibodies directed against various determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0064] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or radiolabel.

[0065] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3), with a hinge region located between the first and second constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) followed by a constant light (CL) domain. The light chains of antibodies can be divided into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0066] "Percentage (%) of amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of sequence identity for the purpose of alignment. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using commonly available computer software, such as BLAST, BLAST-2, ClustalW, Megalign (DNASTAR) software, or FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences to be compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code together with the user manual has been filed with the US Copyright Office, Washington DC, 20559, is registered under U.S. Copyright Registration No. TXU510087, and is described in WO 2001 / 007611.

[0067] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c or later, with the BLOSUM50 comparison matrix. The FASTA program package is described by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448, WR Pearson (1996) "Effective protein sequence comparison", Meth. Enzymol. 266:227-258, and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the program ggsearch(global protein:protein) and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring that global rather than local alignments are performed. Percent amino acid identity is given in the output alignment header.

[0068] The term "CD19," as used herein, refers to rabbit B lymphocyte antigen CD19 (alternative names: differentiation antigen CD19, B lymphocyte surface antigen B4, T cell surface antigen Leu-12). The term encompasses "full-length," unprocessed rabbit CD19 (SEQ ID NO: 02) as well as any form of rabbit CD19 that results from processing in the cell (e.g., by cleavage of the signal peptide), so long as the antibodies described herein bind, such as, for example, SEQ ID NO: 01 and fragments thereof.

[0069] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91.) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of an antibody that binds to the antigen. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628.

[0070] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0071] II. Compositions and Methods The present invention is based, at least in part, on the discovery that rabbit CD19 is a highly valuable pan-rabbit B cell marker.

[0072] More specifically, rabbit CD19 has been found to be an advantageous cell surface marker for the analysis and characterization of rabbit B cells. Labeling rabbit B cells via surface-presented CD19 can improve the enrichment or even sorting of B cells. The improvement is, among other things, more specific labeling, thereby enriching / sorting / putting as single cells, or / and facilitating the process, reducing the number of B cells to be processed, while increasing the number of B cells that produce antigen-specific antibodies.

[0073] The present invention is based at least in part on the discovery that an anti-rabbit CD19 antibody according to the invention does not result in apoptosis of B cells when used as a cell surface marker, as compared to an anti-rabbit CD20 antibody.

[0074] Until the present invention, the only antibodies available for specifically staining rabbit B cells were antibodies against cell surface immunoglobulins such as IgG, IgM, IgA, or human light chains in human transgenic rabbits. No specific B cell markers were available for specifically staining the entire B cell population. The anti-rabbit CD19 antibody according to the present invention makes it possible for the first time to identify and specifically stain the entire rabbit B cell population.

[0075] The rabbit CD19 sequence contains an extended gap in exon #3 compared to other non-rodent mammalian species (see alignment). TIFF0007650802000001.tif72156

[0076] For the isolation of genes from the rabbit genome, the 5' / 3'-UTR regions were extracted from rabbit, human and mouse and compared to obtain regions where PCR primers could be placed (rb_5UTR_primer_region1 (SEQ ID NO: 17), rb_5UTR_primer_region2 (SEQ ID NO: 18), rb_3UTR_primer_region (SEQ ID NO: 19)). Exemplary primers have the sequences of SEQ ID NO: 53 (binding in 3'UTR) and SEQ ID NO: 54 (binding in 5'UTR).

[0077] For the isolation of rabbit CD19 genomic DNA, either the primer combination of rbCD19 UTR forward primer and rbCD19 UTR reverse primer (binding at the 5' / 3'-UTR sequence of rabbit CD19 structural gene: SEQ ID NOs: 20 and 21) or the primer combination of rbCD19 CDS forward primer and rbCD19 CDS reverse primer (binding at the start codon and stop codon of rabbit CD19 structural gene: SEQ ID NOs: 22 and 23) can be used. The respective amplification products have a length of 1831 bp or 1663 bp, respectively.

[0078] The antibody according to the present invention was produced by immunizing mice with an expression plasmid of rabbit CD19. Before harvesting B cells, the immunized mice were boosted with rabbit CD19-presenting cells transfected with rabbit CD19 DNA. The harvested B cells were fused with myeloma cells to produce hybridoma cells.

[0079] The rabbit CD19 expressing cells used for boosting are NIH / 3T3 (ATCC® CRL-1658™) mouse embryonic fibroblasts transfected with rabbit CD19-GPI anchor-FLAG tag fusion polypeptide. Expression of rabbit CD19 on the cell surface works well in NIH / 3T3 cells (about 40% FITC positive cells after 24 hours) and gives a high signal with intracellular staining. Unexpectedly, when using closely related C2C12 (ATCC® CRL-1772™) mouse myoblasts, expression of the construct could not be detected (almost no detectable rabbit CD19 positive cells, about 9% FITC positive cells after 24 hours). Further improvement of rabbit CD19 expression could be achieved by using a reverse transfection approach (see FIG. 1). It has been shown that for CHO and HEK cells, the FLAG tag can be used as a surrogate for detection of rabbit CD19 expressed on the cell surface (see table below).

[0080] TIFF0007650802000002.tif140131 Anti-Flag tag antibody-Alexa488 conjugate = Detection of FLAG tag using anti-Flag tag antibody labeled with Alexa488 Polyclonal anti-mIgG antibody-APC conjugate = Detection of anti-rabbit CD19 antibody with anti-mouse IgG antibody labeled with APC

[0081] Of the 356 hybridoma cells generated, only a single cell, clone 1H2, expressed anti-rabbit CD19 antibodies with (sufficiently good) affinity for native rabbit CD19 on the B cell surface (see FIG. 2 and table below).

[0082] TIFF0007650802000003.tif214158TIFF0007650802000004.tif238158TIFF00076508020 00005.tif238158TIFF0007650802000006.tif238158TIFF0007650802000007.tif238158 TIFF0007650802000008.tif238158TIFF0007650802000009.tif238158TIFF00076508020 00010.tif238158TIFF0007650802000011.tif238158TIFF0007650802000012.tif178158

[0083] The present invention is based at least in part on the finding that a specific immunization protocol must be followed to obtain anti-rabbit CD19 antibody-expressing B cells or hybridomas, respectively. It has been found that the combination of DNA immunization and boosting with cells expressing rabbit CD19 on their surface before harvesting can obtain rabbit CD19-specific antibody-expressing B cells.

[0084] Without being bound by this theory, it is believed that anti-rabbit CD19 antibodies, let alone monoclonal antibodies, have not been available so far due to the sheer difficulty of generating such antibodies. For example, experience has shown that immunization with CD19 only as a recombinant protein or as a recombinant cell line does not result in antibodies that bind to the native CD19 on B cells. Therefore, DNA-only immunizations were performed with a final cell boost, assuming that CD19 was present in the animal as a natural confirmation.

[0085] Provided herein are antibodies against rabbit CD19 that are useful as tools for labeling and enrichment / selection of rabbit B cells.

[0086] Without being bound by this theory, it is postulated that these antibodies may be beneficially used, inter alia, after macrophage depletion and / or for staining of IgM-IgG+CD19+-B cells.

[0087] The present invention is based at least in part on the discovery that CD19 can be used to label B cells for single cell plating or pool selection. All B cells that produce antigen-specific IgG were found to be CD19 positive on the day of plating or selection, respectively. Therefore, CD19 on rabbit cells can be used to select antigen-specific antibody-producing B cells (see FIG. 3 and the table below).

[0088] TIFF0007650802000013.tif127128

[0089] The anti-rabbit CD19 antibody according to the present invention can be used to detect IgG-positive and IgM-positive B cells (all antigen-specific IgG-positive B cells have high levels of CD19). Using the antibody according to the present invention, it was possible to determine for the first time that about 5.4% of the total cells of B cells in the spleen are CD19-positive, and an average of 38.7% of the total cells of B cells in peripheral blood mononuclear cells (PBMCs) are CD19-positive. It must be pointed out that the percentage of rabbit CD19-positive cells in rabbit PBMCs freshly isolated from blood is higher than the total of immunoglobulin-stained B cells, meaning that the anti-rabbit CD19 antibody is an excellent marker for labeling all rabbit B cells. The results are shown in the table below.

[0090] TIFF0007650802000014.tif137128

[0091] Notably, in IgG-sorted B cell populations, it is important that all IgG-positive cells have high levels of CD19 (see data in table below).

[0092] TIFF0007650802000015.tif71128

[0093] The present invention is based at least in part on the discovery that CD19 can be used to distinguish B cells from feeder cells in co-culture of B cells. B cells placed as single cells require the presence of feeder cells in culture for proliferation and cell division. Feeder cells are irradiated before co-culture with B cells to reduce their proliferation and cell division, but their total number and cell size are comparable to those of B cells obtained after co-culture. With the anti-rabbit CD19 antibody according to the present invention, it is now possible to distinguish feeder cells from B cells by simple FACS analysis after co-culture. This allows the total number of B cells to be determined. This allows the normalization of other culture parameters, such as antibody production rate or yield (see Figure 4 and Figure 5).

[0094] Before the present invention, it was not possible to estimate the size of the expanded B cell clones in terms of B cell number after culture, since rabbit IgG as a cell surface B cell marker is not suitable since its expression decreases during culture and therefore cannot be used as a B cell marker. The use of anti-rabbit CD19 antibodies according to the present invention solves this problem, since rabbit CD19 is still expressed on B cells after culture, thereby allowing to count B cells after culture (B cells / well=number of events in CD19+ gate / Facsed volume×sample volume).

[0095] The table below shows the B cell counts (CD19 positive PI-cells) of 36 single wells after culture. The B cell counts were in a wide range. The CD19 positive B cell counts were calculated as follows: counts in FACS gate / 150 (FACSed volume) x 200 (total sample volume).

[0096] TIFF0007650802000016.tif222153

[0097] For each B cell clone, CD19 positive B cells could be detected and counted by excluding live feeder cells. It can be seen that the cell counts (=total number of cells) of the B cell clones are highly heterogeneous over a wide range.

[0098] The size of the B cell clone is a very good surrogate marker for the success of B cell expansion. Moreover, the size of the B cell clone can be correlated with pre-sorted B cell populations and ELISA results, allowing a better characterization of the system.

[0099] Moreover, it is possible to identify slow-growing and low-producing B cells, especially in the presence of a large excess of feeder cells.At the same time, dead feeder cells do not interfere with the staining and analysis / selection process.This specific labeling allows the counting of B cells in the presence of (dead or alive) feeder cells directly in culture (e.g. in the wells of multi-well plates).

[0100] The present invention is based, at least in part, on the discovery that the level of CD19 on the surface of cells at the time of plating positively correlates with the IgG titers obtained after culture. This correlation can be utilized to select high producing B cells immediately after isolation from rabbits, without the need for co-culture.

[0101] The present invention is based, at least in part, on the discovery that CD19 can be utilized to enrich rabbit B cells, thereby increasing the B cell population, e.g., the IgG+ B cell population, for single cell sorting, thereby reducing the number of unwanted cells. Without being bound by theory, this property may result in, among other things, improved sorting results.

[0102] The present invention is based, at least in part, on the discovery that anti-rabbit CD19 antibodies according to the invention can be used to identify rabbit primary B cells.

[0103] Using the antibodies according to the invention it was possible to determine the percentage of CD19 positive B cells in a sample, for example rabbit PBMCs or rabbit splenocytes. The results for PBMCs are shown in the table below. TIFF0007650802000017.tif31128

[0104] Commercially available goat anti-rabbit IgG polyclonal antibody conjugated to a FITC label (AbD Serotec STAR121F) in 0.2%–2% of rabbit PBMCs (Figure 6).

[0105] The cells were double stained with FITC-labeled anti-IgG antibody and APC-labeled anti-rabbit CD19 antibody according to the present invention. The cells were then index-sorted for FSC and IgG. The results show that only CD19 and IgG positive cells produced IgG in the subsequent ELISA. The percentage of sorted CD19 positive cells was then checked. This was used as a measure to estimate how much sorting efficiency can be improved by using the antibody according to the present invention. On average, the efficiency can be improved by about 14% when only sorting IgG and CD19 double positive cells, and in the best case, it can be improved by more than 20% without significant loss of IgG positive cells or antigen-specific cells. The results are shown in the table below.

[0106] TIFF0007650802000018.tif36128

[0107] For the bead-based selection / extraction process, various conditions were tested as follows: - Magnetic bead-based panning with biotinylated antigen and streptavidin beads, followed by FSC and sorting gating of IgG (FITC) and CD19 (APC) double stained cells, and - Magnetic bead-based panning with biotinylated anti-CD19 antibody and streptavidin beads according to the invention, followed by FCS and sorting gating of IgG (FITC) and antigen (APC) double stained cells.

[0108] In both cases, viability staining was performed with 7AAD.

[0109] It was found that panning with anti-CD19 antibodies according to the invention results in an increase (four-fold) in B cell survival. Without being bound by this theory, it is assumed that the increased survival may be caused by reduced cross-linking and activation.

[0110] The present invention is based at least in part on the finding that by using an anti-CD19 antibody according to the invention in a panning step for enriching B cells from a population of cells, a higher percentage of selected B cells produce antigen-specific antibodies as shown by subsequent ELISA. Furthermore, fewer cells are positive in cross-reactivity assays, leading to a higher number of usable clones. This may be associated with increased cell viability.

[0111] The results obtained with B cells from the different immunization campaigns (different antigens) are shown in the table below.

[0112] TIFF0007650802000019.tif90131

[0113] A. Exemplary Anti-Rabbit CD19 Antibodies and Uses Thereof A.1 Exemplary Antibodies Rabbit CD19 has been found to be an advantageous cell surface marker for the analysis and characterization of rabbit B cells. Labeling rabbit B cells via surface-presented CD19 allows for improved B cell sorting. The improvement is, among other things, more specific labeling, thereby sorting / putting as single cells, or / and an easier process, which reduces the number of B cells to be processed, while at the same time increasing the number of B cells that produce antigen-specific antibodies.

[0114] In one aspect, provided herein is an isolated antibody that specifically binds rabbit CD19, comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32 or 33 or 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0115] In one aspect, provided herein is an anti-rabbit CD19 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0116] In one aspect, provided herein is an anti-rabbit CD19 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0117] In one aspect, provided herein is an anti-rabbit CD19 antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0118] In one aspect, provided herein is an isolated antibody that specifically binds to rabbit CD19, comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32 or 33 or 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0119] In one aspect, provided herein is an anti-rabbit CD19 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0120] In one aspect, provided herein is an anti-rabbit CD19 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0121] In one aspect, provided herein is an anti-rabbit CD19 antibody comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0122] In another aspect, the anti-rabbit CD19 antibody described herein comprises (i) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32 or 33 or 34, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, and (ii) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0123] In another aspect, the anti-rabbit CD19 antibody described herein comprises (i) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, and (ii) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0124] In another aspect, the anti-rabbit CD19 antibody described herein comprises (i) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, and (ii) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0125] In another aspect, the anti-rabbit CD19 antibody described herein comprises (i) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 34, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, and (ii) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40.

[0126] In another aspect, an anti-rabbit CD19 antibody is provided that is a humanized antibody. In one embodiment, the humanized anti-rabbit CD19 antibody comprises an HVR as in any of the above-mentioned embodiments and further comprises an acceptor human framework, such as a human immunoglobulin (germline) framework or a human consensus framework.

[0127] In a further aspect, provided herein is an antibody that binds to the same epitope as the anti-rabbit CD19 antibody described herein.For example, in certain embodiments, provided is an antibody that binds to the same epitope as the anti-rabbit CD19 antibody comprising the VH sequence of SEQ ID NO:30 and the VL sequence of SEQ ID NO:26.

[0128] In one embodiment, the anti-rabbit CD19 antibody according to any of the above embodiments is a monoclonal antibody. In one embodiment, the anti-rabbit CD19 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 or IgG4 antibody as defined herein, or other antibody classes or isotypes.

[0129] In one embodiment of all aspects, the antibody comprises: i) optionally a homodimeric Fc region of the human IgG1 subclass, having the mutations P329G, L234A, and L235A; or ii) optionally a homodimeric Fc region of the human IgG4 subclass, having the mutations P329G, S228P, and L235E; or iii) a homodimeric Fc region of the human IgG1 subclass having the mutations (P329G, L234A, L235A) I253A, H310A, and H435A, or the mutations (P329G, L234A, L235A) H310A, H433A, and Y436A, or iv) a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, and Y407V, or b) one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C; or c) a heterodimeric Fc region, wherein one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C; or v) heterodimeric Fc regions of the human IgG1 subclass, in which both Fc region polypeptides contain the mutations P329G, L234A, and L235A; and a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, and Y407V, or b) one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C; or c) one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C; or vi) heterodimeric Fc regions of the human IgG4 subclass, in which both Fc region polypeptides contain the mutations P329G, S228P, and L235E; and a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A, and Y407V, or b) one Fc region polypeptide comprises the mutations T366W and Y349C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C; or c) one Fc region polypeptide comprises the mutations T366W and S354C and the other Fc region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C; or vii) Includes one of iii) in combination with one of vi), v), and vi) (all positions according to the EU index of Kabat).

[0130] In further aspects, the anti-rabbit CD19 antibodies according to any of the above embodiments may incorporate any of the features described in the following sections, either alone or in combination.

[0131] 1. Antibody fragment In certain embodiments, the antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson, PJ, et al., Nat.Med.9 (2003) 129-134. For a review of scFv fragments, see, for example, Plueckthun, A., The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315, and also see WO 93 / 16185, U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptors that bind epitopic residues and have increased in vivo half-lives.

[0132] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific.See, for example, EP 0404097, WO 1993 / 01161, Hudson, PJ et al., Nat.Med.9 (2003) 129-134, and Hollinger, P. et al., Proc.Natl.Acad.Sci.USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat.Med.9 (20039 129-134).

[0133] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516).

[0134] Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0135] 2. Chimeric and humanized antibodies The antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison, SL et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region from a mouse, rat, hamster, rabbit, or non-human primate, e.g., a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0136] A humanized antibody is a chimeric antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, in which the HVR, e.g., CDR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0137] Humanized antibodies and methods for making them are reviewed, for example, in Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and described, for example, in Riechmann, I. et al., Nature 332 (1988) 323-329, Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033, U.S. Patent No. 5,821,337, U.S. Patent No. 7,527,791, U.S. Patent No. 6,982,321, and U.S. Patent No. 7,087,409, Kashmiri, SV et al., Methods 36 (2005) 25-34 (description of specificity determining region (SDR) grafting), Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (description of "surface reconstitution"), Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (description of "FR shuffling"), as well as Osbourn, J. et al., Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (description of a "guided selection" approach to FR shuffling).

[0138] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308), framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289, and Presta, LG et al., J. Immunol., 151 (1993) 2623-2632), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633), and framework regions derived from screening of FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684, and Rosok, MJ et al., J. Biol. Chem. 271 (19969) 22611-22618).

[0139] 3. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., antigen binding).

