ENGINEERING B LYMPHOCYTES BY UTILIZING ENDOGENOUS ACTIVATION-INDUCED CYTIDINE DEAMINASE

Abstract

The present invention provides a method for engineering B lymphocytes by utilizing activation-induced cytidine deaminase of the B lymphocyte. Thereby, use of engineered nucleases, such as Cas nuclease, can be avoided. Engineered B cells are useful to produce customized antibodies and for B cell therapy. Accordingly, the present invention also provides engineered B cells and customized antibodies produced by engineered B cells.

Claims

1. A method for editing the genome of an isolated B lymphocyte comprising the following steps: (i) activating endogenous activation-induced cytidine deaminase of the B lymphocyte; and (ii) introducing a DNA molecule comprising a nucleotide sequence encoding a (poly)peptide of interest into the B lymphocyte.

2. The method according to claim 1, wherein the method does not involve an exogenous nuclease and/or an engineered nuclease, such as a CRISPR nuclease, a zinc finger nuclease, a transcription activator-like nuclease or a meganuclease.

3. The method according to claim 1 or 2, wherein the DNA molecule is a linear or linearized DNA molecule.

4. The method according to any one of claims 1-3, wherein the DNA molecule is a single strand DNA molecule (ssDNA) or a double strand DNA molecule (dsDNA).

5. The method according to claim 4, wherein the DNA molecule is dsDNA molecule.

6. The method according to claim 5, wherein the DNA molecule has blunt ends or overhangs.

7. The method according to any one of claims 1-6, wherein the nucleotide sequence of the DNA molecule encoding the (poly)peptide of interest is codon-optimized.

8. The method according to any one of claims 1-7, wherein the DNA molecule comprises an intronic sequence upstream and/or downstream of the nucleotide sequence encoding the (poly)peptide of interest.

9. The method according to claim 8, wherein the intronic sequence comprises a splice recognition site.

10. The method according to claim 8 or 9, wherein the intronic sequence contains Ig-locus intronic sequences.

11. The method according to claim 10, wherein the intronic sequence comprises an intronic sequence of a J-segment downstream intron and/or an intronic sequence of a CH-upstream intron.

12. The method according to any one of claims 1-11, wherein the DNA molecule comprises a splicing enhancer.

13. The method according to any one of claims 1-12, wherein the genome of the B lymphocyte is edited to express a modified immunoglobulin chain comprising in N- to C-terminal direction: a variable domain, the (poly)peptide of interest and a constant domain.

14. The method according to any one of claims 1-13, wherein the genome of the B lymphocyte is edited to express a modified immunoglobulin chain, wherein an endogenous variable domain is replaced by the (poly)peptide of interest.

15. The method according to any one of claims 1-14, wherein the DNA molecule comprises a nucleotide sequence encoding a cleavage site upstream and/or downstream of the nucleotide sequence encoding the (poly)peptide of interest.

16. The method according to claim 15, wherein the cleavage site is a T2A cleavage site.

17. The method according to any one of claims 1-16, wherein the (poly)peptide of interest comprises or consists of a pathogen binding domain, a V.sub.L domain, or a V.sub.H-V.sub.L domain.

18. The method according to any one of claims 1-17, wherein the (poly)peptide of interest comprises or consists of CD4, dipeptidyl peptidase 4, CD9, or angiotensin-converting enzyme 2 or a fragment or sequence variant thereof.

19. The method according to any one of claims 1-18, wherein the isolated B lymphocyte is a primary B lymphocyte.

20. The method according to any one of claims 1-19, wherein the method comprises obtaining an engineered B lymphocyte, wherein the genome of the B lymphocyte comprises the nucleotide sequence encoding the (poly)peptide of interest.

21. The method according to any one of claims 1-20, wherein the method further comprises a step (iii) of confirming integration of the nucleotide sequence encoding the (poly)peptide of interest into the genome of the B lymphocyte.

22. The method according to any one of claims 1-21, wherein the isolated B lymphocyte is cultured in RPMI or IMDM with 10% MS, 1% NEAA, 1% sodium pyruvate, 1% beta-mercaptoethanol, 1% Glutamax, 1% penicillin/streptomycin, 1% kanamycin, and 1% transferrin.

23. The method according to any one of claims 1-22, wherein the isolated B lymphocyte is cultured at 1×10.sup.5 to 1×10.sup.6 cells/ml, preferably at 2×10.sup.5 cells/ml.

24. The method according to any one of claims 1-23, wherein the B lymphocyte is cultured in a culture medium comprising an activator of activation-induced cytidine deaminase.

25. The method according to claim 24, wherein the activator of activation-induced cytidine deaminase is selected from the group consisting of: a cytokine, an anti-B cell receptor antibody or fragments thereof, a TLR agonist, a CpG-B agonist, an imidazoquinoline compound or a combination of any of said activators.

26. The method according to claim 25, wherein the cytokine is selected from the group consisting of CD40L, IL4, IL2, IL21, BAFF, APRIL, CD30L, TGF-β1, 4-1BBL, IL6, IL7, IL10, IL13, c-Kit, FLT-3, IFNα or any combination thereof.

27. The method according to any one of claims 24-26, wherein the cytokine is administered at a concentrations of 0.01-20 ng/ml.

28. The method according to any one of claims 1-27, wherein the B lymphocyte is cultured in a medium comprising IL4 and/or CD40L.

29. The method according to claim 28, wherein activating of the activation-induced cytidine deaminase is performed by co-culture with a CD40L expressing cell line and addition of IL-4.

30. The method according to claim 29, wherein the concentration of IL-4 (in the final culture medium) is 0.005-0.03 ng/ml, preferably 0.01-0.025 ng/ml, more preferably 0.015-0.02 ng/ml and most preferably 0.16 ng/ml.

31. The method according to claim 29 or 30, wherein the CD40L expressing cell line is K562L.

32. The method according to any one of claims 1-31, wherein introducing the DNA molecule into the B lymphocyte is performed up to 10 days after activating the activation-induced cytidine deaminase, preferably up to 7 days after activating the activation-induced cytidine deaminase, more preferably up to 5 days after activating the activation-induced cytidine deaminase, even more preferably up to 2 days after activating the activation-induced cytidine deaminase and most preferably about 1 day after activating the activation-induced cytidine deaminase.

33. The method according to any one of claims 1-32, wherein the method does not comprise transducing the B lymphocyte with a retrovirus.

34. The method according to any one of claims 1-33, wherein the DNA molecule is introduced by nucleofection.

35. The method according to any one of claims 1-34, wherein the B lymphocyte is reactivated after introducing the DNA molecule into the B lymphocyte.

36. The method according to any one of claims 1-35, wherein B cell stimulating agents, for example as defined in claims 25-31, are applied to the B lymphocyte after introducing the DNA molecule into the B lymphocyte.

37. The method according to any one of claims 1-36, wherein the B lymphocyte is treated with a DNA inhibitor capable of blocking alternative-end joining before introducing the DNA molecule into the B lymphocyte.

38. The method according to any one of claims 1-37, wherein the DNA molecule comprising a nucleotide sequence encoding the (poly)peptide of interest is incubated with a Ku protein, such as Ku70/Ku80, before introducing the DNA molecule into the B lymphocyte.

39. The method according to any one of claims 1-38, wherein the DNA molecule comprises a nuclear localization signal, such as SV40 nuclear localization signal.

40. The method according to any one of claims 1-39, wherein the DNA molecule does not comprise a nucleotide sequence encoding GFP or RFP.

