Method for discovery of alternative antigen specific antibody variants

Abstract

Herein is reported a method for selecting a variant of a parental antibody variable domain encoding nucleic acid, wherein the parental antibody variable domain amino acid sequence encoded by said encoding nucleic acid has at least one developability hot spot, the method comprising the steps of (i) providing a multitude of DNA-containing samples (genomic material of antibody secreting B-cell) each including one or more antibody variable domain encoding nucleic acids; (ii) performing PCR amplification of said antibody variable domain encoding nucleic acids of (i) using consensus sequence-specific primers to obtain amplification products (wherein said consensus sequence-specific primers bind to consensus sequences that are common to a plurality of genes within the genetic loci set, thereby generating a pool of amplification products); (iii) sequencing a plurality of said amplification products obtained in step (ii) in order to determine the relative proportion of each nucleotide at each position in a sequencing read; (iv) performing a sequence alignment between the sequencing read results of (iii) and the parental antibody variable domain encoding nucleic acid; (v) performing a sequence-identity/homology-based ranking of the antibody variable domain encoding nucleic acids in said sequence alignment with the parental antibody variable domain encoding nucleic acid being the perfect/template/reference sequence; and (vi) selecting the variant antibody variable domain encoding nucleic acid based on the sequence ranking of step (v), whereby the variant selected in step (vi) is selected so that the developability hot-spot is removed.

Claims

1. A method for selecting a variant of a reference antibody variable domain encoding nucleic acid, wherein either the variant antibody variable domain amino acid sequence encoded by said variant of the reference antibody variable domain encoding nucleic acid has improved developability compared to the reference antibody variable domain amino acid sequence encoded by said reference antibody variable domain encoding nucleic acid, or wherein in the variant antibody variable domain amino acid sequence encoded by said variant of the reference antibody variable domain encoding nucleic acid at least one amino acid residue that is post-translationally modified has been changed compared to the reference antibody variable domain amino acid sequence encoded by said reference antibody variable domain encoding nucleic acid, wherein the variant and the reference antibody variable domain when paired with the respective other domain bind to the same antigen, the method comprising the following steps: (i) sequencing a plurality of the amplification products obtained in a PCR amplification of the antibody variable domain encoding nucleic acids of a multitude of DNA-containing samples each including one or more antibody variable domain encoding nucleic acids using consensus sequence-specific primers to obtain amplification products; (ii) performing a sequence-identity/homology-based ranking of the antibody variable domain encoding nucleic acids in a sequence alignment between the sequencing results of (i) and the reference antibody variable domain encoding nucleic acid with the reference antibody variable domain encoding nucleic acid being the template sequence; and (iii) selecting the variant antibody variable domain encoding nucleic acid from one of the top 10 sequences of the sequence ranking of step (ii), whereby the variant selected in step (iii) is selected so that the developability has improved and/or at least one amino acid residue that is post-translationally modified has been changed.

2. A method for selecting a variant of a reference antibody variable domain, wherein the reference antibody variable domain has at least one amino acid residue that is post-translationally modified, the method comprising the following steps: aligning antibody variable domain encoding nucleic acids produced by sequencing nucleic acids from a multitude of B-cell clones each producing an antibody specifically binding to the same target as the reference antibody with the sequence of the reference antibody being the template sequence; and selecting a sequence that has the highest structural or/and functional identity or/and similarity to the reference sequence wherein at least one of the at least one amino acid residue that is post-translationally modified of the reference antibody variable domain is replaced by/changed to an amino acid residue that is not post-translationally modified.

3. A method for identifying a variant antibody of a reference antibody specifically binding to the same target/antigen comprising the following steps: i) generating one or more sets of VH or/and VL sequences of a multitude of amino acid sequences or nucleic acid sequences of variant antibodies of the reference antibody specifically binding to the same target as the reference antibody, which have been determined by next generation sequencing, wherein the sequences are aligned with the amino acid sequence or nucleic acid sequence of a reference antibody as template, whereby the reference antibody specifically binds to a target/antigen, based on ) identical length of VH or/and VL, or/and ) identical length of all -sheet framework regions, or/and ) identical length of all HVRs/CDRs, or/and ) identical HVR3/CDR3 sequence, mutations in frameworks and/or HVRs/CDRs with up to 3 amino acid exchanges allowed, or/and ) homologous HVR/CDR3 sequence with up to 2 amino acid exchanges allowed and identical HVR/CDR1 and 2, or/and ) mutations in frameworks and HVR/CDRs are allowed; ii) ranking the aligned sequences in the one or more sets of i) by ) the number of, and/or ) the position(s) of, and/or ) the change of the physico-chemical properties resulting from the, and/or ) the difference of VH/VL orientation resulting from the amino acid difference(s) to the reference antibody sequence; iii) identifying one of the best 10 aligned and ranked antibodies as a variant antibody of a reference antibody, whereby the variant antibody does have at least one amino acid residue that is post-translationally modified less as the reference antibody.

4. The method according to claim 3, wherein step i) further comprises annotating the sequence with the same numbering scheme, which is the Wolfguy numbering scheme.

5. The method according to claim 3, wherein differences in the sequence are annotated in the form: reference antibody amino acid residue-position-variant antibody amino acid residue and are grouped into a mutation tuple.

6. The method according to claim 3, wherein the change of the physico-chemical properties is determined by the change in charge, hydrophobicity and/or size.

7. The method according to claim 3, wherein the change of the physico-chemical properties is determined using a mutation risk score.

8. The method according to claim 7, wherein the mutation risk score is determined based on the following Table, wherein residues that are not explicitly given in this Table are weighted with the value one: TABLE-US-00031 Wolfgulf Wolfguy Index Weight Index Weight 101 0.2 151 2 102 1.1 152 2.6 103 0 153 1.2 104 0.5 154 2.3 105 0.2 155 1.9 106 0.8 156 3.7 107 0.5 157 4 108 0.8 158 4 109 0.4 193 4 110 0.2 194 4 111 0 195 4 112 0.4 196 3.3 113 0 197 3.9 114 0.1 198 2.6 115 0.6 199 3.8 116 0.1 251 3.6 117 0.1 252 4 118 0.2 253 4 119 0.2 254 4 120 1.2 255 3.9 121 0 256 3.4 122 4 287 3.7 123 0 288 2.1 124 2 289 3.6 125 0.5 290 2.3 201 4 291 2.3 202 2.6 292 1 203 2 293 1.2 204 1.7 294 2.4 205 1 295 0.5 206 0 296 4 207 0 297 3.5 208 0.7 298 4 209 1.6 299 3.5 210 2 351 3.5 211 1.3 352 3 212 3.1 353 3 213 0.9 354 3 214 3.1 355 3 301 0.1 356 3 302 1.2 357 3 303 1.7 358 3 304 0.3 359 3 305 1.8 360 3 306 0 361 3 307 2.4 362 3 308 0 363 3 309 1.2 364 3 334 1 365 3 335 1 366 3 336 1 367 3 337 1 382 3 310 0.8 383 3 311 0.9 384 3 312 0.8 385 3 313 2.5 386 3 314 0.1 387 3 315 1.5 388 3 316 0 389 3 317 1.5 390 3 318 0.8 391 3 319 0.1 392 3 320 1.4 393 3 321 0.2 394 3 322 0.3 395 3.5 323 0.2 396 3 324 1.8 397 3.5 325 0.6 398 1.5 326 2.2 399 3 333 1 327 0.6 328 3 329 2.5 330 4 331 2.9 332 2.8 401 3.5 402 2 403 0.5 404 1 405 0.3 406 0.1 407 1 408 0.1 409 1 410 0.1 411 0.1 with positions 101 to 125 corresponding to heavy chain variable domain framework 1, positions 151 to 199 corresponding to CDR-H1, positions 201 to 214 corresponding to heavy chain variable domain framework 2, positions 251 to 299 corresponding to CDR-H2, positions 301 to 332 corresponding to heavy chain variable domain framework 3, positions 351 to 399 corresponding to CDR-H3, positions 401 to 411 corresponding to heavy chain variable domain framework 4.

9. The method according to claim 3, wherein the multitude of amino acid sequences or nucleic acid sequences of variant antibodies specifically binding to the same target as the reference antibody are obtained from B-cells from the same immunization campaign as the reference antibody, wherein the B-cells have been enriched for antigen-specific antibody expressing B-cells.

10. The method according to claim 3, wherein in the variant one or more of the following is removed i) unpaired Cys-residues in the variable domain or the HVR, ii) glycosylation sites, and iii) degradation hot-spots (Asp, Asn or Met).

11. A method for selecting a variant of a reference antibody variable domain, wherein the reference antibody variable domain has at least one amino acid residue that is post-translational modified, the method comprising the steps of the method according to claim 1.

12. A method for producing an antibody comprising the following steps: cultivating a cell comprising the nucleic acid obtained with a method according to claim 1 and all other nucleic acids required for the expression of a functional antibody, recovering the antibody from the cell or the cultivation medium.

13. A cell comprising the nucleic acid obtained with the method according to claim 1.

Description

DESCRIPTION OF THE FIGURES

[0351] FIG. 1 VH (top) and VL (bottom) sequences of the reference antibody 763 and six variants of its VH domain. Framework and CDR classification follows Wolfguy nomenclature. In the VH variants, amino acid substitutions with regard to the reference are shown in light grey. CDRs are shaded grey in the numbering.

[0352] FIG. 2 VH (top) and VL (bottom) sequences of the reference antibody 763 and six variants of its VH domain. Residues forming part of the VH-VL orientation fingerprint have been marked with a gray background. Amino acid substitutions in this region are likely to induce a change VH-VL orientation as compared to the reference antibody. CDRs are shaded grey in the numbering.

[0353] FIGS. 3A-3D EC.sub.50 (M) correlation plot; human versus murine LRP8 binding of all the NGS variants. The controls, the clones identified by screening and panning, (red and blue), and clones, identified by NGS (grey), were plotted; FIG. 3A: Variants for BCC 763, FIG. 3B: Variants for BCC 776, FIG. 3C: Variants for BCC 770, FIG. 3D: Variants for BCC 755.

[0354] FIGS. 4A-4G Absolute EC.sub.50 [nM] values show binding of all NGS variants (white bars) to human CDCP1. Parental controls are shown in black. Successful NGS variants are marked with (*). na=not available, meaning that no EC.sub.50 value could be derived from the binding curve due to weak binding of the variant. FIG. 4A: Variants for CDCP1_105; FIG. 4B: Variants for CDCP1_223; FIG. 4C: Variants for CDCP1_236; FIG. 4D: Variants for CDCP1_284; FIG. 4E: Variants for CDCP1_212; FIG. 4F: Variants for CDCP1_088; FIG. 4G: Variants for CDCP1_234.

[0355] FIG. 5 Scheme of the BCC and NGS workflow.

EXAMPLES

Example 1

Immunization of Rabbits

[0356] A KLH conjugate of a human LRP8 was used for the immunization of the New Zealand White rabbit. Each rabbit was immunized with 500 g of the immunogen, emulsified with complete Freund's adjuvant, at day 0 by intradermal application and 500 g each at days 7, 14, 28, 42 by alternating intramuscular and subcutaneous applications. Thereafter, rabbits received monthly subcutaneous immunizations of 500 g, and small samples of blood were taken 7 days after immunization for the determination of serum titers. A larger blood sample (10% of estimated total blood volume) was taken during the third, fourth and fifth month of immunization (at 5-7 days after immunization), and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B-cells in the B-cell cloning process.

Example 2

Determination of Serum Titers (ELISA)

[0357] Biotinylated human LRP8 was immobilized on a 96-well streptavidin-coated plate at 0.5 g/ml, 100 l/well, in PBS, followed by blocking of the plate with 2% CroteinC in PBS, 200 l/well. Thereafter 100 l/well serial dilutions of antisera, in duplicates, in 0.5% CroteinC in PBS were applied. The detection was done with HRP-conjugated donkey anti-rabbit IgG antibody (Jackson Immunoresearch/Dianova, Cat. No. 711-036-152; 1/16 000), each diluted in 0.5% CroteinC in PBS, 100 l/well. For all steps, plates were incubated for 1 h at 37 C. Between all steps plates were washed 3 times with 0.05 Tween 20 in PBS. Signal was developed by addition of BM Blue POD (peroxidase)-substrate soluble (Roche Diagnostics GmbH, Mannheim, Germany), 100 l/well; and stopped by addition of 1 M HCl, 100 l/well. Absorbance was read out at 450 nm, against 690 nm as reference. Titer was defined as dilution of antisera resulting in half-maximal signal.

Example 3

Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMCs)

[0358] Blood samples were taken of immunized wild-type rabbits (NZW). EDTA containing whole blood was diluted twofold with 1PBS (PAA, Pasching, Austria) before density centrifugation using lympholyte mammal (Cedarlane Laboratories, Burlington, Ontario, Canada) according to the specifications of the manufacturer. The PBMCs were washed twice with 1PBS.

Example 4

Depletion of Macrophages/Monocytes

[0359] The PBMCs were seeded on sterile KLH-coated SA-6-well-plates to deplete macrophages and monocytes through unspecific adhesion and to remove cell binding to KLH. Each well was filled at maximum with 4 ml medium and up to 610E6 PBMCs from the immunized rabbit and were allowed to bind for 1 h at 37 C. and 5% CO2. The cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.

Example 5

Enrichment of B-Cells

[0360] Sterile streptavidin coated 6-well plates (Microcoat, Bernried, Germany) were coated either with 2 g/ml of the biotinylated KLH protein or the biotinylated LRP8/CDCP1 protein in PBS for 3 h at room temperature. Prior to the panning step these 6-well plates were washed three times with sterile PBS. Coated plates were seeded with up to 610E6 PBLs per 4 ml medium and allowed to bind for 1 h at 37 C. and 5% CO2. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1PBS. The remaining sticky cells were detached by trypsin for 10 min. at 37 C. and 5% CO2. Trypsination was stopped with EL-4 B5 medium. The cells were kept on ice until the immune fluorescence staining.