[0140] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. For substitutional mutagenesis, the sites of interest include HVR and FR. Conservative substitutions are shown in the table below under the heading of "preferred substitutions". More substantial changes are shown in Table 1 under the heading of "exemplary substitutions" and are further described below with respect to amino acid side chain classes. Amino acid substitutions are introduced into the antibody of interest, and the product can be screened for the desired activity, for example, retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC.

[0141] TIFF0007650802000020.tif160128

[0142] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0143] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0144] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0145] Modifications (e.g., substitutions) can be made to the HVRs, for example, to improve antibody affinity. Such modifications can be made at "hot spots" of the HVRs, i.e., residues encoded by codons that undergo high frequency mutation during the somatic maturation process (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196), and / or at residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0146] In certain embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such modification does not substantially reduce the ability of antibody to bind to antigen.For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.Such modifications may, for example, be outside the antigen contact residues in HVRs.In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains no more than one, no more than two, or no more than three amino acid substitutions.

[0147] A useful method for identification of residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they have the desired properties.

[0148] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0149] b) Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0150] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32. The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies described herein may be made to generate antibody variants with certain improved properties.

[0151] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues according to Kabat) of the Fc region, although Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication No. 2003 / 0157108, U.S. Patent Application Publication No. 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, U.S. Patent Application Publication No. 2003 / 0115614, U.S. Patent Application Publication No. 2002 / 0164328, U.S. Patent Application Publication No. 2004 / 0093621, U.S. Patent Application Publication No. 2004 / 0132140, U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 01102 82, U.S. Patent Application Publication No. 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO 2005 / 053742, WO 2002 / 031140, Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249, Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545, U.S. Patent Application Publication No. 2003 / 0157108, and WO 2004 / 056312, especially Example 11), and knockout cell lines such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622, Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688, and WO 2003 / 085107).

[0152] Further provided are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. 2005 / 0123546. Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0153] c) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0154] In certain embodiments, antibody variants are provided herein that retain some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important and where certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492, p. 464, Table 3. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063, and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502), U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay may be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996) 163-171; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006:1759-1769).

[0155] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0156] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0157] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0158] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0159] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593, and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434), have been described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0160] For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740, U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0161] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody that can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0162] A. Two Example Uses The anti-rabbit CD-19 antibodies according to the present invention can be used in any method requiring specific labeling and detection of rabbit B cells.

[0163] The individual methods are i) isolating a B cell or B cell clone from a population of B-cells or other cells, whereby the isolated B cell or B cell clone produces an antibody that specifically binds to a target; ii) To co-culture B cells deposited as single cells, iii) To label B cells for counting and selecting / placing; and iv) To produce antibodies This is the method.

[0164] Along with the methods, corresponding uses are also encompassed and disclosed.

[0165] In one aspect, a) obtaining B cells from rabbit blood; b) incubating the B cells with an antibody according to the invention, and c) selecting one or more B cells to which an antibody according to the invention is bound A method for selecting B cells comprising:

[0166] In one embodiment, the method comprises: After step b) and before step c), incubating the B cells in co-culture medium at 37° C. for 1 hour; c) placing (in a separate container) one or more B cells to which an antibody according to the invention is bound; d) co-culturing the plated cells with feeder cells in a co-culture medium; e) selecting the B cells proliferating in step d), thereby selecting the B cells. The method further includes one or more of:

[0167] One aspect described herein is a) co-culturing each of the B cells of the population of B cells placed as single cells by FACS based on the binding of the labeled antibody according to the present invention with mouse EL-4 B5 cells as feeder cells; b) selecting B cell clones which proliferate and secrete antibodies in step a); A method for selecting B cells comprising:

[0168] One aspect described herein is a) co-culturing one or more B cells of the population of B cells deposited in individual containers by FACS based on binding of the labeled antibody according to the invention, optionally in the presence of mouse EL-4 B5 cells as feeder cells and IL-1β, TNFα, IL-10 as a feeder mix, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4, and IL-6; b) selecting B cell clones that produce antibodies that specifically bind to the target antigen; b1) determining the nucleic acid sequences encoding the light and heavy chain variable domains of the antibody by reverse transcription PCR; b2) transfecting a cell with a nucleic acid comprising a nucleic acid sequence encoding the antibody light chain variable domain and the heavy chain variable domain; and c) producing the antibody by culturing cells containing a nucleic acid encoding the antibody or a humanized variant thereof produced by the B cell clone selected in step b) and recovering the antibody from the cells or the culture supernatant. A method for producing an antibody that binds to a target antigen, comprising:

[0169] One aspect described herein is - incubating the plurality of rabbit B cells with an antibody according to the invention conjugated to a detectable label / labeling individual B cells of the plurality of rabbit B cells with an antibody according to the invention conjugated to a detectable label, - selecting / depositing one or more rabbit B cells, on whose surface an antibody according to the invention is bound / labeled either as individual B cells (B cells deposited as single cells) or as a pool of B cells, and - co-culturing said single cell-placed rabbit B cells or said pool of rabbit B cells with feeder cells, - optionally, after co-culture, incubating the resulting cell mixture with an antibody according to the invention conjugated to a detectable label and selecting / locating / counting rabbit B cells which have on their surface bound / labeled with an antibody according to the invention. A method for co-culturing one or more rabbit B cells, comprising:

[0170] One aspect of the present invention is a) co-culturing B cells deposited as single cells or a pool of B cells with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to the invention, and c) selecting one or more cells to which an antibody according to the invention is bound, thereby selecting B cells and removing non-B cells from the mixture of cells; A method for removing non-B cells from a mixture of cells (e.g., a culture), comprising:

[0171] One aspect of the present invention is a) co-culturing B cells deposited as single cells with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to the invention, and c) determining the number of B cells in the culture by counting the number of cells to which the antibody according to the invention is bound. A method for determining the number of B cells in a co-culture of B cells and feeder cells deposited as single cells, comprising:

[0172] In one embodiment of any corresponding aspect or embodiment, the co-culture is performed in the presence of a synthetic feeder mixture comprising IL-1β, TNFα, IL-10, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4 and IL-6.

[0173] In one embodiment of any corresponding aspect or embodiment, the feeder cells are EL-4 B5 cells.

[0174] In one embodiment of all corresponding aspects or embodiments, the number of feeder cells (at the start of the co-culture) is 5×10 per B cell. 4 Less than one.

[0175] In one embodiment of all corresponding aspects or embodiments, the feeder cells are irradiated before co-culture. In one embodiment, the irradiation is at a dose of about 50 Gy or less. In one embodiment, the irradiation is at a dose of 9.5 Gy or less and more than 0 Gy.

[0176] In one embodiment of any corresponding aspect or embodiment, the number of EL-4 B5 cells is 1×10 per B cell. 4 EL-4 B5 cells (whereby, in this embodiment, the radiation dose is 0 Gy). In one embodiment, the number of EL-4 B5 cells is less than 7.5×10 per B cell. 3 Less than 10 EL-4 B5 cells.

[0177] In one embodiment of any corresponding aspect or embodiment, the co-culture is additionally carried out in the presence of a feeder mixture.

[0178] In one embodiment of any corresponding aspect or embodiment, the feeder mixture (cytokine mixture, CM) comprises: i) interleukin-1β and tumor necrosis factor α, ii) interleukin-2 (IL-2) and / or interleukin-10 (IL-10); iii) Staphylococcus aureus Cowan cells (SAC); iv) interleukin-21 (IL-21), and optionally interleukin-2 (IL-2); v) B-cell activating factor of the tumor necrosis factor family (BAFF); vi) interleukin-6 (IL-6), vii) interleukin-4 (IL-4), and viii) thymocyte culture supernatant.

[0179] In one embodiment of any corresponding aspect or embodiment, the feeder mixture comprises: up to about 2ng / ml (mouse) IL-1β; up to about 2ng / ml (mouse) TNFα; up to about 50ng / ml (mouse) IL-2; up to about 10 ng / ml (mouse) IL-10, and up to about 10 ng / ml (mouse) IL-6; or any part thereof.

[0180] In one embodiment of any corresponding aspect or embodiment, the feeder mixture comprises: 5.5~14×10 8 up to about 2ng / ml (mouse) IL-1β with IU / mg; 2.3~2.9×10 8 TNFα (mouse) up to about 2 ng / ml with U / mg; 6 to 7 (preferably 6.3) x 10 6 up to about 50 ng / ml (mouse) IL-2 with IU / mg; 6~7.5×10 5 up to about 10 ng / ml (mouse) IL-10 with IU / mg; and 9.2~16.1×10 8 (mouse) IL-6 up to about 10 ng / ml with U / mg; or any part thereof.

[0181] In one embodiment of any corresponding aspect or embodiment, the ratio of the feeder mixture is in the range of 1.0 to 0.015 times the concentration of each of said IL-1β, TNFα, IL-2, IL-10, and IL-6.

[0182] In one embodiment of any corresponding aspect or embodiment, the ratio of the feeder mixture is selected from the group of ratios consisting of 0.75x, 0.5x, 0.32x, 0.25x, 0.1x, 0.066x, 0.032x, 0.015x, 0.01x, 0.0075x, and 0.0038x the respective concentrations of IL-1β, TNFα, IL-2, IL-10, and IL-6.

[0183] In one embodiment of any corresponding aspect or embodiment, the feeder mixture further comprises about 0.01 ng / ml to 1.0 ng / ml of phorbol myristate acetate (PMA). In one embodiment, the feeder mixture further comprises about 0.01 ng / ml to 0.5 ng / ml of phorbol myristate acetate.