41. The method according to any one of claims 1-40, wherein the DNA molecule comprises a promoter.

42. The method according to any one of claims 1-41, wherein the DNA molecule comprises a transcription unit.

43. The method according to any one of claims 1-42, wherein about 24 hours after activation of the activation-induced cytidine deaminase of the B lymphocyte, the B lymphocyte is treated with a nuclease inhibitor, such as Mirin.

44. The method according to any one of claims 1-43, wherein the B lymphocyte is a human B lymphocyte.

45. An engineered B lymphocyte obtainable by the method according to any one of claims 1-44.

46. An engineered B lymphocyte comprising an edited immunoglobulin gene locus comprising a heterologous insert comprising a nucleotide sequence encoding a (poly)peptide of interest inserted in its switch region.

47. The B lymphocytes according to claim 45 or 46, wherein the B lymphocyte is human.

48. The B lymphocyte according to any one of claims 45-47, wherein the switch region of an immunoglobulin gene locus of the B lymphocyte comprises a cleavage site, in particular a T2A cleavage site.

49. The B lymphocyte according to any one of claims 45-48, wherein the switch region of an immunoglobulin gene locus of the B lymphocyte comprises a nucleotide sequence encoding a pathogen binding domain, a V.sub.H domain, or a V.sub.L domain.

50. The B lymphocyte according to any one of claims 45-49, wherein the switch region of an immunoglobulin gene locus of the B lymphocyte comprises a nucleotide sequence encoding CD4, dipeptidyl peptidase 4, CD9, or angiotensin-converting enzyme 2 or a fragment or sequence variant thereof.

51. The B lymphocyte according to any one of claims 45-50, wherein the B lymphocyte does not express a fluorescent reporter protein.

52. The B lymphocyte according to any one of claims 45-51 for use in medicine.

53. The B lymphocyte for use according to claim 52, wherein the B lymphocyte is engineered according to any one of claims 1-44.

54. The B lymphocyte for use according to claim 52 or 53, wherein the engineered B lymphocyte is administered to a patient.

55. The B lymphocyte for use according to claim 54, wherein the patient receiving the engineered B lymphocyte is the same patient from whom the B lymphocyte was isolated prior to engineering.

56. Method for B cell therapy comprising the following steps: (a) isolating a B lymphocyte from a patient; (b) engineering the B lymphocyte according to any one of claims 1-44; and (c) administering the engineered B lymphocyte to the patient.

57. A cell line of B lymphocytes according to any one of claims 45-51.

58. A method for generating an antibody or a fragment thereof comprising a (heterologous) (poly)peptide of interest, the method comprising the following steps: (1) providing an engineered B lymphocyte or a B cell line according to any one of claims 45-51 and 57, wherein the B lymphocyte comprises an edited immunoglobulin gene locus comprising a heterologous insert comprising a nucleotide sequence encoding the (poly)peptide of interest inserted in its switch region; (2) culturing the engineered B lymphocyte or the B cell line; and (3) isolating the antibody or the fragment thereof comprising the (heterologous) (poly)peptide of interest from the B cell culture.

59. The method according to claim 58, wherein the engineered B lymphocyte is obtained by a method according to any one of claims 1-44.

60. The method according to claim 58 or 59 further comprising characterization of the antibody or antibody fragment, wherein characterization comprises Performing functional assays to determine the function of the antibody or antibody fragment; Performing binding assays to determine the binding specificity of the antibody or antibody fragment and/or the binding partner/epitope recognized by the antibody or antibody fragment; and/or Performing neutralization assays to determine the ability of the antibody or antibody fragment to neutralize a toxin or a pathogen.

61. Antibody obtainable by the method according to any one of claims 58-60.

62. A composition comprising the B lymphocyte according to any one of claims 45-51 or the antibody according to claim 61.

63. The composition according to claim 62 further comprising a pharmaceutically acceptable carrier.

64. The composition according to claim 62 or 63 for use in medicine.

65. A method for immunotherapy comprising administration of the antibody according to claim 61, the engineered B cell according to any one of claims 45-51 or the composition according to claim 62 or 63 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0357] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0358] FIG. 1 shows a schematic overview of AID mediated B cell engineering of the antibody switch region on chromosome 14 by integration of an extra exon element ((poly)peptide of interest) generating antibodies comprising a desired specificity ((poly)peptide of interest).

[0359] FIG. 2 shows schematic examples of engineered genes encoding antibody chains obtainable from B cells engineered according to the present invention and schematic drawings of the respective antibodies. (A) Examples including additional inserts in the antibody's elbow region (between the variable and constant regions). (B) Examples including a T2A protease cleavage site, e.g. to replace the original variable region of the antibody. V, D, J—original V, D, genes. Constant—original constant domain(s). T2A—introduced T2A cleavage site. V.sub.H—introduced heavy chain variable region. V.sub.L—introduced light chain variable region. Receptor domain—introduced receptor domain.

[0360] FIG. 3 provides a schematic overview over Examples (exp) for detecting genomic LAIR1 insertions (Example 1; exp 1), LAIR1-Ab expressing primary B cell production and selection by sorting (Example 2; exp 2) or by high throughput screening (Example 3; exp 3). The number of days indicates how many days after stimulation nucleofection was performed, the remarks in the column “nucleofection” indicate features of the nucleic acid used for nucleofection and the remarks in the column “screening” indicate what type of screening was performed.

[0361] FIG. 4 shows for Example 1 (A) the design of switch region PCR and (B) the results of detection of codon optimized LAIR1 (including partial integrations) in long switch-μ-region PCR amplicons by MinION sequencing technology after nucleofection of double stranded (dsDNA) LAIR1 substrates.

[0362] FIG. 5 shows for Example 2 (A) LAIR1 and IgM surface co-staining of a B cell line generated according to the present invention, which expresses LAIR1-containing antibodies, selected post nucleofection by FACS sorting in comparison to negative (MME17) and positive (MMJS) control B cell lines. (B) Bead pull down and FACS analysis of artificial LAIR1-containing antibodies secreted by B cell lines nucleofected with a LAIR1 wildtype and a LAIR1 CH1/J6 intron optimized substrate.

[0363] FIG. 6 shows for Example 2 (A) LAIR1 and IgM specific western blots of culture supernatants. (B) PCR amplification using switch-μ-forward and LAIR1-reverse primers of genomic DNA isolated from engineered B cell lines expressing recombinant LAIR1-containing antibodies. (C) PCR product sequence alignment of switch-region and 5′ LAIR1-insert covering region highlighting switch region in gray, LAIR1 intron light gray with splice acceptor site in bold and LAIR1 exon in black.

[0364] FIG. 7 shows for Example 3 (A) frequency of engineered B cell lines expressing recombinant LAIR1-containing antibodies detected by high throughput screening of 60 000 and 35 000 cells, respectively. Cells were nucleofected with either LAIR1 wildtype substrate or a CH1/J6 intron optimized version. Screening conditions in II) were optimized by decreasing cell seeding numbers while increasing cultivation time to achieve higher antibody concentrations in culture supernatants. (B) Example of bead screenings of two 384 well culture plates measuring MFI ration of IgM captured by anti-LAIR1 versus control beads. Open circles show positive controls and the rectangle a culture secreting artificial LAIR1-containing antibodies.

[0365] FIG. 8 shows for Example 4 (A) H2AX staining indicating DNA double strand breaks after irradiation of PBMCs (FACS plots) and (B) primary B cells post CD40L/IL4 stimulation and AID induction. MFI=mean fluorescence intensity.