EL-4 B5 medium

[0361] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM Glutamine, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM beta-mercaptoethanol (Gibco, Paisley, Scotland).

Example 6

Immune Fluorescence Staining and Flow Cytometry

[0362] An anti-IgG antibody FITC conjugate (AbD Serotec, Dsseldorf, Germany) was used for single cell sorting. For surface staining, B-cells pre-treated with a depletion step and an enrichment step (Example 4 and 5) were incubated with the anti-IgG antibody FITC conjugate in PBS (phosphate buffered saline solution) and incubated for 45 min. in the dark at 4 C. After staining the cells were washed two times with ice cold PBS. Finally, the labelled B-cells were resuspended in ice cold PBS and immediately subjected to the FACS analyses. Propidium iodide in a concentration of 5 g/ml (BD Pharmingen, San Diego, Calif., USA) was added prior to the FACS analyses to discriminate between dead and live cells.

[0363] A Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences, USA) were used for single cell sort.

Example 7

B-Cell Cultivation

[0364] The cultivation of the single sorted B-cells was done according to a method described by Seeber et al. (Seeber, S., et al., PLoS One, 9 (2014) e86184.). Briefly, single sorted rabbit B-cells were incubated in 96-well plates with 200 l/well EL-4 B5 medium containing Pansorbin Cells (1:100,000) (Calbiochem (Merck), Darmstadt, Germany), 5% rabbit thymocyte supernatant (MicroCoat, Bernried, Germany) and gamma-irradiated murine EL-4 B5 thymoma cells (510E5 cells/well) for 7 days at 37 C. in the incubator. The supernatants of the B-cell cultivation were removed for screening and the remaining cells were harvested immediately and frozen at 80 C. in 100 l RLT buffer (Qiagen, Hilden, Germany).

Example 8

Enzyme-Linked Immunosorbent Assay (ELISA)

Human Antigen:

[0365] The antigen, biotinylated human LRP8, was incubated with 5 L sample containing the anti-LRP8 antibody at a concentration of 250 ng/mL in a total volume of 25 L in PBS, 0.5% BSA and 0.05% Tween in a 384 w microtiterplate (Maxisorb (with Streptavidin, Nunc). After 1.5 hrs. incubation at 25 C. unbound antibody was removed by washing 6 times with 90 L PBS (dispense and aspiration). The antigen-antibody complex was detected by an anti-rabbit antibody conjugated to POD (ECL anti-rabbit IgG-POD, Cat. No. NA9340V; POD=peroxidase). 20-30 min after adding 35 L POD-substrate 3,3,5,5-tetramethyl benzidine (TMB; Piercenet, Cat. No. 34021) the optical density was determined at 370 nm. The EC.sub.50 value was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.

Murine Antigen:

[0366] The antigen, biotinylated murine LRP8, was incubated with 5 L sample containing the anti-LRP8 antibody at a concentration of 250 ng/mL in a total volume of 25 L in PBS, 0.5% BSA and 0.05% Tween in a 384 w microtiterplate (Maxisorb (with Streptavidin, Nunc). After 1.5 hrs. incubation at 25 C. unbound antibody was removed by washing 6 times with 90 L PBS (dispense and aspiration). The antigen-antibody complex was detected by an anti-rabbit antibody conjugated to POD (ECL anti-rabbit IgG-POD, Cat. No. NA9340V). 20-30 min after adding 35 L POD-substrate 3,3,5,5-tetramethyl benzidine (TMB, Piercenet, Cat. No. 34021) the optical density was determined at 370 nm. The EC.sub.50 value was calculated with a four parameter logistic model using GraphPad Prism 6.0 software.

Example 9

PCR Amplification of V-Domains for SLIC Cloning

[0367] Total RNA was prepared from B-cell lysates (resuspended in RLT buffer (Qiagen, Cat. No. 79216) using the NucleoSpin 8/96 RNA kit (Macherey&Nagel; Cat. No. 740709.4, 740698) according to manufacturer's protocol. RNA was eluted with 60 l RNase free water. 6 l of RNA was used to generate cDNA by reverse transcriptase reaction using the Superscript III First-Strand Synthesis SuperMix (Invitrogen, Cat. No. 18080-400) and an oligo dT-primer according to the manufacturer's instructions. All steps were performed on a Hamilton ML Star System. 4 l of cDNA were used to amplify the immunoglobulin heavy and light chain variable regions (VH and VL) with the AccuPrime SuperMix (Invitrogen, Cat. No. 12344-040) in a final volume of 50 l using the primers rbHC. up and rbHC. do for the heavy chain and rbLC. up and rbLC. do for the light chain:

TABLE-US-00026 rbHC.up (SEQIDNO:30) AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC rbHC.do (SEQIDNO:31) CCATTGGTGAGGGTGCCCGAG rbLC.up (SEQIDNO:32) AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC rbLC.do (SEQIDNO:33) CAGAGTRCTGCTGAGGTTGTAGGTAC

[0368] All forward primers were specific for the signal peptide (of respectively VH and VL) whereas the reverse primers were specific for the constant regions (of respectively CH1 and CL). The PCR conditions for the RbVH+RbVL were as follows: hot start at 94 C. for 5 min.; 35 cycles: 20 sec. at 94 C.; 20 sec. at 70 C.; 45 sec. at 68 C.; final extension at 68 C. for 7 min.

[0369] 8 l of the 50 l PCR solution were loaded on a 48 E-Gel 2% (Invitrogen, Cat. No. G8008-02). Positive PCR reactions were purified using the NucleoSpin Extract II kit (Macherey&Nagel; Cat. No. 740609250) according to manufacturer's protocol and eluted in 50 l elution buffer. All purification steps were performed on a Hamilton ML Starlet System. 5 l of purified VH and VL PCR solutions were used for DNA-sequencing.

Example 10

[0370] NGS VH-PCR from PBMCs and Antigen-Enriched B-Cells

[0371] 4.210E6 PBMCs and 1.210E6 antigen-enriched B-cells were resuspended in 300 RLT Buffer (Qiagen; Cat. No. 79216). Total RNA was prepared from B-cell lysates using RNeasy Mini or Micro kit (Qiagen; Cat. No. 74134) according to manufacturer's protocol. RNA was eluted in 50 l and 30 l RNase free water, respectively, for PBMCs and antigen-enriched B-cells. 6 l of RNA was used to generate cDNA by reverse transcriptase reaction using the Superscript III First-Strand Synthesis SuperMix (Invitrogen, Cat. No. 18080-400) and an oligo dT-primer according to the manufacturer's instructions.

[0372] 50-80 ng of cDNA were used to amplify the immunoglobulin heavy chain variable regions (VH) with the AccuPrime SuperMix (Invitrogen, Cat. No. 12344-040) in a final volume of 50 l using the primers rbHCfinal_FS.up and rbHC_shortCH1_fs2.do:

TABLE-US-00027 rbHCfinal_FS.up (SEQIDNO:34) ATGGAGACTGGGCTGCGCTGGCTTC rbHC_shortCH1_fs2.do (SEQIDNO:35) GGGAAGACTGATGGAGC

[0373] The forward primer is specific for the signal peptide VH whereas the reverse primers specific for the constant regions is. The PCR conditions were as follows: Hot start at 94 C. for 3 min.; 22 cycles: 20 sec. at 94 C.; 20 sec. at 68 C.; 40 sec. at 68 C.; final extension at 68 C. for 5 min. Totally 6 PCR reactions were performed on each cell pool sample.

[0374] 8 l of one PCR reaction were loaded on a 12 E-Gel 2% (Invitrogen, Cat. No. G521802). All PCR reactions respectively for the 2 B-cell libraries (PBMC-library; antigen-enriched B-cell-library) were purified with one column using the NucleoSpin Extract II kit (Macherey&Nagel; Cat. No. 740609) according to manufacturer's protocol and eluted in 50 l elution buffer. 5 l of cleaned VH PCR solutions were used for DNA-MiSeq sequencing.

Example 11

Template Preparation for NGS Sequencing

[0375] Paired-Ends Run 2300 Base: Minimal DNA amount for Samples: 100 ng, good 500 ng

[0376] The NGS sequencing was run on MiSeq from Illumina. After purification on AMPure XP beads PCR templates were assessed on a DNA1000 Agilent BioAnalyzer Chip. The library preparation was performed using the TruSeq Nano DNA Sample Preparation Kit according to manufacturer's protocol.

[0377] The final libraries were quantified using qPCR technology. qPCR reactions were prepared according to the KAPA SYBR FAST qPCR protocol and run using the Roche Light Cycler 480. The samples were pooled and contrasted with PhiX.

[0378] In more detail, the libraries were analyzed by a paired-end Illumina MiSeq sequencing run with Illumina sequencing primers.