[0184] In one embodiment of all corresponding aspects or embodiments, the (co-)culture of one or more B cells comprises: - co-culturing one or more B cells with EL-4 B5 cells in the presence of a feeder mixture, Prior to the co-culture, the EL-4 B5 cells are irradiated at a dose of 9.5 Gy or less; The number of EL-4 B5 cells (at the beginning of the coculture) was 4 × 10 per B cell. 4 fewer than EL-4 B5 cells, The feeder mixture comprises: 5.5~14×10 8 up to about 2ng / ml (mouse) IL-1β with IU / mg; 2.3~2.9×10 8 TNFα (mouse) up to about 2 ng / ml with U / mg; 6 to 7 (preferably 6.3) x 10 6 up to about 50 ng / ml (mouse) IL-2 with IU / mg; 6~7.5×10 5 up to about 10 ng / ml (mouse) IL-10 with IU / mg; and 9.2~16.1×10 8 (mouse) IL-6 up to about 10 ng / ml with U / mg; or 0.75x, 0.5x, 0.32x, 0.25x, 0.1x, 0.066x, 0.032x, 0.015x, 0.01x, 0.0075x, or 0.0038x the respective concentrations of IL-1β, TNFα, IL-2, IL-10, and IL-6; The feeder mixture further comprises about 0.01 ng / ml to 1.0 ng / ml of phorbol myristate acetate.

[0185] In one embodiment of any corresponding aspect or embodiment, the feeder mixture comprises Staphylococcus aureus strain Cowan cells (SAC) and thymocyte culture supernatant.

[0186] In one embodiment of all corresponding aspects or embodiments, the method is for co-culture of one B cell. In one preferred embodiment, the one B cell is a B cell deposited as a single cell.

[0187] In one embodiment of any corresponding aspect or embodiment, the co-culture is for 5 to 10 days. In one preferred embodiment, the co-culture is for about 7 days.

[0188] One aspect described herein is a method for producing antibodies, comprising the co-culture method described herein.

[0189] All methods and uses described herein are - co-culturing (individually) the feeder cells and the B cells (either individual B cells placed as single cells or pooled B cells) in a co-culture medium supplemented with the feeder mixture; Includes.

[0190] The result of the co-culture is a B cell clone, ie, a population of B cells that are the descendants of a single B cell.

[0191] In one embodiment of any corresponding aspect or embodiment, the method described herein further comprises, prior to the co-cultivation step, - placing the B cells of the population of B cells, with or without a fluorophore bound thereto, in one or more individual containers, in contact with a labeled antibody according to the invention, thereby Further includes:

[0192] In one embodiment of any corresponding aspect or embodiment, the method described herein further comprises, prior to the co-cultivation step, - contacting those B cells of the population of B cells, with a labeled antibody according to the invention, whereby the fluorophore is bound and / or not bound to the B cell, as single B cells. Further includes:

[0193] In one embodiment of any corresponding aspect or embodiment, the method described herein further comprises, prior to the co-cultivation step, - placing these B cells of the population of B cells in contact with an antibody according to the invention and with one to four additional antibodies that each specifically bind to a different B cell surface antigen, these antibodies being labeled as single cells with one to five fluorescent dyes, whereby each antibody is conjugated to a different fluorescent dye; Further includes:

[0194] Labeling is by contacting the B cell population (sequentially or simultaneously) with fluorescently labeled antibodies, resulting in a preparation of labeled B cells, each of which binds to a different B cell surface marker / target.

[0195] Plating involves introducing the labeled B cell preparation into a flow cytometer and plating the cells as single cells labeled with one to three fluorescent labels. Because it is possible to incubate the cells with many more fluorescent dyes, similar to those used to select cells in a cell sorter, cells can be selected for the presence of certain surface markers and (optionally) simultaneously selected for the absence of other surface markers.

[0196] Labeling and single cell placement is performed to reduce the complexity of the B cell population by depleting B cells that are unlikely to produce antibodies with the intended characteristics. The labeled antibodies bind to specific polypeptides displayed on the surface of the B cells, thus providing a positive selection label. Similarly, it is also possible to select cells that are only labeled with a reduced number of fluorescent dyes compared to the number of labeled antibodies with which the B cells were incubated, for example cells with one fluorescent label out of two (i.e., incubation with two fluorescently labeled antibodies was performed, but only one of them binds to the B cells). Using a microfluidic sorting device, it is possible to identify and separate the target B cells based on the binding / non-binding of the fluorescently labeled antibodies to individual B cells of the B cell population. Simultaneous with the selection, the amount of labeling can also be determined.

[0197] In one embodiment of any corresponding aspect or embodiment, the method described herein further comprises incubating the population of B cells without / in the absence of feeder cells in co-culture medium during / before placing the single cells. In one embodiment, the incubation is at about 37° C. In one embodiment, the incubation is for about 0.5 to about 2 hours. In one embodiment, the incubation is for about 1 hour. In one preferred embodiment, the incubation is for about 1 hour at about 37° C.

[0198] In one embodiment of any corresponding aspect or embodiment, the method described herein further comprises centrifuging the single cell-laying B cells after the laying step and before the co-culturing step, but after the addition of the EL-4 B5 feeder cells. Without being bound by this theory, it is assumed that this increases physical contact between the feeder cells and the B cells. In one embodiment, the centrifugation is for about 1 minute to about 30 minutes. In one embodiment, the centrifugation is for about 5 minutes. In one embodiment, the centrifugation is for about 100 x g to about 1,000 x g. In one preferred embodiment, the centrifugation is for about 5 minutes at about 300 x g.

[0199] In one embodiment of all corresponding aspects or embodiments, the method for selecting / obtaining B cells (clones) further comprises the steps of: a) labeling B cells of a population of B cells with (1 to 5) fluorescent dyes (optionally by incubating the B cell population with 2 to 5 fluorescently labeled antibodies that specifically bind to 2 to 5 different predefined B cell surface markers), one of which is (conjugated to) an antibody according to the invention; b) optionally incubating the labeled cells in co-culture medium; c) placing those B cells of the population of B cells labeled with at least one (1 to 5) fluorescent dyes (and, optionally, not labeled with other fluorescent dyes) as single cells on EL-4 B5 feeder cells irradiated with a dose of 9.5 Gy or less; d) optionally, centrifuging the deposited B cell / feeder cell mixture as single cells; e) co-culturing each B cell deposited as a single cell with the feeder cells (individually) in a co-culture medium supplemented with the feeder mixture; f) Selecting the B cell clones that proliferate and secrete antibodies in step e).

[0200] In one embodiment of any corresponding aspect or embodiment, the method for producing an antibody that specifically binds to a target further comprises the steps of: a) labeling B cells of a population of B cells with (1 to 5) fluorescent dyes (optionally by incubating the B cell population with 2 to 5 fluorescently labeled antibodies that specifically bind to 2 to 5 different predefined B cell surface markers), one of which is (conjugated to) an antibody according to the invention; b) optionally incubating the cells in co-culture medium; c) placing those B cells of the population of B cells labeled with at least one (1 to 5) fluorescent dyes (and, optionally, not labeled with other fluorescent dyes) as single-cell EL-4 B5 feeder cells irradiated with a dose of 9.5 Gy or less; d) optionally, centrifuging the deposited B cell / feeder cell mixture as single cells; e) co-culturing each B cell deposited as a single cell (individually) with a feeder cell in a co-culture medium supplemented with the feeder mixture; f) selecting the B cell clones of step e) which secrete antibodies; g) i) obtaining one or more nucleic acids encoding variable domains of antibodies secreted from the B cell clones selected in step g); ii) if the B cell clone is not a human B cell clone, providing nucleic acid encoding each of the variable domains, and iii) introducing the nucleic acid(s) into one or more expression vectors in frame with a nucleic acid sequence encoding a constant region; h) culturing cells (optionally selected from CHO and BHK cells) transfected with one or more expression vectors of step g) and recovering the antibody from the cells or the culture supernatant, thereby producing the antibody.

[0201] In one embodiment of any corresponding aspect or embodiment, the method for producing an antibody further comprises the steps of: a) labeling B cells of a population of B cells with (1 to 5) fluorescent dyes (optionally by incubating the B cell population with 2 to 5 fluorescently labeled antibodies that specifically bind to 2 to 5 different predefined B cell surface markers), one of which is (conjugated to) an antibody according to the invention; b) optionally incubating the cells in co-culture medium; c) placing those B cells of the population of B cells labeled with at least one (1 to 5) fluorescent dyes (and, optionally, not labeled with other fluorescent dyes) as single cells on EL-4 B5 feeder cells irradiated with a dose of 9.5 Gy or less; d) optionally, centrifuging the deposited B cell / feeder cell mixture as single cells; e) co-culturing each B cell deposited as a single cell (individually) with a feeder cell in a co-culture medium supplemented with the feeder mixture; f) determining the binding specificity of the antibodies secreted into the culture medium of the co-cultured B cells for each supernatant individually; g) selecting the B cell clones of step f) based on the binding properties of the secreted antibodies; h) obtaining one or more nucleic acids encoding the variable domains of the antibodies secreted from the B cell clones selected in step g) by reverse transcription PCR and nucleotide sequencing (and thereby obtaining nucleic acids encoding the monoclonal antibody variable light and heavy chain domains); i) if the B cell is a non-human B cell, in which case the variable light and heavy chain variable domains are humanized, providing nucleic acids encoding the humanized variable domains; j) introducing nucleic acids encoding monoclonal antibody variable light and heavy chain variable domains (in frame with nucleic acids encoding antibody constant domains) into one or more expression vectors for expressing (human or humanized) antibodies; k) introducing the expression vector(s) into a mammalian cell (optionally selected from CHO cells and BHK cells); l) culturing the cells and recovering the antibody from the cells or the cell culture supernatant, thereby producing the antibody.

