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Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

Kügler J, Wilke S, Meier D, Tomszak F, Frenzel A, Schirrmann T, Dübel S, Garritsen H, Hock B, Toleikis L, Schütte M, Hust M - BMC Biotechnol. (2015)

Bottom Line: We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display.For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate.Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

View Article: PubMed Central - PubMed

Affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. jonas.kuegler@tu-bs.de.

ABSTRACT

Background: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.

Results: The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.

Conclusion: The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

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Related in: MedlinePlus

Comparision of pHAL14 and pHAL30.(A) Comparision of the production of soluble antibodies. SN supernatant, PE periplasmatic extract, OS osmotic shock fraction, Eluate after IMAC purification, OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments analyzed: D1.3, HT186-D11 and TM43-E10, antigens: lysozyme, Muc-1 and OmpD. (B) Comparision of scFv-phage antigen binding. OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments D1.3, HT186-D11, SH313-B5 and TM43-E10. antigens: lysozyme, MUC1, CD30 and OmpD.
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Fig2: Comparision of pHAL14 and pHAL30.(A) Comparision of the production of soluble antibodies. SN supernatant, PE periplasmatic extract, OS osmotic shock fraction, Eluate after IMAC purification, OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments analyzed: D1.3, HT186-D11 and TM43-E10, antigens: lysozyme, Muc-1 and OmpD. (B) Comparision of scFv-phage antigen binding. OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments D1.3, HT186-D11, SH313-B5 and TM43-E10. antigens: lysozyme, MUC1, CD30 and OmpD.

Mentions: A prerequisite for a good up to date antibody gene library is a phagemid that allows both, a high display level of functional antibody fragments on phage and a good expression of soluble antibodies for screening. The new design of the phagemid generation pHAL30 (Figure 1) is based on the pHAL14 [31]. In the first optimization step and independent of kappa or lambda light chains, the tag order in the pHAL30 phagemid was changed from the initial His/Myc-tag in pHAL14 to Myc/His tag. For evaluation of the expression capacity of both phagemids, three different soluble scFv antibody fragments were produced in 150 mL E.coli XL-1 Blue MRF’ culture for 4 h at 25°C and 250 rpm. Functional soluble scFv antibody fragments were quantified by antigen ELISA in the production supernatant, the periplasmatic extract, the osmotic shock fraction and the eluate after IMAC purification (Figure 2A). Except for the osmotic shock preparation, the production level with the pHAL30 phagemid was always significantly improved. After IMAC purification of the supernatants, the yields of HT186-D11 were 1 mg/L for pHAL14 and 2 mg/L for pHAL30, in case of TM43-E10 the yields were 1 mg/L for both vectors and for D1.3 the yields increased from 0.6 mg/L for pHAL14 to 1 mg/L for pHAL30. The production yields in the periplasmic fraction of scFvs HT186-D11 and TM43-E10 after IMAC purification were improved from 1.6 mg/L, and 0.9 mg/L with pHAL14 to 3.9 mg/L and 2.4 mg/L, respectively, with pHAL30.Figure 1


Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

Kügler J, Wilke S, Meier D, Tomszak F, Frenzel A, Schirrmann T, Dübel S, Garritsen H, Hock B, Toleikis L, Schütte M, Hust M - BMC Biotechnol. (2015)

Comparision of pHAL14 and pHAL30.(A) Comparision of the production of soluble antibodies. SN supernatant, PE periplasmatic extract, OS osmotic shock fraction, Eluate after IMAC purification, OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments analyzed: D1.3, HT186-D11 and TM43-E10, antigens: lysozyme, Muc-1 and OmpD. (B) Comparision of scFv-phage antigen binding. OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments D1.3, HT186-D11, SH313-B5 and TM43-E10. antigens: lysozyme, MUC1, CD30 and OmpD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352240&req=5

Fig2: Comparision of pHAL14 and pHAL30.(A) Comparision of the production of soluble antibodies. SN supernatant, PE periplasmatic extract, OS osmotic shock fraction, Eluate after IMAC purification, OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments analyzed: D1.3, HT186-D11 and TM43-E10, antigens: lysozyme, Muc-1 and OmpD. (B) Comparision of scFv-phage antigen binding. OD450nm-620nm optical density at 450 nm (620 nm reference), scFv antibody fragments D1.3, HT186-D11, SH313-B5 and TM43-E10. antigens: lysozyme, MUC1, CD30 and OmpD.
Mentions: A prerequisite for a good up to date antibody gene library is a phagemid that allows both, a high display level of functional antibody fragments on phage and a good expression of soluble antibodies for screening. The new design of the phagemid generation pHAL30 (Figure 1) is based on the pHAL14 [31]. In the first optimization step and independent of kappa or lambda light chains, the tag order in the pHAL30 phagemid was changed from the initial His/Myc-tag in pHAL14 to Myc/His tag. For evaluation of the expression capacity of both phagemids, three different soluble scFv antibody fragments were produced in 150 mL E.coli XL-1 Blue MRF’ culture for 4 h at 25°C and 250 rpm. Functional soluble scFv antibody fragments were quantified by antigen ELISA in the production supernatant, the periplasmatic extract, the osmotic shock fraction and the eluate after IMAC purification (Figure 2A). Except for the osmotic shock preparation, the production level with the pHAL30 phagemid was always significantly improved. After IMAC purification of the supernatants, the yields of HT186-D11 were 1 mg/L for pHAL14 and 2 mg/L for pHAL30, in case of TM43-E10 the yields were 1 mg/L for both vectors and for D1.3 the yields increased from 0.6 mg/L for pHAL14 to 1 mg/L for pHAL30. The production yields in the periplasmic fraction of scFvs HT186-D11 and TM43-E10 after IMAC purification were improved from 1.6 mg/L, and 0.9 mg/L with pHAL14 to 3.9 mg/L and 2.4 mg/L, respectively, with pHAL30.Figure 1

Bottom Line: We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display.For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate.Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

View Article: PubMed Central - PubMed

Affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Spielmannstr. 7, 38106, Braunschweig, Germany. jonas.kuegler@tu-bs.de.

ABSTRACT

Background: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.

Results: The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.

Conclusion: The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Show MeSH
Related in: MedlinePlus