[0379] All reagents were thawed at RT just before starting experiment. The reagent cartridge was thawed in a water bath. The cartridge was inverted several times to ensure mixing of reagents and all air bubbles were removed by hitting the cartridge on the bench. 1 mL of 0.2 M NaOH was prepared by adding 200 l 1 M NaOH to 800 L laboratory-graded water. The prepared solution was vortexed, spun down and stored on ice. Flow cell was brought to RT, and carefully washed with laboratory-graded water, dried using kimtech precision wipes and inserted into the sequencer following the instructions. 5 l of 4 nM DNA library pool was mixed with freshly diluted 0.2 M NaOH, vortexed briefly and spun down on a table top centrifuge. The solution was incubated for 5 min. at Room temperature and 990 L pre-chilled HT1 was added and mixed by briefly vortexing. The resulting 20 M denatured library in 1 mM NaOH was stored on Ice until further use. To obtain 600 l of a 12 M library, 360 L of the 20 M denatured library was diluted with 240 l pre-chilled HT1, inverted several times to mix and then pulse centrifuged. The resulting 12 M library was stored on ice until further use. To prepare 4 nM PhiX library, 2 L of the 10 nM PhiX library control was added to 3 l of 10 mM Tris-HCl, pH 8.5 with 0.1% Tween 20. The dilution was briefly vortexed and pulse centrifuged. To denature the PhiX Control 5 l freshly diluted 0.2 M NaOH was added to the 5 L of the prepared 4 nM PhiX library, vortexed briefly and spun down on a table top centrifuge. The solution was incubated for 5 min. at Room temperature and 990 L pre-chilled HT1 was added and mixed by briefly vortexing. To obtain a 12.5 M PhiX library, 375 L of the 20 M denatured PhiX solution was mixed with 225 L Pre-chilled HT1, briefly vortexed and pulse centrifuged. 520 L 12 M Sample library and 80 L 12.5 M PhiX were combined to create a library with 15% PhiX control spike-in. The combined sample library and PhiX control were stored on ice until loaded onto the MiSeq reagent cartridge.

Example 12

Bioinformatics Analysis of NGS Sequences for Identification of Clonally Related VH Variants

[0380] Data from Illumina MiSeq consist of two paired and usually overlapping reads per sequence. All data have been analyzed using the following workflow: [0381] Assembly of paired reads by FLASH (any other software tool should work as well) [0382] FLASH available from http://ccb.jhu.edu/software/FLASH/ [0383] Using Flash v1.2.10 with DEFAULT PARAMETERS (no outies, min overlap 10 bp, max overlap 65 bp) [0384] Result: Overlapped sequences without Illumina adaptors [0385] Extraction of antibody variable domains: [0386] Translating DNA to all 6 ORFs [0387] For each ORF: [0388] Searching peptide sequence for FW1 by comparing to a consensus FW1 sequence and counting the difference. Continuing if that value is above a defined threshold. [0389] Alike searching for FW2, trying to connect to FW1 (area in between is CDR1) [0390] Alike searching for FW3, trying to connect to FW2 (area in between is CDR2) [0391] Alike searching for FW4, trying to connect to FW3 (area in between is CDR3) [0392] Usually, in just 1 of the 6 ORFs a variable domain with the above described procedure can be identified. If multiples are found, a score that described the distance to the consensus is calculated and best ORF is selected. [0393] For variable domains found, several values are. Most importantly, the closest germlines were detected by simply aligning the variable domain sequence to the available germline repertoire provided by IMGT and report the best hit. By this it also reports per sequence the V/D/J Germlines that are most likely to be the origin of those sequences. [0394] Result: Table with one row per sequence containing all information about the contained variable domain. [0395] Additional Analysis performed: Calculated # Mutations for each CDR/FR on DNA/PEP level compared to the reference sequences.

Example 13

[0396] Transient Transfection of NGS VH Variants with Parental VL

[0397] For recombinant expression of NGS variants, PCR-products coding for parental VL of B-cell clones were cloned as cDNA into expression vectors by the overhang cloning method (Haun, R. S., et al., BioTechniques 13 (1992) 515-518; Li, M. Z., et al., Nature Methods 4 (2007) 251-256) in an expression cassette containing the rabbit constant region to accept the VL region. The expression vectors contained an expression cassette consisting of a 5 CMV promoter including intron A, and a 3 BGH poly adenylation sequence. In addition to the expression cassette, the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E. coli. Furthermore, the expression vector contained the rabbit kappa LC constant region to accept the VL regions.

[0398] Linearized expression plasmids coding for the kappa constant region and VL inserts were amplified by PCR using overlapping primers. Purified PCR products were incubated with T4 DNA-polymerase which generated single-strand overhangs. The reaction was stopped by dCTP addition. In the next step, plasmid and insert were combined and incubated with recA which induced site specific recombination. The recombined plasmids were transformed into E. coli. The next day the grown colonies were picked and tested for correct recombined plasmid by plasmid preparation, restriction analysis and DNA-sequencing.

[0399] Selected NGS VH variants were synthesized (Gene Art, Regensburg, Germany) and cloned as cDNA into expression vectors. The expression vectors contained an expression cassette consisting of a 5 CMV promoter including intron A, and a 3 BGH poly adenylation sequence. In addition to the expression cassette, the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E. coli. Furthermore, the expression vector contained the rabbit IgG constant region designed to accept the VH regions.

[0400] For antibody expression, 500 ng of the isolated HC and LC plasmids were transiently co-transfected into 2 ml (96-well plate) of FreeStyle HEK293-F cells (Invitrogen, Cat. No. R790-07) by using 239-Free Transfection Reagent (Novagen) following procedure suggested by Reagent supplier. After 1-week cultivation the HEK supernatants were harvested, filtered (1.2 m Supor-PALL) and purified with MabSelectSuRe (50 l, GE Healthcare). Columns were equilibrated with 1PBS. Samples were eluted with 2.5 mM HCl, pH 2.6 and neutralized with 10PBS.

Example 14

Staining Procedure for Antigen (CDCP1) Specific Sort

[0401] The cells from the macrophage depletion step were used to perform the antigen specific sort. In a first round the cells were incubated with the biotinylated CDCP1 antigen at a concentration of 5 g/ml on ice. After two washing steps the biotinylated and cell-bound CDCP1 was detected with an A647-streptavidin conjugate (Invitrogen). In parallel, the anti-IgG FITC (AbD Serotec, Dsseldorf, Germany) and the anti-IgM PE (BD Pharmingen) antibodies were added. The stained cells were washed two times.

[0402] Finally, the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses. The cell gate used for the single cell sorting was: rbIgM/rbIgG /CDCP1.

Example 15

Screening Hu CDCP1 Binders

[0403] Nunc Maxisorb streptavidin coated plates (MicroCoat, Cat. No. #11974998001) were coated with 25 l/well biotinylated human CDCP1-AviHis fusion protein at a concentration of 200 ng/ml and incubated at 4 C. over night. After washing (290 l/well with PBST-buffer (Phosphate Buffered Saline Tween-20)) 25 l anti-CDCP1 antibody samples were added and incubated for one hour at RT. After washing (390 l/well with PBST-buffer) 25 l/well goat-anti-human IgG-HRP conjugate (Millipore, Cat. No. AP502P) was added in 1:1,000 dilution and incubated at RT for one hour on a shaker. After washing (490 l/well with PBST-buffer) 25 l/well TMB substrate (Roche Diagnostics GmbH, Cat. No. 11835033001) was added and incubated until OD reached 1.5-2.5. The reaction was stopped by the addition of 25 l/well 1 N HCl. Measurement took place at 370/492 nm.

Example 16

[0404] NGS VH-PCR from PBMCs and Antigen-Enriched (Panning Sample) B-Cells

[0405] 4*10E6 PBMCs and 352 antigen-enriched B-cells were resuspended in 350 l RLT Buffer (Qiagen, Cat. No. 79216). Total RNA was prepared from B-cells lysate using RNeasy Mini or Micro kit (Qiagen, Cat. No. 74134) according to manufacturer's protocol. RNA was eluted in 50 l and 30 l RNase free water, respectively, for PBMCs and antigen-enriched B-cells. 6 l of RNA was used to generate cDNA by reverse transcriptase reaction using the Superscript III First-Strand Synthesis SuperMix (Invitrogen, Cat. No. 18080-400) and an oligo dT-primer according to the manufacturer's instructions.

[0406] 50-80 ng of cDNA were used to amplify the immunoglobulin heavy chain variable regions (VH) with the AccuPrime SuperMix (Invitrogen, Cat. No. 12344-040) in a final volume of 50 l using the primers rbHCfinal_FS.up and rbHC_shortCH1_fs2.do:

TABLE-US-00028 rbHCfinal_FS.up (SEQIDNO:34) ATGGAGACTGGGCTGCGCTGGCTTC rbHC_shortCH1_fs2.do (SEQIDNO:35) GGGAAGACTGATGGAGC

[0407] The forward primer is specific for the signal peptide VH whereas the reverse primers specific for the constant regions is. The PCR conditions were as follows: hot start at 94 C. for 3 min; 20 and 29 cycles (respectively for PBMC and antigen-enriched samples) of 20 sec. at 94 C.; 20 sec. at 68 C.; 40 sec. at 68 C.; final extension at 68 C. for 5 min. Totally 6-8 PCR reactions were performed each cell pool sample.

TABLE-US-00029 CDCP1: 3 wt-rabbits (5571, 5565, 5566) Sample ID Description Animal PCRs Cycles G1 (5571) PBMC 5571 6 50 l PCR a 20 (Lympholite) 1 l cDNA G2 (5565) PBMC 5565 6 50 l PCR a 20 (Lympholite) 1 l cDNA G3 (5566) PBMC 5566 6 50 l PCR a 20 (Lympholite) 1 l cDNA G4 (5565) M dep. + 5565 4 50 l PCR a 29 AG Sort 4 l cDNA G5 (5566) M dep. + 5566 4 50 l PCR a 29 AG Sort 4 l cDNA G6 (5571) M dep. + 5571 4 50 l PCR a 29 AG Sort 4 l cDNA

TABLE-US-00030 Sequencing results: number of rabbit VH sequences and clusters H3 total non- bad VH H3 Clusters name sequences pairable sequence OK Clusters n > 3 G1 1,021,345 149,110 44,692 827,543 28,487 8,506 G2 1,040,062 146,080 47,609 846,373 21,842 4,544 G3 1,406,058 245,445 75,106 1,085,507 30,947 6,876 G4 1,019,453 147,601 41,484 830,368 4,774 1,349 G5 1,166,355 171,698 60,861 933,796 4,847 1,055 G6 1,166,807 173,888 55,718 937,201 3,753 806

Example 17

[0408] HEK Transient Transfection of NGS VH Variants with Parental VL

[0409] For recombinant expression of NGS variants, PCR-products coding for parental VL of B-cell clones were cloned as cDNA into expression vectors by the overhang cloning method (Haun, R. S., et al., BioTechniques 13 (1992) 515-518; Li, M. Z., et al., Nature Methods 4 (2007) 251-256). The expression vectors contained an expression cassette consisting of a 5 CMV promoter including intron A, and a 3 BGH poly adenylation sequence. In addition to the expression cassette, the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E. coli. Furthermore, the expression vector contained the rabbit kappa LC constant region to accept the VL regions.

[0410] Linearized expression plasmids coding for the kappa constant region and VL inserts were amplified by PCR using overlapping primers. Purified PCR products were incubated with T4 DNA-polymerase which generated single-strand overhangs. The reaction was stopped by dCTP addition. In the next step, plasmid and insert were combined and incubated with recA which induced site specific recombination. The recombined plasmids were transformed into E. coli. The next day the grown colonies were picked and tested for correct recombined plasmid by plasmid preparation, restriction analysis and DNA-sequencing.

[0411] Selected NGS VH variants were synthesized (Gene Art, Regensburg, Germany) and cloned as cDNA into expression vectors. The expression vectors contained an expression cassette consisting of a 5 CMV promoter including intron A, and a 3 BGH poly adenylation sequence. In addition to the expression cassette, the plasmids contained a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E. coli. Furthermore, the expression vector contained the rabbit IgG constant region designed to accept the VH regions.

[0412] For antibody expression, 500 ng of the isolated HC and LC plasmids were transiently co-transfected into 2 ml (96-well plate) of HEK293-F cells (Invitrogen, Cat. No. R790-07) by using 239-Free Transfection Reagent (Novagen) following procedure suggested by Reagent supplier. After 1-week cultivation the HEK supernatants were harvested, filtered (1.2 m Supor-PALL) and purified with MabSelectSuRe (50 l, GE Healthcare). Columns were equilibrated with 1PBS. Samples were eluted with 2.5 mM HCl, pH 2.6 and neutralized with 10PBS.

Example 18

Human CDCP1 Binding ELISA

[0413] Nunc Maxisorb streptavidin coated plates (MicroCoat, Cat. No. #11974998001) were coated with 25 l/well biotinylated human CDCP1-AviHis fusion protein at a concentration of 200 ng/ml and incubated at 4 C. over night. After washing (290 l/well with PBST-buffer) 25 l anti-CDCP1 antibody samples were added in a 1:2 dilution series starting at 5 g/ml. Plates were incubated one hour at RT. After washing (390 l/well with PBST-buffer) 25 l/well of a mix of goat-anti-human IgG-HRP conjugate (Jackson, Cat. No. 109-036-098) and donkey-anti-rabbit IgG (GE Healthcare, Cat. No. NA9340V, Lot #389592,) was added in 1:9,000 dilution and incubated at RT for one hour on a shaker. After washing (490 l/well with PBST-buffer) 25 l/well TMB substrate (Roche, Cat. No. 11835033001) was added and incubated until OD reached 1.5-2.5. The reaction was stopped by addition of 25 l/well 1 N HCl. Measurement took place at 370/492 nm.