[0202] In one embodiment of any corresponding aspect or embodiment, obtaining one or more nucleic acids encoding variable domains of secreted antibodies from the B cell clone further comprises the steps of: - extracting total RNA from the antibody-producing B cell clones; - Poly A Extracted + A step of performing single-stranded cDNA synthesis / reverse transcription of mRNA; - performing PCR using a set of species-specific primers; - optionally a PCR primer removal / PCR product purification step, - optionally sequencing the PCR products.

[0203] In one embodiment of all corresponding aspects or embodiments, introducing the nucleic acid encoding the monoclonal antibody variable light and / or heavy chain variable domains into an expression vector for expression of a (human or humanized) antibody further comprises the steps of: - a T4 polymerase incubation step of the variable light and heavy chain variable domains, - Linearization and amplification of the expression vector, - a T4 polymerase incubation step of the amplified expression vector; - sequence- and ligation-independent cloning of nucleic acids encoding variable domains into the amplified expression vector; and - preparing the vector(s) from a pool of E. coli cells transformed with the vector.

[0204] In one embodiment of any corresponding aspect or embodiment, the method further comprises, immediately prior to the labeling step: - incubating the population of B cells with a (target) antigen that is soluble, fluorescently labeled, or immobilized on a solid surface, and recovering (only) those B cells that bind to the (immobilized) antigen. Further includes:

[0205] In one embodiment of any corresponding aspect or embodiment, the population of B cells is obtained from the blood of the rabbit at least 4 days after immunization, hi one embodiment, the population of B cells is obtained from the blood of the rabbit 4 days up to 13 days after immunization.

[0206] In one embodiment of any corresponding aspect or embodiment, the population of B cells is obtained from blood by density gradient centrifugation.

[0207] In one embodiment of any corresponding aspect or embodiment, the B cell is a mature B cell.

[0208] In one embodiment of any corresponding aspect or embodiment, single cells are placed (individually) in wells of a multi-well plate.

[0209] In one embodiment of all corresponding aspects or embodiments, the feeder mixture is a natural thymocyte culture supernatant (TSN) or a defined and / or synthetic feeder mixture. In one embodiment, the thymocyte culture supernatant is obtained from thymocytes of the thymus of a young animal.

[0210] In one embodiment of any corresponding aspect or embodiment, the feeder mixture is a defined and / or synthetic feeder mixture. In one embodiment, the defined and / or synthetic feeder mixture comprises: i) interleukin-1β and tumor necrosis factor α, and / or ii) interleukin-2 (IL-2) and / or interleukin-10 (IL-10), and / or iii) Staphylococcus aureus Cowan cells (SAC), and / or iv) interleukin-21 (IL-21) and, optionally, interleukin-2 (IL-2), and / or v) B-cell activating factor of the tumor necrosis factor family (BAFF), and / or vi) interleukin-6 (IL-6), and / or vii) Interleukin-4 (IL-4) Includes.

[0211] In one embodiment of any corresponding aspect or embodiment, the feeder mixture comprises IL-1β and TNF-α, and one or more selected from IL-10, IL-21, SAC, BAFF, IL-2, IL-4, and IL-6.

[0212] In one embodiment of any corresponding aspect or embodiment, the feeder mixture comprises IL-1β, TNF-α, IL-10, SAC, and IL-2.

[0213] In one embodiment of any corresponding aspect or embodiment, the B cell population is a rabbit B cell population and the feeder mixture is thymocyte culture supernatant.

[0214] In one embodiment of any corresponding aspect or embodiment, the B cell population is a rabbit B cell population and the feeder mixture consists of IL-1β, TNF-α, and any two of IL-2, IL-6, and IL-10.

[0215] In one embodiment of any corresponding aspect or embodiment, the B cell population is a rabbit B cell population and the feeder mixture consists of IL-1β, TNF-α, IL-6, and IL-10.

[0216] In one embodiment of any corresponding aspect or embodiment, the B cell population is a rabbit B cell population and the feeder mixture comprises IL-1β, TNF-α, IL-10, SAC, and IL-2 or IL-6.

[0217] In one embodiment of any corresponding aspect or embodiment, the B cell population is a rabbit B cell population and the feeder mixture comprises IL-1β, TNF-α, IL-21, and at least one of IL-2, IL-10, and IL-6.

[0218] In one embodiment of any corresponding aspect or embodiment, the antibody is a monoclonal antibody.

[0219] In one embodiment of any corresponding aspect or embodiment, the labelling is of B cell surface IgG.

[0220] In one preferred embodiment of all corresponding aspects or embodiments, the incubation is with a fluorescently labeled antibody according to the invention, a fluorescently labeled anti-IgG antibody, and a fluorescently labeled anti-IgM antibody (labeling is of cell surface IgG and cell surface IgM), and the selection is the selection of cells positive for cell surface CD19, cell surface IgG, and negative for cell surface IgM (CD19 + IgG + IgM - (resulting in the deposition of B cells as single cells).

[0221] In one embodiment of all corresponding aspects or embodiments, the incubation is with a fluorescently labeled antibody according to the invention, a fluorescently labeled anti-IgG antibody, and a fluorescently labeled anti-light chain antibody (labeling is of cell surface IgG and cell surface antibody light chain), and the selection is selection of cells positive for cell surface CD19, cell surface IgG, and cell surface antibody light chain (resulting in placing IgG+LC+B cells as single cells).

[0222] In one embodiment of any corresponding aspect or embodiment, the incubation is performed in addition with a fluorescently labeled anti-light chain antibody (the labeling is of the cell surface antibody light chain in addition to the other two labels) and the selection is of cells positive for the cell surface antibody light chain (resulting in placing LC+ B cells as single cells).

[0223] In one preferred embodiment of all corresponding aspects or embodiments, the incubation is with a fluorescently labeled antibody according to the invention, a fluorescently labeled anti-IgG antibody, and a fluorescently labeled anti-IgM antibody (labeling is of cell surface CD19, cell surface IgG, and cell surface IgM), and the selection is the selection of cells that are positive for cell surface CD19 and IgG and negative for cell surface IgM (CD19 + IgG+ IgM - This results in the deposition of B cells as single cells, whereby a population of B cells is incubated with a (target) antigen immobilized on a solid surface, and (only) the B cells that bound to the immobilized antigen are recovered and subjected to incubation with a fluorescently labeled antibody.

[0224] In one embodiment of any corresponding aspect or embodiment, the co-culture is carried out in a co-culture medium comprising RPMI 1640 medium supplemented with 10% (v / v) FCS, 1% (w / v) 200 mM glutamine solution containing penicillin and streptomycin, 2% (v / v) 100 mM sodium pyruvate solution, and 1% (v / v) 1 M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethanesulfonic acid (HEPES) buffer. In one embodiment, the co-culture medium further comprises 0.05 mM β-mercaptoethanol.

[0225] In a further method, the antibody according to the present invention or its rabbit CD19 binding fragment is immobilized on a solid phase and used to capture rabbit CD19 positive B cells. The solid surface can be any surface, including a multi-well plate or a well of beads, especially magnetic beads. These immobilized antibodies according to the present invention can be used in a similar manner to the labeled antibodies according to the present invention, i.e. to selectively bind rabbit CD19 positive B cells. Those skilled in the art know how to replace the FACS-based step in the above method with a bead-based approach.

[0226] For example, one aspect of the present invention is a) obtaining B cells from rabbit blood; b) incubating the B cells with an antibody according to the invention immobilized on beads; c) washing the beads to remove unbound B cells; and d) optionally recovering the B cells from the beads, thereby selecting one or more B cells to which an antibody according to the invention is bound. A method for selecting B cells comprising:

[0227] In one embodiment of any corresponding aspect or embodiment, the beads are magnetic beads. In a further embodiment, the method comprises a step bc) of binding the beads to a magnet after step b) and before step c).

[0228] In one embodiment of any corresponding aspect or embodiment, the method comprises: d) optionally incubating the B cells in co-culture medium at 37° C. for 1 hour; e) placing one or more B cells or beads into individual containers; f) co-culturing the plated cells with feeder cells in a co-culture medium; g) selecting the B cells proliferating in step f), thereby selecting the B cells The method further includes one or more of:

[0229] One aspect described herein is a) co-culturing each B cell of the enriched population of B cells obtained from an original population of B cells by binding to an antibody according to the invention, itself bound to a solid surface, with mouse EL-4 B5 cells as feeder cells; b) selecting B cell clones which proliferate and secrete antibodies in step a); A method for selecting B cells comprising:

[0230] One aspect described herein is a) co-culturing one or more B cells of an enriched population of B cells, obtained from an original population of B cells by binding to an antibody according to the invention itself bound to a solid surface, optionally in the presence of mouse EL-4 B5 cells as feeder cells and IL-1β, TNFα, IL-10 as a feeder mix, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4 and IL-6; b) selecting a B cell clone that produces an antibody that specifically binds to the target antigen; b1) determining the nucleic acid sequences encoding the light and heavy chain variable domains of said antibody by reverse transcription PCR; b2) transfecting a cell with a nucleic acid comprising a nucleic acid sequence encoding said antibody light chain variable domain and said heavy chain variable domain; c) producing the antibody by culturing cells containing a nucleic acid encoding the antibody or a humanized variant thereof produced by the B cell clone selected in step b) and recovering the antibody from the cells or the culture supernatant. A method for producing an antibody that binds to a target antigen, comprising:

[0231] One aspect described herein is - incubating a large number of rabbit B cells with an antibody according to the invention bound to a solid surface / labeling individual B cells of the large number of rabbit B cells with an antibody according to the invention bound to a solid surface, - selecting / depositing one or more rabbit B cells, either as individual B cells (B cells deposited as single cells) or as a pool of B cells, on the surface of which an antibody according to the invention is bound; and - co-culturing said single cell-placed rabbit B cells or said pool of rabbit B cells with feeder cells, - optionally, after co-culture, incubating the resulting cell mixture with an antibody according to the invention conjugated to a detectable label and selecting / locating / counting rabbit B cells which have on their surface bound / labeled with an antibody according to the invention. A method for co-culturing one or more rabbit B cells, comprising:

[0232] B. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-human CD19 antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell or a lymphocytic cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making an anti-rabbit CD19 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and, optionally, recovering the antibody from the host cell (or host cell culture medium).

[0233] For recombinant production of anti-rabbit CD19 antibodies, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

[0234] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and may be further purified.

[0235] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414, and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0236] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0237] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0238] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney cell lines (e.g., 293 or 293 cells, as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells, as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT060562), e.g., Mather, JP et al., Annals Other useful mammalian host cell lines include TRI cells, MRC5 cells, and FS4 cells, as described in NYAcad.Sci.383 (1982) 44-68. - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0239] C. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the anti-rabbit CD19 antibodies provided herein is useful for detecting the presence of rabbit CD19-presenting cells in biological samples.The term "detecting" as used herein encompasses quantitative or qualitative detection.In certain embodiments, the biological sample comprises cells or tissues, such as blood, serum or plasma.

[0240] In one embodiment, there is provided an anti-rabbit CD19 antibody for use in a method of diagnosis or detection, aspects of which are outlined above.

[0241] In certain embodiments, a labeled anti-rabbit CD19 antibody is provided. Labels include, but are not limited to, directly detectable labels or moieties (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties (such as enzymes or ligands) via, for example, enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131I, rare earth chelates or those coupled with fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that use hydrogen peroxide to oxidize dye precursors, such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0242] III. Description of Sequence Listing SEQ ID NO: 01: Rabbit CD19 amino acid sequence without the signal peptide SEQ ID NO: 02 Rabbit CD19 amino acid sequence with signal peptide SEQ ID NO: 03 Rabbit CD19 cDNA SEQ ID NO: 04 Hamster CD19 amino acid sequence SEQ ID NO: 05 Mouse CD19 amino acid sequence SEQ ID NO: 06 Rat CD19 amino acid sequence SEQ ID NO: 07 Squirrel CD19 amino acid sequence SEQ ID NO: 08 Marmoset CD19 amino acid sequence SEQ ID NO: 09 Rhesus monkey CD19 amino acid sequence SEQ ID NO: 10 Human CD19 amino acid sequence SEQ ID NO: 11 Feline CD19 amino acid sequence SEQ ID NO: 12 Naked mole rat CD19 amino acid sequence SEQ ID NO: 13 Guinea pig CD19 amino acid sequence SEQ ID NO: 14 Porcine CD19 amino acid sequence SEQ ID NO: 15 Canine CD19 amino acid sequence SEQ ID NO: 16 GAP consensus amino acid sequence SEQ ID NO: 17 Primer SEQ ID NO:18 Primer SEQ ID NO: 19 Primer SEQ ID NO: 20 Primer SEQ ID NO:21 Primer SEQ ID NO:22 Primer SEQ ID NO:23 Primer SEQ ID NO: 24 Antibody 1H2 light chain amino acid sequence SEQ ID NO: 25 Antibody 1H2 light chain leader peptide amino acid sequence SEQ ID NO: 26: Antibody 1H2 light chain variable domain amino acid sequence SEQ ID NO: 27: Antibody 1H2 light chain constant region amino acid sequence SEQ ID NO: 28 Antibody 1H2 heavy chain amino acid sequence SEQ ID NO: 29 Antibody 1H2 heavy chain leader peptide amino acid sequence SEQ ID NO: 30 Antibody 1H2 heavy chain variable domain amino acid sequence SEQ ID NO: 31 Antibody 1H2 heavy chain constant region amino acid sequence SEQ ID NO: 32 Antibody 1H2 heavy chain HVR-1 variant 1 SEQ ID NO: 33 Antibody 1H2 heavy chain HVR-1 variant 2 SEQ ID NO: 34 Antibody 1H2 heavy chain HVR-1 variant 3 SEQ ID NO: 35 Antibody 1H2 heavy chain HVR-2 variant 1 SEQ ID NO: 36 Antibody 1H2 heavy chain HVR-2 variant 2 SEQ ID NO: 37 Antibody 1H2 heavy chain HVR-3 SEQ ID NO: 38 Antibody 1H2 light chain HVR-1 SEQ ID NO: 39 Antibody 1H2 light chain HVR-2 SEQ ID NO: 40 Antibody 1H2 light chain HVR-3 SEQ ID NO: 41 Human kappa light chain constant region amino acid sequence SEQ ID NO: 42 Human lambda light chain constant region amino acid sequence SEQ ID NO: 43 Human IgG1 heavy chain constant region amino acid sequence: Caucasian allotype SEQ ID NO: 44 Human IgG1 heavy chain constant region amino acid sequence: Afro-American allotype SEQ ID NO: 45 Human IgG1 heavy chain constant region amino acid sequence: LALA variant SEQ ID NO: 46 Human IgG1 heavy chain constant region amino acid sequence: LALAPG variant SEQ ID NO: 47 Human IgG4 heavy chain constant region amino acid sequence SEQ ID NO: 48 Human IgG4 heavy chain constant region amino acid sequence: SPLE variant SEQ ID NO: 49 Human IgG4 heavy chain constant region amino acid sequence: SPLEPG variant SEQ ID NO: 50 Mouse kappa light chain constant region amino acid sequence SEQ ID NO: 51 Human lambda light chain constant region amino acid sequence SEQ ID NO: 52 GPI anchor amino acid sequence SEQ ID NO:53 3'UTR primer SEQ ID NO:54 5'UTR primer [Brief description of the drawings]

[0243] IV. Description of the Figures [Figure 1A] Figure 1: FACS analysis of recombinant mouse cells transfected with an expression plasmid for rabbit CD19 fused to a FLAG tag. Expression of rabbit CD19 was indirectly confirmed by cell surface staining with a FITC-labeled anti-FLAG tag antibody. A: NIH / 3T3, regular transfection with Lipofectamine, 24 hours post-transfection; B: C2C12, regular transfection with Lipofectamine, 24 hours post-transfection; C: NIH / 3T3, reverse transfection with Lipofectamine, 48 hours post-transfection. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A]Figure 2: FACS analysis of rabbit PBMCs to identify hybridoma supernatants that bind rabbit CD19. Rabbit PBMCs were double stained with FITC-labeled anti-rabbit IgM antibody and hybridoma supernatants. Unlabeled mouse antibodies of individual hybridoma supernatants were developed with PE-labeled anti-mouse IgG antibody. A: Positive hybridoma supernatants that bind rabbit CD19 on rbIgM-positive B cells; B: Exemplarily, negative hybridoma supernatants that show no binding to rabbit IgM-positive B cells. [Figure 2B] See legend to Figure 2A. [Figure 3A] Figure 3: Use of rabbit CD19 as a B cell marker using purified and Alexa647-labeled anti-rabbit CD19 antibody. Comparison of FACS plots of the gates used during single B cell sorting with plots generated by the index sorting approach revealed that antigen-specific and IgG-secreting B cells were highly rabbit CD19 positive. A: FACS plot of rabbit IgG-FITC labeled B cells (gate P8) used for single cell sorting. Rabbit IgM-PE labeling of rabbit B cells was used as counterstain; B: Use of anti-rabbit CD19 antibody-based labeling (gate P5) for characterization of sorted B cells; C: FACS plot generated by the index sorting approach with rabbit IgG and antigen-specific ELISA data showing the rabbit IgG gate; D: FACS plot generated by the index sorting approach with rabbit IgG and antigen-specific ELISA data showing the rabbit CD19 gate. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A]Figure 4: FACS analysis of B cells and feeder cells after 7 days of B cell culture. A: FACS plot showing all cells distributed via cell size (FSC, forward scatter) and cell complexity (SSC, side scatter); B: FACS plot showing all cells distributed via rabbit CD19 staining (Alexa647) and dead cells (propidium iodide); C: Accurate count of B cells in wells containing few B cells. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A] Figure 5: FACS analysis of B cells and feeder cells after 7 days of B cell culture. A: FACS plot showing all cells distributed via cell size (FSC) and cell complexity (SSC); B: FACS plot showing all cells distributed via rabbit CD19 staining (Alexa647) and dead cells (propidium iodide); C: Accurate count of B cells in wells containing a large number of B cells. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] Figure 6: A: FACS plot of peripheral blood lymphocytes (PBLs) from rabbit showing cell size (FSC) and cell complexity (SSC). B: FACS plot of PBLs from rabbit showing cell size (FSC) and rabbit IgG stained cell population (=gate P5) in the FITC channel. [Figure 7A] Figure 7: A: FACS plot showing B cell viability after antigen enrichment with magnetic beads. B: FACS plot showing B cell viability after CD19+ B cell enrichment. [Figure 7B] See legend to Figure 7A. EXAMPLES

[0244] V. Working Examples The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0245] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but such descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

[0246] Example 1 Immunization and generation of mouse anti-rabbit CD19 antibody (hybridoma) NMRI mice obtained from Charles River Laboratories International, Inc. were used for immunization. Animals were housed in an AAALACi-accredited animal facility in accordance with Appendix A, "Guidelines for animal facilities and care." All animal immunization protocols and experiments were approved by the Upper Bavarian State Government (permit number AZ 55.2-1-54-2531-83-13) and were performed in accordance with the German Animal Welfare Act and Directive 2010 / 63 of the European Parliament and Council.