[0366] FIG. 9 shows for Example 4 (A) % of surviving and GFP expressing primary B cells two days after NEON nucleofection of a pMAX-GFP control plasmid. (B) FACS plots show gating strategies of mock nucleofectants and condition d) (2150V, 10 ms, 2 pules) which was used for further screening experiments.

[0367] FIG. 10 shows the principle and results for Example 5. (A) In vitro switch insertions are dependent on c-NHEJ. (B) naïve sorted B cells were stimulated with CD40L and IL4 and cultivated for 9 days in the presence of inhibitors for c-NHEJ (SCR7), a-NHEJ (Olaparib) or reverse transcriptases (ddl/AZT). Natural switch inserts were detected in 50,000 in vitro IgG+ switched B cells by MinION sequencing technology.

[0368] FIG. 11 shows a schematic representation of intron optimization for splice site recognition.

EXAMPLES

[0369] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Generation of B Cells Engineered According to the Present Invention and Expressing Recombinant Antibodies

[0370] The underlying rationale of Examples 1-3 was to demonstrate that the immunoglobulin switch region of isolated human B cells can be targeted for genetic modification and subsequently results in production of recombinant antibodies. As an example, several experiments were successfully conducted to generate engineered human primary B cells producing an antibody with an inserted LAIR1 domain (Examples 1-3).

[0371] Methods:

[0372] B cell isolation, simulation and nucleofection. Primary human B cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic cell sorting with anti-CD19 microbeads from Miltenyi Biotec. The 100 000/ml B cells were plated in 12 well. CD40L expressing, irradiated K562L cells were added to the B cells in a 1:2 ratio. Human recombinant IL4 was added at 16 ng/ml. The following day cells were re-stimulated with 8 ng/ml IL4. The nucleofection was either performed at day 1 after cell seeding, 4 h after IL4 re-stimulation or they were further cultivated, re-stimulated every 3 days with 8 ng/ml IL4 and nucleofected at indicated time points. For nucleofection the B cells were harvested and 2×10.sup.6 B cells were nucleofected with 1 μg DNA using a NEON® device according to manufacturers' instructions and 2150V, 10 ms and 2 pulses.

[0373] DNA nucleofection products. Codon optimized LAIR1 was ordered by gene synthesis from GenScript® using the company own codon optimization tool. For ssDNA generation, the “Long single strand DNA (LsODN) Preparation Kit” (funakoshi) was used. Codon optimized LAIR1 was cloned into pLSODN-1 vector. Restriction enzyme digestion of the vector was performed to generate either ssDNA or blunt/sticky-end dsDNA of codon optimized LAIR1.

[0374] Sequence analysis. gDNA was isolated from nucleofected B cells 7 days after nucleofection using a commercial kit (QIAGEN). Switch region PCRs on gDNA were performed using LongAmp Taq Polymerase (New England Biolabs) in 50 μl reaction volumes with incubation for 3 min at 95° C., followed by 30 cycles of 95° C. for 40 s, 60° C. for 30 s, 65° C. for 3 min and a final extension for 10 min at 65° C. The upstream switch-μ forward primer S-μ-FW (cacccttgaaagtagcccatgccttcc; SEQ ID NO: 96) was combined with S-γ-REV (cctgcctcccagtgtcctgcattacttctg; SEQ ID NO: 97). Instead, the switch-μ region of nucleofected B cell gDNA was amplified combining the S-μ-FW primer with S-μ-REV (ggaacgcagtgtagactcagctgagg; SEQ ID NO: 98). The PCR reaction was performed using Herculase II Fusion DNA Polymerases (Agilent) with 1 M betaine and 3% DMSO in a 50 μl volume at 98° C. for 4 min followed by 30 cycles of 98° C. for 40 s, 58° C. for 30 s and 72° C. for 4 min, with a final extension for 10 min at 72° C. An overview of the design of switch region PCR is provided in FIG. 4 A. Size-selected, purified switch amplicons from oligoclonal B cell cultures were sequenced by MinION/Oxford Nanopore Technology (ONT). Barcodes were introduced by the addition of recommended BC-sequences to S-μ and S-γ primers and PCR amplification. The sequencing library was prepared using the Nanopore 2D sequencing kit SQK-LSK207, followed by loading onto Nanopore flow cells FLO-MIN106 and sequencing with the MinION Mk1B sequencer for up to 20 h.

[0375] The DNA substrates used in the first experiment comprised a ssDNA and dsDNA version with codon optimized LAIR1 exon and wildtype flanking intronic sequences having the following nucleotide sequence:

TABLE-US-00009 [SEQ ID NO: 99] TTGTGAGCAAGTCTCAGGGTCCTCACTGTCAACTGGGAAAAAACTCTGC AGTGATGAGAATCACATGCACGTAGAAGGTGCAGGAGGCGTGGGAATGT TCTAAGGTTGGGCTGTGGTCATGGCTGCATAACTCTATAAAATTGCTAA AATCCCTGAATTGTGATGCTAAAATGACGTGTGTGGCATGGTGACTTCC TACAGTGGACGCTGAGATCCTGCTCTGCTTCCCTCCT AAGATCTGCC CAGACCCTCCATCTCGGCTGAGCCAGGCACCGTGATCCCCCTGGGGAGC CATGTGACTTTCGTGTGCCGGGGCCCGGTTGGGGTTCAAACATTCCGCC TGGAGAGGGACAGTAGATCCACATACAATGATACTGAAGATGTGTCTCA AGCTAGTCCATCTGAGTCAGAGGCCAGATTCCGCATTGACTCAGTAAGA GAAGGAAATGCCGGGCTTTATCGCTGCATCTATTATAAGCCCCCTAAAT GGTCTGAGCAGAGTGACTACCTGGAGCTGCTGGTGAAAG GAGGACGT CACCTGGGCCCTGCCCCAGTCTCAGCTCGACCCTCGAGCTTGTCCCCAG GT

[0376] (nucleotide sequence encoding a polypeptide of interest is shown underlined; 5′ and 3′ splice recognition sites are shown in bold and italics)

[0377] Results

[0378] In general, nucleofection with both, ssDNA and dsDNA substrates, resulted in successful integration of the nucleic acid substrate into the B cell genome. As an example, FIG. 4B shows results obtained with dsDNA.

Example 2: Further Investigation of B Cell Lines Engineered According to the Present Invention and Expressing Recombinant Antibodies

[0379] To provide proof for productive insertion and expression of LAIR1 containing antibodies, primary B cells were nucleofected with a dsDNA LAIR1 wildtype substrate and, were screened by cell sorting for LAIR1 and IgM co-staining. As the natural LAIR1 receptor is downregulated after Epstein-Barr virus (EBV) immortalization, in this experimental setting EBV lines were generated to distinguish the natural receptor from an engineered B cell receptor.

[0380] To prepare LAIR1 wildtype (wt) products for nucleofection human wildtype LAIR1 was PCR amplified from human genomic DNA (gDNA) using the following primers (LAIR1_IN_FW ccacctccaaacggcaggcatcc (SEQ ID NO: 100); LAIR1_INTR_REV ccaaaggccgcatgaccatcacgc (SEQ ID NO: 101)). Chimeric DNA products containing a LAIR1 exon and introns deriving from the human immunoglobulin locus were generated by first amplifying single products with primers IgM-CH1-IN-fw cctcagctgagtctacactgcgttcc (SEQ ID NO: 102), IgM-CH1-IN-rev ctgaggacccgcaggacaaaagagaaaggg (SEQ ID NO: 103), J6-lN-fw ggtcaccgtctcctcaggtaagaatggcc (SEQ ID NO: 104), J6_IN-REV gccttttcagtttcggtcagcctcgc (SEQ ID NO: 105) and then fusing them by PCR with overlapping primers to a LAIR1 wt amplicon with LAIR1-CH1-FW gcgggtcctcagaagatctgcccagaccc (SEQ ID NO: 106) and LAIR1-J6-REV ggccattcttacctttcaccagcagctccagg (SEQ ID NO: 107). Optimized versions were generated using primers LAIR1-CH1-opt-FW gcgggtcctcaggggaagatctgcccagaccc (SEQ ID NO: 108), and LAIR1-J6-REV ggccattcttacctgaggagacggctttcaccagcagctccagg (SEQ ID NO: 109). To minimize mutations introduced by the polymerase during amplification, the Q5® High-Fidelity DNA Polymerase (New England Biolabs) with high proofreading activity was used applying the standard PCR amplification program.

[0381] B cells were isolated, stimulated and nucleofected as described above. One day after nucleofection, the B cells were immortalized with Epstein-Barr virus (EBV) by 4 h virus incubuation rotating at 37° C. as previously described (Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo M R, Murphy B R, Rappuoli R, Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 2004 August; 10(8):871-5. Epub 2004 Jul. 11) B cells were washed and plated in bulk at 1×10.sup.6/ml into 24 well cultures in the presence of CpG-DNA (2.5 μg/ml). One week after immortalization and downregulation of the B cell own LAIR1 wildtype receptor, the B cells were selected for LAIR1 and IgM co-expression by first labeling them with monoclonal anti-LAIR1 PE-conjugated (clone DX26, BD Bioscience, 550811) and anti-IgM APC-conjugated antibodies (Jackson ImmunoResearch, 109-606-129) followed by FACS-sorting. The cells which co-expressed LAIR1 and IgM were plated in 96U wells, expanded for two weeks and then repeatedly selected by FACS-sorting. Genomic analysis of gDNA isolated from the cell line and switch-μ region PCR amplification was performed as described above, followed by Sanger sequencing.

[0382] To confirm secretion of LAIR1 containing antibodies by EBV immortalized B cells, the culture supernatants were analyzed by western blot analysis. Supernatants were diluted in water and incubated with 4× sample loading buffer (Life Technologies) and 10× reducing agent (Life Technologies) for 10 min at 70° C. The samples were loaded to a precast gel with a 4-12% acrylamide gradient (Invitrogen). Proteins were transferred to PVDF membranes by the iBlot2 apparatus (Life Technologies) followed by blocking for 1 h at room temperature with 3% BSA in TBS. The membrane was incubated with different combinations of primary and secondary antibodies diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to wash the membrane between incubations. IgM isotypes were stained with 10 μg/ml unlabelled goat anti-human IgM (Southern Biotech, 2020-01) and 8 ng/ml donkey anti-goat HRP (Jackson ImmunoResearch, 705-036-147). LAIR1-containing antibodies were detected with a polyclonal goat anti-human LAIR1antibody (R&D) at 2 μg/ml was combined with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a Las4000 imager (General Electric Company).

[0383] FIG. 5 shows the results of FACS analysis. FIG. 5A shows that the B cells were generated successfully expressing LAIR1-IgM on their surface. The integration of the LAIR1 domain into secreted antibodies was confirmed by a bead capture assay (FIG. 5B) and western blot analysis (FIG. 6A) as described above. Furthermore, successful integration was also achieved using a substrate where a LAIR1 wildtype exon was flanked by Immunoglobulin-locus intronic regions namely the J-segment downstream intron and the CH1-upstream intron (named LAIR1 CH1/J6) having the following sequence:

TABLE-US-00010 [SEO ID NO: 110] CCTCAGCTGAGTCTACACTGCGTTCCCCATCACACTCACCCTCCCTATA CTCACTCCCAGGCCTGGGTTGTCTGCCTGGGGAGACTTCAGGGTAGCTG GAGTGTGACTGAGCTGGGGGCAGCAGAAGCTGGGCTGGAGGGACTCTAT TGGCTGCCTGCGGGGTGTGTGGCTCCAGGCTTCACATTCAGGTATGCAA CCTGGGCCCTCCAGCTGCATGTGCTGGGAGCTGAGTGTGTGCAGCACCT ACGTGCTGATGCCTCGGGGGAAAGCAGGCCTGGTCCACCCAAACCTGAG CCCTCAGCCATTCTGAGCAGGGAGCCAGGGGCAGTCAGGCCTCAGAGTG CAGCAGGGCAGCCAGCTGAATGGTGGCAGGGATGGCTCAGCCTGCTCCA GGAGACCCCAGGTCTGTCCAGGTGTTCAGTGCTGGGCCCTGCAGCAGGA TGGGCTGAGGCCTGCAGCCCCAGCAGCCTTGGACAAAGACCTGAGGCCT CACCACGGCCCCGCCACCCCTGATAGCCATGACAGTCTGGGCTTTGGAG GCCTGCAGGTGGGCTCGGCCTTGGTGGGGCAGCCACAGCGGGACGCAAG TAGTGAGGGCACTCAGAACGCCACTCAGCCCCGACAGGCAGGGCACGAG GAGGCAGCTCCTCACCCTCCCTTTCTCTTTTGTCCTGCGGGTCCTC A AGATCTGCCCAGACCCTCCATCTCGGCTGAGCCAGGCACCGTGATCCCC CTGGGGAGCCATGTGACTTTCGTGTGCCGGGGCCCGGTTGGGGTTCAAA CATTCCGCCTGGAGAGGGACAGTAGATCCACATACAATGATACTGAAGA TGTGTCTCAAGCTAGTCCATCTGAGTCAGAGGCCAGATTCCGCATTGAC TCAGTAAGAGAAGGAAATGCCGGGCTTTATCGCTGCATCTATTATAAGC CCCCTAAATGGTCTGAGCAGAGTGACTACCTGGAGCTGCTGGTGAAAG AAGAATGGCCACTCTAGGGCCTTTGTTTTCTGCTACTGCCTGTGGGG TTTCCTGAGCATTGCAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCT GGGCGGACTGGCCAGGAGGGGATGGGCACTGGGGTGCCTTGAGGATCTG GGAGCCTCTGTGGATTTTCCGATGCCTTTGGAAAATGGGACTCAGGTTG GGTGCGTCTGATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCACATTT GGACGAGATGCCTGAACAAACCAGGGGTCTTAGTGATGGCTGAGGAATG TGTCTCAGGAGCGGTGTCTGTAGGACTGCAAGATCGCTGCACAGCAGCG AATCGTGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTGTCAACC TGCATCTTAAATTCTTTATTGGCTGGAAAGAGAACTGTCGGAGTGGGTG AATCCAGCCAGGAGGGACGCGTAGCCCCGGTCTTGATGAGAGCAGGGTT GGGGGCAGGGGTAGCCCAGAAACGGTGGCTGCCGTCCTGACAGGGGCTT AGGGAGGCTCCAGGACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAAAA GGATTGTTTATCTTAGGAGGCATGCTTACTGTTAAAAGACAGGATATGT TTGAAGTGGCTTCTGAGAAAAATGGTTAAGAAAATTATGACTTAAAAAT GTGAGAGATTTTCAAGTATATTAATTTTTTTAACTGTCCAAGTATTTGA AATTCTTATCATTTGATTAACACCCATGAGTGATATGTGTCTGGAATTG AGGCCAAAGCAAGCTCAGCTAAGAAATACTAGCACAGTGCTGTCGGCCC CGATGCGGGACTGCGTTTTGACCATCATAAATCAAGTTTATTTTTTTAA TTAATTGAGCGAAGCTGGAAGCAGATGATGAATTAGAGTCAAGATGGCT GCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGCAGGTCACCG CGAGAG

[0384] (nucleotide sequence encoding a polypeptide of interest is shown underlined; 5′ and 3′ splice recognition sites are shown in bold and italics)

[0385] The genomic insertion of the LAIR1 wildtype sequence into the switch region was confirmed by a specific PCR reaction and sequence analysis (FIG. 6 B, C).

Example 3: Further Investigation of B Cell Lines Engineered According to the Present Invention and Expressing Recombinant Antibodies

[0386] To evaluate the frequency of successfully nucleofected cells producing LAIR1-containing antibodies, 10-30 cells per well cultures in 384 well formats were screened by LAIR1-capture bead assay.

[0387] B cell isolation, stimulation, nucleofection and EBV immortalization was performed as described above. After virus incubation, the B cells were plated at 10 or 30 cells/well in presence of 25 000 irradiated, autologous PBMCs as feeder cells and CpG-DNA (2.5 μg/ml). After 2 weeks of cultivation the supernatants of the cells were analyzed for secretion of LAIR1-containing antibodies by a two-determinant bead-based immunoassay. Therefore, anti-goat IgG microbeads (Spherotech) were coated with either goat anti-human LAIR1 (R&D Systems, AF2664) or the control antibody goat anti-human EGF (R&D Systems, AF-259-NA) for 20 min at room temperature. SYBR Green I (ThermoFisher Scientific) was added at 40× to the LAIR1 antibody coating solution to distinguish LAIR1-coated from control beads. The beads were washed, mixed, and incubated with the supernatant of immortalized B cells for 30 min at room temperature. Bead captured, LAIR1 containing antibodies were detected using 2.5 μg/ml Alexa Fluor 647-conjugated donkey anti-human IgM (Jackson ImmunoResearch, 709-606-073).

[0388] Results are shown in FIG. 7. The results confirm a frequency of one productive insertion in about 12 000 primary B cells (FIG. 7 A, B).

Example 4: Optimization of Nucleofection Time Point and Condition

[0389] In this example the optimal time point and conditions of nucleofection was investigated.

[0390] B cell were isolated from PBMCs by Magnetic cell sorting with anti-CD19 beads and stimulated with CD40L expressing K562L cells and IL4 as described above. To assess the induction of DNA double strand breaks the cells were harvested at indicated time points, fixed with 3.7% formaldehyde, permabilized with 90% methanol and stored at −20° C. At the day of analysis, the cells were stained with rabbit anti-H2AX (Histone H3, clone D1H2, #12167S, cell signaling) at 0.25 μg/ml and analyzed by flow cytometry. To control for antibody staining specificity a staining with irradiated or untreated PBMCS was performed.

[0391] B cells were nucleofected 1-10 days after culture initiation. Results are shown in FIG. 8. As shown by staining of the H2AX histone marker in FIG. 8B, the maximum of DNA double strand breaks is achieved starting at days 2-3.

[0392] Next, B cells were nucleofected under distinct conditions with the Neon® Transfection System (Thermo Fisher Scientific) at 2150V 10 ms 1 pulse, 2150V 15 ms 1 pulse, 2150V 20 ms 1 pulse, 2150V 10 ms 2 pulse, 2400V 10 ms 1 pulse, 2400V 15 ms 1 pulse, 2150V 20 ms 1 pulse, 2500V 10 ms 1 pulse and 2500V 15 ms 1 pulse. Results are shown in FIG. 9. Under all conditions successful nucleofection was achieved. The best results were obtained using 2150V, 10 ms, 2 pulses.

Example 5: Influence of c-NHEJ and a-EJ on Insert Acquisition in the Switch Region

[0393] To increase engineering efficiency, an in vitro system was used to study the influence of c-NHEJ and a-EJ on acquisition of natural inserts.

[0394] To this end, B cells were isolated by magnetic beads using anti-CD19 beads, followed by FACS-sorting and selection of naïve B cells (IgM.sup.+ IgD.sup.+ CD27.sup.− IgG.sup.− IgA.sup.−). Cells were plated in 48 well plates at concentrations of 50 000 B cells/ml and stimulated with 25 000 irradiated K562L/ml and 8 ng/ml IL4. The DNA repair inhibitor Olaparip (4 nM), SCR7 (100 nM) or DMSO (1:100) as control were added to the culture medium. At day 3 and day 6 the culture medium was replaced which fresh medium supplemented with IL4 and inhibitors. Cells were harvested at day 10 and stained with fluorescently labeled anti-CD19 and anti-IgG antibodies. Switched IgG B cell were sorted by flow cytometry and gDNA was isolation using a commercial kit. Genomic DNA of 50 000 sorted cells were used for γ-switch region PCR amplification and MINION sequencing as described above. Inserts frequencies were analyzed with a bioinformatics pipeline (Pieper K, Tan J, Piccoli L, Foglierini M, Barbieri S, Chen Y, Silacci-Fregni C, Wolf T, Jarrossay D, Anderle M, Abdi A, Ndungu F M, Doumbo O K, Traore B, Tran T M, Jongo S, Zenklusen I, Crompton P D, Daubenberger C, Bull P C, Sallusto F, Lanzavecchia A: Public antibodies to malaria antigens generated by two LAIR1 insertion modalities. Nature. 2017. Aug. 31; 548(7669):597-601).

[0395] The general principle is shown in FIG. 10A and results are shown in FIG. 10B. The results show that natural switch inserts depend on the c-NHEJ DNA repair pathway as insert frequencies in presence of SCR7 are decreased while elevated when Olaparib was added to the culture medium (FIG. 10 A, B). Therefore, B cell engineering in presence of the inhibitor Olaparib can increase engineering efficiency. Likewise, the pre-incubation of the DNA substrate with the DNA-binding proteins Ku70/80, the first event in c-NHEJ mediated repair, and nucleofection of the DNA-Ku70/80 protein complex can increase the number of successful integrations (FIG. 10 A).

TABLE-US-00011 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks SEQ ID NO: 1 AGGTAAGT 3′ splice site SEQ ID NO: 2 YNCTGAC branch site wherein Y may be C or T and N may be any nucleotide selected from A, G, C and T SEQ ID NO: 3 GTAGTGAGGG intronic splicing SEQ ID NO: 4 GTTGGTGGTT intronic splicing enhancer SEQ ID NO: 5 AGTTGTGGTT intronic splicing enhancer SEQ ID NO: 6 GTATTGGGTC intronic splicing enhancer SEQ ID NO: 7 AGTGTGAGGG intronic splicing enhancer SEQ ID NO: 8 GGGTAATGGG intronic splicing enhancer SEQ ID NO: 9 TCATTGGGGT intronic splicing enhancer SEQ ID NO: 10 GGTGGGGGTC intronic splicing enhancer SEQ ID NO: 11 GGTTTTGTTG intronic splicing enhancer SEQ ID NO: 12 TATACTCCCG intronic splicing enhancer SEQ ID NO: 13 GTATTCGATC intronic splicing enhancer SEQ ID NO: 14 GGGGGTAGG intronic splicing enhancer SEQ ID NO: 15 GTAGTTCCCT intronic splicing enhancer SEQ ID NO: 16 GTTAATAGTA intronic splicing enhancer SEQ ID NO: 17 TGCTGGTTAG intronic splicing enhancer SEQ ID NO: 18 ATAGGTAACG intronic splicing enhancer SEQ ID NO: 19 TCTGAATTGC intronic splicing enhancer SEQ ID NO: 20 TCTGGGTTTG intronic splicing enhancer SEQ ID NO: 21 CATTCTCTTT intronic splicing enhancer SEQ ID NO: 22 GTATTGGTGT intronic splicing enhancer SEQ ID NO: 23 GGAGGGTTT intronic splicing enhancer SEQ ID NO: 24 TTTAGATTTG intronic splicing enhancer SEQ ID NO: 25 ATAAGTACTG intronic splicing enhancer SEQ ID NO: 26 TAGTCTATTA intronic splicing enhancer SEQ ID NO: 27 CGAGGAGGCAGCTCCTCACCCTCCCTTTCTCTTT intronic sequence TGTCCTGCGGGTCCTCAG SEQ ID NO: 28 CGAAGGGGGCGGGAGTGGCGGGCACCGGGC intronic sequence TGACACGTGTCCCTCACTGCAG SEQ ID NO: 29 TCCGCCCACATCCACACCTGCCCCACCTCTGACT intronic sequence CCCTTCTCTTGACTCCAG SEQ ID NO: 30 CCACAGGCTGGTCCCCCCACTGCCCCGCCCTCA intronic sequence CCACCATCTCTGTTCACAG SEQ ID NO: 31 TGGGCCCAGCTCTGTCCCACACCGCGGTCACAT intronic sequence GGCACCACCTCTCTTGCAG SEQ ID NO: 32 GGACACCTTCTCTCCTCCCAGATTCCAGTAACTC intronic sequence CCAATCTTCTCTCTGCAG SEQ ID NO: 33 AGGGACAGGCCCCAGCCGGGTGCTGACACGTC intronic sequence CACCTCCATCTCTTCCTCAG SEQ ID NO: 34 GGCCCACCCTCTGCCCTGAGAGTGACCGCTGTA intronic sequence CCAACCTCTGTCCCTACAG SEQ ID NO: 35 TGGGCCCAGCTCTGTCCCACACCGCAGTCACAT intronic sequence GGCGCCATCTCTCTTGCAG SEQ ID NO: 36 AGATACCTTCTCTCTTCCCAGATCTGAGTAACTC intronic sequence CCAATCTTCTCTCTGCAG SEQ ID NO: 37 ACGCATCCACCTCCATCCCAGATCCCCGTAACTC intronic sequence CCAATCTTCTCTCTGCAG SEQ ID NO: 38 ACGCGTCCACCTCCATCCCAGATCCCCGTAACT intronic sequence CCCAATCTTCTCTCTGCAG SEQ ID NO: 39 ACGCATCCACCTCCATCCCAGATCCCCGTAACTC intronic sequence CCAATCTTCTCTCTGCAG SEQ ID NO: 40 ACGCATCCACCTCCATCCCAGATCCCCGTAACTC intronic sequence CCAATCTTCTCTCTGCAG SEQ ID NO: 41 GACCCACCCTCTGCCCTGGGAGTGACCGCTGT intronic sequence GCCAACCTCTGTCCCTACAG SEQ ID NO: 42 TGGGCCCAGCTCTGTCCCACACCGCGGTCACAT intronic sequence GGCACCACCTCTCTTGCAG SEQ ID NO: 43 AGACACCTTCTCTCCTCCCAGATCTGAGTAACTC intronic sequence CCAATCTTCTCTCTGCAG SEQ ID NO: 44 AGGGACAGGCCCCAGCCGGGTGCTGACGCATC intronic sequence CACCTCCATCTCTTCCTCAG SEQ ID NO: 45 GGCCCACCCTCTGCCCTGGGAGTGACCGCTGT intronic sequence GCCAACCTCTGTCCCTACAG SEQ ID NO: 46 GTGAGTCTGCTGTCTGGGGATAGCGGGGAGCC intronic sequence AGGTGTACTGGGCCAGGCAA SEQ ID NO: 47 GTGAGTCCCACTGCAGCCCCCTCCCAGTCTTCT intronic sequence CTGTCCAGGCACCAGGCCA SEQ ID NO: 48 GTAAGATGGCTTTCCTTCTGCCTCCTTTCTCTGG intronic sequence GCCCAGCGTCCTCTGTCC SEQ ID NO: 49 GTGAGTCCTCACAACCTCTCTCCTGCTTTAACTC intronic sequence TGAAGGGTTTTGCTGCAT SEQ ID NO: 50 GTGAGTCCTCACCACCCCCTCTCTGAGTCCACTT intronic sequence AGGGAGACTCAGCTTGCC SEQ ID NO: 51 GTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTG intronic sequence CTACTGCCTGTGGGGTTT SEQ ID NO: 52 CATGGTGACTTCCTACAGTGGACGCTGAGATCC intronic sequence TGCTCTGCTTCCCTCCTAG SEQ ID NO: 53 GTGAGGACGTCACCTGGGCCCTGCCCCAGTCT intronic sequence CAGCTCGACCCTCGAGCTTG SEQ ID NO: 54 LEVLFQGP cleavage tag SEQ ID NO: 55 DDDDK cleavage tag SEQ ID NO: 56 IEGR cleavage tag SEQ ID NO: 57 ENLYFQG cleavage tag SEQ ID NO: 58 LVPRGS cleavage tag SEQ ID NO: 59 DX.sub.1EX.sub.2NPGP self-processing site wherein X.sub.1 is Val or Ile, and X.sub.2 may be any (naturally occurring) amino acid SEQ ID NO: 60 EGRGSLLTCGDVEENPGP self-processing site SEQ ID NO: 61 VKQTLNFDLLKLAGDVESNPGP self-processing site SEQ ID NO: 62 ATNFSLLKQAGDVEENPGP self-processing site SEQ ID NO: 63 GSGATNFSLLKQAGDVEENPGP self-processing site SEQ ID NO: 64 RKRRGSGATNFSLLKQAGDVEENPGP self-processing site SEQ ID NO: 65 SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK twin StrepTag aa SEQ ID NO: 66 GLNDIFEAQKIEWHE AviTag SEQ ID NO: 67 KRRWKKNFIAVSAANRFKKISSSGAL Calmodulin-tag SEQ ID NO: 68 EEEEEE polyglutamate tag SEQ ID NO: 69 GAPVPYPDPLEPR E-tag SEQ ID NO: 70 DYKDDDDK FLAG-tag SEQ ID NO: 71 YPYDVPDYA HA-tag SEQ ID NO: 72 HHHHHH His-tag SEQ ID NO: 73 EQKLISEEDL Myc-tag SEQ ID NO: 74 TKENPRSNQEESYDDNES NE-tag SEQ ID NO: 75 KETAAAKFERQHMDS S-tag SEQ ID NO: 76 MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQ SBP-tag GQREP SEQ ID NO: 77 SLAELLNAGLGGS Softag 1 SEQ ID NO: 78 TQDPSRVG Softag 3 SEQ ID NO: 79 WSHPQFEK Strep-tag SEQ ID NO: 80 CCPGCC TC tag SEQ ID NO: 81 GKPIPNPLLGLDST V5 tag SEQ ID NO: 82 YTDIEMNRLGK VSV-tag SEQ ID NO: 83 DLYDDDDK Xpress tag SEQ ID NO: 84 TDKDMTITFTNKKDAE Isopeptag SEQ ID NO: 85 AHIVMVDAYKPTK SpyTag SEQ ID NO: 86 KLGDIEFIKVNK SnoopTag SEQ ID NO: 87 EVHTNQDPLD Ty1 tag SEQ ID NO: 88 EDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRL mutated LAIR1 ERERNYLYSDTEDVSQTSPSESEARFRIDSVNAGNA fragment aa GLFRCIYYKSRKWSEQSDYLELVVK SEQ ID NO: 89 DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSE PD-1 fragment aa SFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRF RVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAP KAQIKESLRAELRVT SEQ ID NO: 90 EQVSTPEIKVLNKTQENGTCTLILGCTVEKGDHVAY SLAM fragment aa SWSEKAGTHPLNPANSSHLLSLTLGPQHADNIYICT VSNPISNNSQTFSPWPGCRTDPS SEQ ID NO: 91 MAQVQLVESGGGLVQAGGSLTLSCAASGSTSRSY T3-VHH aa ALGWFRQAPGKEREFVAHVGQTAEFAQGRFTISR DFAKNTVSLQMNDLKSDDTAIYYCVASNRGWSPS RVSYWGQGTQVTVSS SEQ ID NO: 92 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSRVGVG TT39.7-scFv aa WIRQPPGKALEWLSLIYWDDEKHYSPSLKNRVTISK DSSKNQVVLTLTDMDPVDTGTYYCAHRGVDTSG WGFDYWGQGALVTVSSGGGGSGGGGSGGGGS QSALTQPASVSGSPGQSITISCSGAGSDVGGHNFV SWYQQYPGKAPKLMIYDVKNRPSGVSYRFSGSKSG YTASLTISGLQAEDEATYFCSSYSSSSTLIIFGGGTRLT VL SEQ ID NO: 93 QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIG F4-VHH aa WFRQAPGKEREAVSCISGSSGSTYYPDSVKGRFTISR DNAKNTVYLQMNSLKPEDTAVYYCATIRSSSWGG CVHYGMDYWGKGTQVTVSS SEQ ID NO: 94 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMN MPE8-scFv aa WVRQAPGKGLEWVSSISASSSYSDYADSAKGRFTIS RDNAKTSLFLQMNSLRAEDTAIYFCARARATGYSSI TPYFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQ SVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVH WYQQLPGTAPKLLIYDNNNRPSGVPDRFSASKSGT SASLAITGLQAEDEADYYCQSYDRNLSGVFGTGTK VTVL SEQ ID NO: 95 TGGTTGCTGACTAATTGAGATGCATGCTTTGCAT SV40 nuclear ACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCC localization signal ACACCTGGTTGCTGACTAATTGAGATGCATGCTT TGCATACTTCTGCCTGCTGGGGAGCCTGGGGA CTTTCCACACC SEQ ID NO: 96 cacccttgaaagtagcccatgccttcc primer SEQ ID NO: 97 cctgcctcccagtgtcctgcattacttctg primer SEQ ID NO: 98 ggaacgcagtgtagactcagctgagg primer SEQ ID NO: 99 TTGTGAGCAAGTCTCAGGGTCCTCACTGTCAAC DNA substrate TGGGAAAAAACTCTGCAGTGATGAGAATCACAT GCACGTAGAAGGTGCAGGAGGCGTGGGAATG TTCTAAGGTTGGGCTGTGGTCATGGCTGCATAA CTCTATAAAATTGCTAAAATCCCTGAATTGTGAT GCTAAAATGACGTGTGTGGCATGGTGACTTCCT ACAGTGGACGCTGAGATCCTGCTCTGCTTCCCT CCTAGAAGATCTGCCCAGACCCTCCATCTCGGC TGAGCCAGGCACCGTGATCCCCCTGGGGAGCC ATGTGACTTTCGTGTGCCGGGGCCCGGTTGGG GTTCAAACATTCCGCCTGGAGAGGGACAGTAG ATCCACATACAATGATACTGAAGATGTGTCTCAA GCTAGTCCATCTGAGTCAGAGGCCAGATTCCGC ATTGACTCAGTAAGAGAAGGAAATGCCGGGCTT TATCGCTGCATCTATTATAAGCCCCCTAAATGGT CTGAGCAGAGTGACTACCTGGAGCTGCTGGTG AAAGGTGAGGACGTCACCTGGGCCCTGCCCCA GTCTCAGCTCGACCCTCGAGCTTGTCCCCAGGT SEQ ID NO: 100 ccacctccaaacggcaggcatcc primer SEQ ID NO: 101 ccaaaggccgcatgaccatcacgc primer SEQ ID NO: 102 cctcagctgagtctacactgcgttcc primer SEQ ID NO: 103 ctgaggacccgcaggacaaaagagaaaggg primer SEQ ID NO: 104 ggtcaccgtctcctcaggtaagaatggcc primer SEQ ID NO: 105 gccttttcagtttcggtcagcctcgc primer SEQ ID NO: 106 gcgggtcctcagaagatctgcccagaccc primer SEQ ID NO: 107 ggccattcttacctttcaccagcagctccagg primer SEQ ID NO: 108 gcgggtcctcaggggaagatctgcccagaccc primer SEQ ID NO: 109 ggccattcttacctgaggagacggctttcaccagcagctccagg primer SEQ ID NO: 110 CCTCAGCTGAGTCTACACTGCGTTCCCCATCACACTC DNA substrate ACCCTCCCTATACTCACTCCCAGGCCTGGGTTGTCTG CCTGGGGAGACTTCAGGGTAGCTGGAGTGTGACTG AGCTGGGGGCAGCAGAAGCTGGGCTGGAGGGACT CTATTGGCTGCCTGCGGGGTGTGTGGCTCCAGGCTT CACATTCAGGTATGCAACCTGGGCCCTCCAGCTGCAT GTGCTGGGAGCTGAGTGTGTGCAGCACCTACGTGCT