Notes+11-17-12+do+1558

Construction of a rationally designed antibody platform for sequencing-assisted selection notes PNAS paper 2012

aDivision of Health Sciences and Technology, Harvard–Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; cDepartment of Genetics, Harvard University Medical School, and dDivision of Genetics, Howard Hughes Medical Institute, Brigham and Women’s Hospital, Boston, MA 02115; and eBiophysics Program, Graduate School of Arts and Sciences, Harvard University, Cambridge, MA 02139

q CDRs 1 and 2 are encoded in the germ line, and are thus more constrained in their diversity. L3 is characterized by “junctional diversity,” formed during the recombination of two gene segments (V and J).

I didn't realize the 1st 2 CDRs are encoded by the germline.

q Like others before, we therefore constructed a highly diverse antibody library within a single variable-domain framework

mathematical model of antibody–antigen interaction

rationally designed CDRs

q Among these domains, the most highly enriched were the heavy chain VH1–69 and the λ-light chain VL1–44.

so some framework domains seem to work better in e coli than others

q This behavior of CDR amino acid sequences can be captured in a two-state hiddenMarkov model (HMM).

2 states are "contact" and "noncontact"

An important feature of HMMs is that the state of each position depends upon its nearest neighbor.

q The transformation efficiency of bacterial cells with plasmid DNA is a significant barrier to construction of molecular libraries with a complexity greater than ∼1010.

in vitro ribosome display

emerging cancer antigen poliovirus receptor-related 4 (PVRL4)

rationally designed human antibodies

q Finally, all CDR sequences were passed through a series of three filters to maximize their utility.

q 71% of CDR contacts are made by amino acids in these three CDRs

the sites near the framework were often non-contact sites there were blocks of contact sites contacts often consisted of tyrosine and tryptophan more proline in L3 and more glutamic acid in H3

Whereas the length of L3 sequences was fixed at 13 residues, 1,000 H3 sequences were randomly chosen for each length from 9 to 21 amino acids long.

type IIS restriction site (I'm not familiar with the different types of restriction sites)

They used the SapI restriction enzyme and noted that q The 3-nt 5′ SapI overhang on each sublibrary ensured that the H3 reading frames would remain intact.

H2 has special structural features they made an HMM to avoid changing structural a.a.

they removed certain restriction sites immunogenicity was decreased avoided features that would interfere with industrial scale production

they selected against the cancer antigen PVRL4

they used Illumina sequencing

they saw CDR recombination

q Synthetic biology has yet to deliver antibody production platforms that rival vertebrate immune systems in both product quality and manufacturing convenience (20).

wow they think we can outperform animal immune systems q However, we anticipate that along with the maturation of gene-synthesis technologies and the affordability of high-throughput DNA sequencing will also come advances in antibody production pipelines that outperform animal immune systems in all regards (21).

they just have to use a single set of primers, but they have reduced framework diversity

they think that normal heavy chain and light chain libraries require more steps to isolate the clone of interest and sequence it

q library vs.- library deconvolution strategies,

q not all of the scFvs predicted to bind PVRL4 were found to do so by FACS analysis.