[0247] Six to eight week old NMRI mice (n=5) were subjected to plasmid DNA-based immunizations over a period of three months. For this purpose, a plasmid DNA encoding rabbit CD19 as a single-stranded molecule was used. Prior to spleen harvest for hybridoma fusion, mice were boosted with NIH / 3T3 cells (ATCC CRL-1658) transiently transfected with the same vector for expression of rabbit CD19.

[0248] For the first immunization, animals were anesthetized with isoflurane and immunized intradermally (id) with 100 μg of plasmid DNA in sterile H2O at a single site on the shaved back proximal to the tail. After id application, the spot was electroporated on an ECM830 electroporation system (BTX Harvard Apparatus) using the following parameters: 2 times 1000 V / cm for 0.1 ms, separated by 125 ms intervals, followed by 4 times 287.5 V / cm for 10 ms, also separated by 125 ms intervals. Booster immunizations were performed in a similar manner on days 14, 28, 49, 63, and 77. Six weeks after the final immunization, mice were injected intravenously (iv) and intraperitoneally (ip) with 0.9×10E6 NIH / 3T3 cells, transiently transfected for expression of rabbit CD19 and dissolved in sterile PBS, respectively. After 72 hours, spleens were aseptically harvested and prepared for hybridoma generation.

[0249] Fusion of spleen cells from immunized mice was performed according to standard protocols: the myeloma cell line P3X63-Ag8653 was incubated at 3 × 10 5 The cells were cultured to a cell density of 1000 cells / mL. The cells were then harvested (1,000 rpm, 10 min, 37°C) and washed with 50 mL of RPMI (37°C). After a second centrifugation step under the same conditions, the cells were resuspended in 50 mL of RPMI (37°C) and then stored on ice. Sterile spleens removed from immunized mice were sieved through a cell strainer (70 μm) to obtain single cells. The single cell culture was transferred to a 15 mL tube and incubated on ice for 10 min. After incubation, the cell suspension supernatant was transferred to a 50 mL tube, harvested (250xg, 10 min, 4°C) and resuspended in 15 mL of RPMI medium.

[0250] After detection of cell density, spleen cells and myeloma cells were mixed in a ratio of 5:1 and centrifuged (250xg, 10 min, 37°C). Spleen cells 10 81 mL of polyethylene glycol (PEG) was added per well with gentle shaking, and the samples were incubated at 37° C. and 6% CO2 for at least 30 min. After incubation, cells were harvested at 250×g (37° C.) for 10 min and resuspended in 20 mL of RPMI medium. Finally, all fusion samples were transferred to microtiter plates (MTP, 200 μl / well), incubated (37° C., 6% CO2), and used for further analysis.

[0251] Example 2 Hybridoma screening and cell biological function evaluation of anti-CD19 antibodies High-throughput FACS analysis for screening antibodies against rabbit CD19 Hybridoma supernatants were characterized by mouse IgG ELISA. Primary screening of IgG-containing culture supernatants was performed by ELISA using a standard protocol: streptavidin-coated 384-well MTPs were incubated with a biotinylated polyclonal anti-mouse Fcg region antibody (pAK <mfcg>S-IgH-(IS)-Bi(XOSu)). 50 μl / well of supernatant (diluted 1:600) was applied and incubated for 60 min at room temperature. Afterwards, samples were washed three times with 0.9% (w / v) NaCl, 0.05% (v / v) Tween20, 0.2% (v / v) BronidoxL. For detection of IgG, samples were incubated with peroxidase-conjugated AffiniPure goat anti-mouse F(ab')2 fragment (diluted 1:15,000, 50 μl / well) and incubated for 60 min at room temperature. After washing as above, ABTS solution (1 mg / mL, 50 μl / well) was added and incubated for another 20 min. Readout was performed at wavelengths of 405 / 492 nm on an X Read Plus Reader (Tecan). Only IgG-positive hybridoma supernatants were subjected to antigen-specific high-throughput FACS analysis.

[0252] FACS analysis was applied to screen the hybridomas to identify those secreting antibodies against rabbit CD19. All IgG-producing hybridomas were screened by FACS analysis of rabbit peripheral blood mononuclear cells (PBMCs) double stained with rabbit IgM-binding antibodies for B cell identification.

[0253] Freshly isolated rabbit PBMCs were incubated on ice with FITC-labeled anti-rabbit IgM antibody (Southern Biotech) and IgG-positive hybridoma supernatants. After 45 min of incubation, PBMCs were washed once with ice-cold PBS and resuspended in PE-labeled anti-mouse IgG antibody (Invitrogen), which binds to the mouse IgG of the hybridoma supernatants. After another 45 min of incubation on ice, cells were washed once again with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. DAPI-HCl (Cayman) at a concentration of 2 μg / ml was added before FACS analysis to distinguish dead and live cells. A Becton Dickinson FACS Canto II device with a computer and FACSDiva software (BD Biosciences) were used for the analysis.

[0254] After identifying hybridoma supernatants that bind to rabbit IgM-positive B cells, rabbit CD19 specificity was confirmed by FACS analysis of CHO or HEK293 cells transfected with rbCD19 expression plasmid. Cells transfected with rabbit CD19 were used as positive cells, and untransfected CHO or HEK293 cells were used as negative control cells. Staining for rabbit CD19 was performed as described in Example 7.

[0255] Example 3 Expression of CD19-binding antibodies Sequences encoding the antibody variable domains are produced by gene synthesis.

[0256] All sequences are verified by sequencing. All sequences are cloned into vectors that allow selection and propagation in E. coli (e.g., origin of replication from vector pUC18, β-lactamase for ampicillin resistance). These vectors additionally contain cassettes that allow expression in mammalian cells (e.g., origin of replication (oriP) of Epstein-Barr virus (EBV), immediate early enhancer and promoter from human cytomegalovirus (HCMV), and polyadenylation sequences).

[0257] All gene segments encoding the light and heavy chains of antibodies contain DNA sequences encoding signal peptides. TIFF0007650802000021.tif11128 precedes. Proteins are expressed by transiently transfected human embryonic kidney HEK293 cells in suspension. These cells are cultured at 37 °C and 8% CO2. On the day of transfection, cells are transfected at 1-2 × 10 6 The cells are seeded at a density of viable cells / mL in fresh medium. Equimolar amounts of both heavy and light chain plasmid DNA are transfected simultaneously. Cell culture supernatants are harvested 7 days after transfection, centrifuged (14,000×g for 45 min at 4° C.), and subsequently filtered through a 0.22 μm filter. These supernatants can be frozen and stored at −20° C. prior to purification.

[0258] Example 4 Purification of CD19-binding antibodies The cell-free hybridoma supernatant was applied to a pre-equilibrated (phosphate buffered saline, PBS) Protein A affinity column (MabSelect (商標) The antibody is loaded onto a column (SuRe, GE Healthcare, 8 x 100 mm). After washing (PBS, 5 column volumes) the antibody is eluted with 25 mM citric acid / NaOH (pH 3.0). The eluate is adjusted to pH 5.5 with 1 M Tris and incubated overnight at 4°C. A final filtration (0.45 μm) is then performed.

[0259] Example 5 Providing CD19 ECD-expressing cells and binding of antibodies thereto For extracellular display, human PSCA GPI anchor sequence Cells were transfected with a plasmid encoding rabbit CD19 (SEQ ID NO: 01) extracellular domain (ECD) fused to TIFF0007650802000022.tif5131. For transfection of NIH / 3T3 cells for immunization, 1x10E7 cells were seeded in three T175 flasks. Cells were then reverse transfected in solution using lipofectamine. Transfection efficiency was determined to 40% positive cells after 48 hours using FITC anti-FLAG antibody (abcam) and FACS (Figure 1C). To confirm CD19 specificity by FACS analysis, CHO or HEK293 cells were transfected with rbCD19. 3x10E5 HEK293 and 1x10E5 CHO cells were transfected using lipofectamine. Expression was confirmed after 48 hours by FACS using FITC anti-FLAG antibody (abcam).

[0260] Example 6 Anti-rabbit CD19 antibody conjugated to a detectable label Anti-rabbit CD19 antibody in phosphate buffer (pH 8.5) was adjusted to a protein concentration of approximately 5 mg / ml. D-Biotinyl-aminocaproic acid-N-hydroxysuccinimide was dissolved in DMSO and added to the antibody solution at a molar ratio of 1:5. After 60 min, the reaction was stopped by adding L-lysine, and the excess of labeling reagent was removed by dialysis against 50 mM potassium phosphate buffer containing 150 mM NaCl, pH 7.5.

[0261] Example 7 2. Labeling of B Cells with Conjugated Anti-Rabbit CD19 Antibody, Including the Index-sorting Approach B cells were stained with anti-IgG FITC (1:200, AbD Serotec), anti-IgM PE (1:200 BD Pharmingen), and anti-CD19 A647 (1:200, Roche) antibodies in DPBS supplemented with normal mouse serum (1:20, Southern Biotech) for 30 min in the dark (4 °C). Cells were subsequently washed and resuspended in ice-cold DPBS. Live / dead discrimination was achieved by adding propidium iodide (PI) (BD Pharmingen) at a concentration of 0.5 μg / ml shortly before single cell sorting on a Becton Dickinson FACSAria equipped with a computer and FACSDiva software (BD Biosciences). Single IgG+ and IgG+IgM+ cells were sorted into 96-well plates. The index sort tool of the FACSAria was applied to preserve the CD19 expression of each sorted cell. Cells were cultured as described in Example 9. After 7 days of culture, the supernatants were used to determine the number of IgG-producing and antigen-specific clones by ELISA. A plugin was developed in FlowJo that combined the ELISA data with the FACS index sorting data. This plugin adds the IgG-positive and antigen-specific wells from the ELISA to the fluorescence data from anti-rabbit IgG, anti-rabbit IgM, and anti-rabbit CD19 staining, and thus the IgG-producing and antigen-specific clones can be visualized in FlowJo. The results show that all IgG-producing and all antigen-specific clones are IgG and CD19 double positive. Furthermore, by checking the percentage of sorted double-positive cells, the sorting efficiency (more specifically sorted cells) can be improved by about 14-20%.

[0262] Example 8 Immunofluorescence staining and flow cytometry prior to B cell culture Anti-IgG FITC (AbD Serotec) and anti-huCk PE (BD Bioscience) antibodies were used for single cell sorting. For surface staining, cells from the depletion and enrichment steps were incubated with anti-IgG FITC and anti-huCk PE antibodies in PBS for 45 min in the dark at 4°C. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Propidium iodide (BD Pharmingen) at a concentration of 0.5 μg / ml was added before FACS analysis to discriminate between dead and live cells. A Becton Dickinson FACSAria equipped with a computer and FACSDiva software (BD Biosciences) was used for single cell sorting.

[0263] Example 9 B cell culture Rabbit B cell culture was performed according to the method described in Seeber et al. (2014). Briefly, single cell sorted rabbit B cells were incubated in 96-well plates with 200 μl / well of EL-4 B5 medium containing Pansorbin cells (1:100000) (Calbiochem) and a synthetic cytokine mixture as described in this table: TIFF0007650802000023.tif31128

[0264] In addition, 0.35 ng / μl phorbol myristate acetate and gamma-irradiated (4 Gy) mouse EL-4 B5 thymoma cells (2×10E5 cells / well) were used, and the cells were cultured in an incubator at 37° C. for 7 days. The supernatant of the B cell culture was removed for screening, and the remaining cells were immediately dissolved in 100 μl of RLT buffer (Qiagen) and frozen at −80° C.

[0265] Example 10 Measurement of CD19+ B cells in blood and spleen Blood was subjected to density gradient centrifugation for isolation of PBMCs. Spleens were mashed and centrifuged before lysing red blood cells with regular red blood cell lysis buffer according to the manufacturer's instructions. Cells from blood or spleens were plated at up to 6 × 10 in 1 ml medium on sterile 6-well plates. 6 PBMCs were seeded at a concentration of 1000 PBMCs / well. Macrophage depletion occurs by non-specific adhesion to the cell culture plate during 1 hour incubation at 37°C in an incubator. Isolated PBMCs were stained and washed as described in Example 7. Live / dead discrimination was achieved by DAPI (Biomol) at a concentration of 0.1 μg / ml shortly before analysis of cell populations on a Becton Dickinson FACSCanto equipped with a computer and FACSDiva software (BD Biosciences). Analysis of CD19 positive B cells in blood and spleen was performed using FlowJo v10.0.7.

[0266] Example 11 Counting B cells after co-culture B cells were sorted and co-cultured with feeder cells as described in Example 7 and Example 9. After 7 days of culture, the 96-well culture plates were centrifuged at 300xg for 5 minutes, the medium was removed, and the pellet was resuspended in ice-cold DPBS containing anti-CD19 A647 antibody (1:400, Roche) and supplemented with normal mouse serum (1:20, Southern Biotech) for 30 minutes of incubation in the dark (4°C). Subsequently, the cells were washed and resuspended in a defined volume of ice-cold DPBS. Live / dead discrimination was achieved by adding DAPI (Biomol) at a concentration of 0.1 μg / ml shortly before analysis of the cell populations on a Becton Dickinson FACSCanto equipped with a computer and FACSDiva software (BD Biosciences). For accurate calculation of the total B cell number per well, it is important to set a defined analysis volume on the FACSCanto and take into account the total sample volume. Analysis was performed using FlowJo v10.0.7, and the number of CD19+ cells was used to calculate the cell number of B cells, taking into account the total sample volume. This method allows for the enumeration of B cells within B cell clones for the first time after our in-house B cell cloning approach, since, for example, cell surface IgG decreases during culture.

[0267] Example 12 Enrichment of CD19-positive B cells using the antibodies according to the present invention PBMCs from immunized rabbits were isolated from blood as described in Example 10, stained and cultured as described in Examples 8 and 9. Half of the cells were treated as follows: biotinylated anti-rabbit CD19 was incubated with PBMCs at 1:500 dilution for 15 min at 4°C. PBMCs were washed with cold MACS buffer (Miltenyi) and then incubated with streptavidin MACS beads (Miltenyi) according to the manufacturer's instructions. Cells were washed with MACS buffer and subsequently purified using Miltenyi LS columns according to the manufacturer's instructions. Purified cells were incubated with fluorescently labeled antigen and anti-rabbit IgG FITC antibody (Southern Biotech) for 15 min at 4°C, followed by washing. The other half of the cells was first incubated with biotinylated antigen (1:10000) for 15 min at 4°C. Cells were washed and then incubated with streptavidin MACS beads according to the manufacturer's instructions. The cells were washed and subjected to an LS column. The cells were then incubated with fluorescently labeled anti-rabbit CD19 and anti-rabbit IgG antibodies. Finally, the PBMCs were resuspended in ice-cold PBS and immediately sorted on a BD Aria III. 7-AAD (BD Pharmingen) was added according to the manufacturer's instructions to distinguish between dead and live cells. The first half of the cells were gated for size, antigen positivity, and IgG positivity. The second half of the cells were gated for size, CD19 positivity, and IgG positivity. The cells were single-cell sorted and cultured as described in Example 9. After one week of incubation, ELISA was used to determine the number of IgG-positive, specific, and cross-reactive clones. Enrichment with anti-rabbit CD19 resulted in an increase in cell viability (Figure 7), an increase in the number of IgG-producing clones, and an increase in the number of antigen-specific clones. Furthermore, the number of cross-reactive clones was reduced.< / mfcg>

Claims

1. An antibody that binds to rabbit CD19, (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 33, or 34; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35 or 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40 An antibody comprising:

2. The antibody of claim 1 which is a monoclonal antibody.

3. The antibody of claim 1 or 2, which is a chimeric or humanized antibody.

4. 3. The antibody of claim 1 or 2, comprising a heavy chain variable domain of SEQ ID NO: 30 and a light chain variable domain of SEQ ID NO:

26.

5. Full length antibody, or Fab, Fab', Fab'-SH, F(ab') ２ 5. The antibody of claim 1 , which is an antibody fragment selected from the group consisting of an Fv, an scFv fragment, and a diabody.

6. The antibody of claim 1 , conjugated to a detectable label.

7. The antibody of claim 6 , wherein the detectable label is a fluorescent dye.

8. 1. A method for selecting rabbit B cells, comprising: a) incubating a multiplicity of rabbit cells with an antibody according to any one of claims 1 to 7; and b) selecting one or more B cells to which an antibody according to any one of claims 1 to 7 is bound, thereby selecting rabbit B cells. A method comprising:

9. After step b) and before step c), incubating the rabbit B cells in co-culture medium at 37° C. for 1 hour; and / or c) placing as single cells one or more rabbit B cells to which the antibody according to any one of claims 1 to 7 is bound, and / or d) co-culturing the single-cell-placed rabbit B cells with feeder cells in a co-culture medium; and / or e) selecting the rabbit B cells proliferating in step d), thereby selecting the rabbit B cells. The method of claim 8 , further comprising one or more of:

10. 1. A method for removing rabbit non-B cells from a culture of rabbit cells, comprising: a) co-culturing rabbit B cells, deposited either as single cells or as a pool of cells, with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to any one of claims 1 to 7, and c) selecting one or more rabbit B cells to which an antibody according to any one of claims 1 to 7 is bound, thereby removing rabbit non-B cells. A method comprising:

11. 1. A method for determining the number of rabbit B cells in a co-culture of rabbit B cells plated as single cells and feeder cells, comprising: a) co-culturing rabbit B cells, deposited as single cells, with feeder cells; b) incubating the cells from the co-culture obtained in step a) with an antibody according to any one of claims 1 to 7, and c) determining the number of rabbit B cells in the culture by counting the number of cells to which the antibody according to any one of claims 1 to 7 is bound. A method comprising:

12. 1. A method for co-culturing one or more rabbit B cells, comprising: - incubating a large number of rabbit B cells with an antibody according to any one of claims 1 to 7 or labelling individual rabbit B cells of the large number of rabbit B cells with an antibody according to any one of claims 1 to 7, - selecting or depositing one or more rabbit B cells having an antibody according to any one of claims 1 to 7 bound to its surface or labelled either as individual rabbit B cells (rabbit B cells deposited as single cells) or as a pool of rabbit B cells; and - co-culturing said single cell-placed rabbit B cells or said pool of rabbit B cells with feeder cells; A method comprising:

13. 13. The method of any one of claims 9 to 12, wherein the co-culture is carried out in the presence of a synthetic feeder mixture comprising IL-1β, TNFα, IL-10, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4, and IL-6.

14. a) obtaining B cells from rabbit blood; b) incubating the B cells with an antibody according to any one of claims 1 to 5 bound to beads; c) removing unbound B cells; and d) Recovering bound B cells from said beads, thereby selecting one or more B cells. A method for selecting B cells, comprising:

15. e) placing one or more of the recovered B cells into individual containers; f) co-culturing the plated cells with feeder cells in a co-culture medium; and g) selecting the B cells proliferating in step f), thereby selecting the B cells. The method of claim 14 further comprising:

16. 16. The method of claim 15, further comprising the step of incubating the collected B cells in co-culture medium at 37° C. for 1 hour prior to step e).

Images