GATGCCTCGGGGGAAAGCAGGCCTGGTCCACCCAA ACCTGAGCCCTCAGCCATTCTGAGCAGGGAGCCAGG GGCAGTCAGGCCTCAGAGTGCAGCAGGGCAGCCAG CTGAATGGTGGCAGGGATGGCTCAGCCTGCTCCAG GAGACCCCAGGTCTGTCCAGGTGTTCAGTGCTGGGC CCTGCAGCAGGATGGGCTGAGGCCTGCAGCCCCAG CAGCCTTGGACAAAGACCTGAGGCCTCACCACGGCC CCGCCACCCCTGATAGCCATGACAGTCTGGGCTTTG GAGGCCTGCAGGTGGGCTCGGCCTTGGTGGGGCAG CCACAGCGGGACGCAAGTAGTGAGGGCACTCAGAA CGCCACTCAGCCCCGACAGGCAGGGCACGAGGAGG CAGCTCCTCACCCTCCCTTTCTCTTTTGTCCTGCGGGT CCTCAGAAGATCTGCCCAGACCCTCCATCTCGGCTGA GCCAGGCACCGTGATCCCCCTGGGGAGCCATGTGA CTTTCGTGTGCCGGGGCCCGGTTGGGGTTCAAACAT TCCGCCTGGAGAGGGACAGTAGATCCACATACAATG ATACTGAAGATGTGTCTCAAGCTAGTCCATCTGAGTC AGAGGCCAGATTCCGCATTGACTCAGTAAGAGAAGG AAATGCCGGGCTTTATCGCTGCATCTATTATAAGCCC CCTAAATGGTCTGAGCAGAGTGACTACCTGGAGCTG CTGGTGAAAGGTAAGAATGGCCACTCTAGGGCCTTT GTTTTCTGCTACTGCCTGTGGGGTTTCCTGAGCATTG CAGGTTGGTCCTCGGGGCATGTTCCGAGGGGACCT GGGCGGACTGGCCAGGAGGGGATGGGCACTGGGG TGCCTTGAGGATCTGGGAGCCTCTGTGGATTTTCCGA TGCCTTTGGAAAATGGGACTCAGGTTGGGTGCGTCT GATGGAGTAACTGAGCCTGGGGGCTTGGGGAGCCA CATTTGGACGAGATGCCTGAACAAACCAGGGGTCTT AGTGATGGCTGAGGAATGTGTCTCAGGAGCGGTGTC TGTAGGACTGCAAGATCGCTGCACAGCAGCGAATCG TGAAATATTTTCTTTAGAATTATGAGGTGCGCTGTGTG TCAACCTGCATCTTAAATTCTTTATTGGCTGGAAAGAG AACTGTCGGAGTGGGTGAATCCAGCCAGGAGGGAC GCGTAGCCCCGGTCTTGATGAGAGCAGGGTTGGGG GCAGGGGTAGCCCAGAAACGGTGGCTGCCGTCCTG ACAGGGGCTTAGGGAGGCTCCAGGACCTCAGTGCC TTGAAGCTGGTTTCCATGAGAAAAGGATTGTTTATCTT AGGAGGCATGCTTACTGTTAAAAGACAGGATATGTTT GAAGTGGCTTCTGAGAAAAATGGTTAAGAAAATTATG ACTTAAAAATGTGAGAGATTTTCAAGTATATTAATTTTT TTAACTGTCCAAGTATTTGAAATTCTTATCATTTGATTA ACACCCATGAGTGATATGTGTCTGGAATTGAGGCCA AAGCAAGCTCAGCTAAGAAATACTAGCACAGTGCTG TCGGCCCCGATGCGGGACTGCGTTTTGACCATCATA AATCAAGTTTATTTTTTTAATTAATTGAGCGAAGCTGG AAGCAGATGATGAATTAGAGTCAAGATGGCTGCATG GGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGC AGGTCACCGCGAGAG SEQ ID NO: 111 AAGATCTGCCCAGACCCTCCATCTCGGCTGAGC Nucleotide sequence CAGGCACCGTGATCCCCCTGGGGAGCCATGTG encoding (poly)peptide ACTTTCGTGTGCCGGGGCCCGGTTGGGGTTCA of interest AACATTCCGCCTGGAGAGGGACAGTAGATCCA CATACAATGATACTGAAGATGTGTCTCAAGCTA GTCCATCTGAGTCAGAGGCCAGATTCCGCATTG ACTCAGTAAGAGAAGGAAATGCCGGGCTTTATC GCTGCATCTATTATAAGCCCCCTAAATGGTCTGA GCAGAGTGACTACCTGGAGCTGCTGGTGAAAG SEQ ID NO: 112 TTGTGAGCAAGTCTCAGGGTCCTCACTGTCAAC intronic sequence TGGGAAAAAACTCTGCAGTGATGAGAATCACAT GCACGTAGAAGGTGCAGGAGGCGTGGGAATG TTCTAAGGTTGGGCTGTGGTCATGGCTGCATAA CTCTATAAAATTGCTAAAATCCCTGAATTGTGAT GCTAAAATGACGTGTGTGGCATGGTGACTTCCT ACAGTGGACGCTGAGATCCTGCTCTGCTTCCCT CCTAG SEQ ID NO: 113 GTGAGGACGTCACCTGGGCCCTGCCCCAGTCT intronic sequence CAGCTCGACCCTCGAGCTTGTCCCCAGGT SEQ ID NO: 114 CCTCAGCTGAGTCTACACTGCGTTCCCCATCACA intronic sequence CTCACCCTCCCTATACTCACTCCCAGGCCTGGGT TGTCTGCCTGGGGAGACTTCAGGGTAGCTGGA GTGTGACTGAGCTGGGGGCAGCAGAAGCTGG GCTGGAGGGACTCTATTGGCTGCCTGCGGGGT GTGTGGCTCCAGGCTTCACATTCAGGTATGCAA CCTGGGCCCTCCAGCTGCATGTGCTGGGAGCT GAGTGTGTGCAGCACCTACGTGCTGATGCCTCG GGGGAAAGCAGGCCTGGTCCACCCAAACCTGA GCCCTCAGCCATTCTGAGCAGGGAGCCAGGGG CAGTCAGGCCTCAGAGTGCAGCAGGGCAGCCA GCTGAATGGTGGCAGGGATGGCTCAGCCTGCT CCAGGAGACCCCAGGTCTGTCCAGGTGTTCAGT GCTGGGCCCTGCAGCAGGATGGGCTGAGGCCT GCAGCCCCAGCAGCCTTGGACAAAGACCTGAG GCCTCACCACGGCCCCGCCACCCCTGATAGCCA TGACAGTCTGGGCTTTGGAGGCCTGCAGGTGG GCTCGGCCTTGGTGGGGCAGCCACAGCGGGA CGCAAGTAGTGAGGGCACTCAGAACGCCACTC AGCCCCGACAGGCAGGGCACGAGGAGGCAGC TCCTCACCCTCCCTTTCTCTTTTGTCCTGCGGGTC CTCAG SEQ ID NO: 115 GTAAGAATGGCCACTCTAGGGCCTTTGTTTTCTG intronic sequence CTACTGCCTGTGGGGTTTCCTGAGCATTGCAGG TTGGTCCTCGGGGCATGTTCCGAGGGGACCTG GGCGGACTGGCCAGGAGGGGATGGGCACTGG GGTGCCTTGAGGATCTGGGAGCCTCTGTGGATT TTCCGATGCCTTTGGAAAATGGGACTCAGGTTG GGTGCGTCTGATGGAGTAACTGAGCCTGGGGG CTTGGGGAGCCACATTTGGACGAGATGCCTGA ACAAACCAGGGGTCTTAGTGATGGCTGAGGAA TGTGTCTCAGGAGCGGTGTCTGTAGGACTGCAA GATCGCTGCACAGCAGCGAATCGTGAAATATTT TCTTTAGAATTATGAGGTGCGCTGTGTGTCAACC TGCATCTTAAATTCTTTATTGGCTGGAAAGAGAA CTGTCGGAGTGGGTGAATCCAGCCAGGAGGGA CGCGTAGCCCCGGTCTTGATGAGAGCAGGGTT GGGGGCAGGGGTAGCCCAGAAACGGTGGCTG CCGTCCTGACAGGGGCTTAGGGAGGCTCCAGG ACCTCAGTGCCTTGAAGCTGGTTTCCATGAGAA AAGGATTGTTTATCTTAGGAGGCATGCTTACTGT TAAAAGACAGGATATGTTTGAAGTGGCTTCTGA GAAAAATGGTTAAGAAAATTATGACTTAAAAATG TGAGAGATTTTCAAGTATATTAATTTTTTTAACTG TCCAAGTATTTGAAATTCTTATCATTTGATTAACA CCCATGAGTGATATGTGTCTGGAATTGAGGCCA AAGCAAGCTCAGCTAAGAAATACTAGCACAGTG CTGTCGGCCCCGATGCGGGACTGCGTTTTGACC ATCATAAATCAAGTTTATTTTTTTAATTAATTGAG CGAAGCTGGAAGCAGATGATGAATTAGAGTCA AGATGGCTGCATGGGGGTCTCCGGCACCCACA GCAGGTGGCAGGAAGCAGGTCACCGCGAGAG