Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2013 Sep-Oct;5(5):690-8.
doi: 10.4161/mabs.25592. Epub 2013 Jul 2.

Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies

Sophie Venet  1 , Marie Kosco-Vilbois, Nicolas Fischer
Affiliations
Comparative Study

Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies

Sophie Venet et al. MAbs. 2013 Sep-Oct.

Abstract

The plasticity of natural immunoglobulin repertoires can be exploited for the generation of phage display libraries. Secondary lymphoid organs, such as the spleen and the lymph nodes, constitute interesting sources of diversity because they are rich in B cells, part of which can be affinity matured. These organs, however, differ in their anatomical structure, reflecting the different fluids they drain, which affects the B cell repertoires. The CDRH3 repertoires from these organs, extracted from naïve or immunized mice, were compared in the context of phage display libraries using human antibody framework families. Deep sequencing analysis revealed that all libraries displayed different CDRH3 repertoires, but the one derived from lymph nodes of naïve mice was the most diverse. Library performance was assessed by in vitro selection. For both organs, immunization increased substantially the frequency of molecules able to bind to the immunogen. The library derived from lymph nodes from naïve mice, however, was the most effective in generating diverse and high affinity candidates. These results illustrate that the use of a biased CDRH3 repertoire increases the performance of libraries, but reduces the clonal diversity, which may be detrimental for certain strategies.

Keywords: antibody repertoire; immune library; next generation sequencing; phage display; scFv; secondary lymphoid organs.

PubMed Disclaimer

Figures

None
Figure 1. Capture of murine CDRH3 into a library of human scFv. (A) BalbC mice, divided in three groups, were either kept naïve or immunized with hIFNγ or hCCL5. (B) After sacrifice, the spleen and the lymph nodes were kept separated and VH repertoires were recovered by PCR. CDRH3 were then amplified from the VH pool by PCR, along with recognition sites for FokI, a type IIS restriction enzyme, were added to the inserts. CDR3 are represented in gray, CDR1 and 2 in white. (C) Murine inserts were then digested with FokI and (D) ligated to the human acceptor scFv library, itself digested with BsmBI. This second type IIS enzyme permitted the removal of a non-diversified stuffer sequence (“S”) at the location of CDRH3 and the generation of compatible cohesive ends for the incorporation of CDRH3. The acceptor library also contains synthetic diversity at the location of the CDRL3 (“L3”) and tags (“T,” a c-myc and a His tag) at the scFv C-terminal for purification purposes. Libraries featuring mouse CDRH3 were analyzed by NGS, which covered the CDRH3 and part of VH framework 3 (“NGS”). (E) The fusion to gIII allowed the expression of scFv at the surface of M13 phage for phage display selection.
None
Figure 2. Assessment of the murine nature of CDRH3 repertoires. (A) Length profile of CDRH3 represented as the percentage of unique CDRH3 in function of their length in amino acids. (B) Amino acid composition profile of unique CDRH3 of all lengths. The few stop codons found are likely due to cloning or sequencing errors. This analysis was performed by NGS.
None
Figure 3. Evaluation of CDRH3 redundancy. Evaluation by NGS of CDRH3 repartition in the context of human scFv libraries. The percentage of total CDRH3 is represented in function of their frequency according to a color code. For each library, one million clones were analyzed. The values on the histograms are the number of unique CDRH3, i.e., the number of CDRH3 with a different amino acids sequence, corresponding to each section. (A) represents the level of redundancy of lymph nodes derived libraries while (B) represents the repartition of CDRH3 from spleens derived libraries.
None
Figure 4. Overlap between CDRH3 repertoires. Evaluation of the unique CDRH3 sequences in common between libraries. The analysis included one million clones for each library (NGS). Diversity of unique CDRH3 (i.e., all CDRH3 with a different amino acids sequence) is represented by circles which size is proportional to the size of the sample. Naïve libraries are represented in red, libraries biased against hIFNγ in blue and libraries biased against hCCL5 in green. For each library, the number of unique CDRH3 is described with the same color code, U refers to “unique sequences.” Unique sequences in common between libraries are symbolized by the overlap between circles. Numbers in black represent the number of unique sequences corresponding to each section determined by a black line. (A) represents the overlap between both naïve libraries, (B) represents the overlap between the spleen derived libraries, and (C) represents the overlap between the lymph nodes derived libraries.
None
Figure 5. Screening of selection outputs from lymph nodes libraries in the scFv format. For each library, 528 random clones from the selection round 3 were independently cultured and tested by scFv ELISA against hIFNγ. Revelation was performed via HRP and read at 450nm. The frequency of clones is provided according to their level of absorbance at 450nm. Clones were classified according to their level of absorbance relative to a positive control, i.e., displaying absorbance values above 70% of the signal of a positive control scFv (absorbance ~1.6), between 10% and 70% (absorbance 0.2 and 1.6), and below 10%.
None
Figure 6. Relative performance of most amplified binders. Representation of all the scFv defined as binders to hIFNγ from each library and with a frequency defined by deep sequencing above 1% at the selection round 3. Clones are grouped according to their library of origin and represented by their EC50 in nM (defined by ELISA). None of the binders found from the library LN / hCCL5 having a frequency above 1%, the library is not represented in this graph.
None
Figure 7. Evaluation of the cross specificity of clones. A selection of clones from each LN library was tested for their cross reactivity on IFNγ. Purified scFv were tested at four concentrations (1100, 110, 11 and 1.1 nM, n = 2) for their specificity to a panel of IFNγ from different species, i.e., human, rhesus monkey, mouse and rabbit, and streptavidin as a negative control. Revelation was performed via HRP and read at 450 nm. All clones above 1% frequency at the selection round 3 were tested.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990;348:552–4. doi: 10.1038/348552a0. - DOI - PubMed
    1. Mazor Y, Van Blarcom T, Mabry R, Iverson BL, Georgiou G. Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat Biotechnol. 2007;25:563–5. doi: 10.1038/nbt1296. - DOI - PubMed
    1. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JMW, Yeung YA, et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol. 2003;21:163–70. doi: 10.1038/nbt785. - DOI - PubMed
    1. Hanes J, Plückthun A. In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A. 1997;94:4937–42. doi: 10.1073/pnas.94.10.4937. - DOI - PMC - PubMed
    1. Thie H, Meyer T, Schirrmann T, Hust M, Dübel S. Phage display derived therapeutic antibodies. Curr Pharm Biotechnol. 2008;9:439–46. doi: 10.2174/138920108786786349. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources