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Second and third generation therapeutic and diagnostic proteinsDirected evolution of new proteins

Connect phenotype (typically a binding) to genotype(typically DNA)

DisplayerSize Number

Membrane display (Rice paper) High LowYeast surface displayE. coli surface displayPhage displayRibosome display Low High

Class 24 last updated 11/30/11 1:00 AM

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Nature Biotechnology  21, 163 - 170 (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface

display libraryMichael J. Feldhaus, et al. and K. Dane Wittrup

Screening for natural antibodies using yeast

Aga2 becomes associated with Aga1 in the cell.Aga1 becomes covalently anchored to the cell wall

C-myc

FACS

c-myc

yeast

3

B

μAv Bead-labeled cells retained on

magnetic column

First, purification to get numbers down to < 108 for FACS: magnetic (20 nm) microbeads

Av

Av

Biotin-

www.miltenyibiotec.com

Avidin-coated magnetic bead

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Created a general scFv library from 58 people using PCR primers from various classes of Igs.

Cloned in E. coli to maintain complexity of 109.

Cloned into yeast for surface expression maintaining complexity

Screen for antigen binding by 2-steps:

1) Too many cells for initial FACS, so: Magnetic bead capture (avidin micromagnetic beads [20 nm], bind via biotinylated antigen) isolate on column

2) FACS

Found Abs vs. many antigens tried:EGF, EGFR, p53 peptides, fluorescein, etc.

Kd ~ nM range, often.

c-m

yc la

bel

Ag label

EGF: epidermal growth factor

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Immunize mice spleen cells mRNA PCR VH and VL cDNAs reconstruct full length H&L chain genes in one plasmid. Clone 107. E. coli with ProteinA/memb. anchor fusion protein (“ZZ”) displayed outside inner membrane Lysozyme+EDTA spheroplasts add fluorescent antigen FACS clone E. coli cellsrescue gene

Fortunately,100 pM avidity,10x affinity. ZZ Divalency?

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Mazor, Y., Van Blarcom, T., Mabry, R., Iverson, B.L., and Georgiou, G. 2007. Nat Biotechnol 25: 563-565.

Outer memb.,permea-bilized

Inner memb.

Periplasm

Fluorescent antigenProteinA-E.coli

memb. anchor fusion protein

cytoplasm

Yields regular Abs, no fusion protein.

No hybridoma used

Fc

Vector: lac prom- Hconst-Hvar – Lconst.-Lvar, dicistronic

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PA = B. anthacis protective antigen

On time Off time

E. Coli carrying a gene for a membrane anchor – protein A fusion protein

ProteinA binds Fc of IgG

7Phage display for a human antibody

RT-PCR the rearranged VH and VL cDNAs

Reassemble full antibody molecules

8Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides

Knappik et al, and Andreas Pluckthun and Bernhard VirnekasJ. Mol. Biol., 296: 57-86 (2000)

CDR3 targeted hereVL VH

CDRs bkgds coloredFramework gray

CDR = complementarity-determining region

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Starting material here = HuCAL:

Fully synthetic human combinatorial antibody library based on modular consensus frameworks and CDRs randomized with trinucleotides.

These are single chain antibodies (scFv’s).

Consensus sequences of framework of 7 heavy chain V-regions X consensus sequences of framework of 7 light chain V-regions = 49 combinations

Plus: codon randomized CDR3’s by inserting synthetic oligos beween restriction sites. CDR3 = site of VJ (light chain) and VDJ (heavy chain) joinings.

Knappik et al, JMB, 296: 57-86 (2000)CDR = complementarity determining region

CDR3 20AA linker CDR3

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Found 95% of human Abs are represented by 7 VH and 7 VL exons

Constructed: Human Combinatorial Antibody Library (HuCAL) as scFv’s

E. coli (2 x 109 recombinants [!])Made all 49 (7x7) combinations of framework regions [but not represented equally]Mutagenized with triplets to put in all AA substitutions into a run of 6 spots in CDR3Simulated the AA frequencies found in natural Abs.

} cf.

11Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides

Knappik et al, and Andreas Pluckthun and Bernhard VirnekasJ. Mol. Biol., 296: 57-86 (2000)

CDR3 targeted hereVL VH

CDRs bkgds coloredFramework gray

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Figure 7. Comparison between design and experimental composition of CDR3 libraries used. For each position of the CDR3 region (numbering according to Kabat et al., 1991; for HCDR3 the position before H101 is numbered 100z, the length variable region is numbered from H95 to H100s), the amino acid composition in the planned libraries (P, left columns) is compared with the composition found from sequencing 257 clones of the initial libraries (F, right columns). The TRIM mixture indicates the mixtures of trinucleotides used in the oligonucleotide synthesis (see Table 3 of the Supplementary Material). Occupied indicates the number of amino acids encoded by the respective mixture and found in the sequenced clones, respectively.

Knappik et al, JMB, 296: 57-86 (2000)

Different amino acid combinations achieved after insertion of synthetic oligonucleotides.

Planned Found

AAs

There’s a similar table for VL

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Kd (nM) on time (t1/2) off time (t1/2)

From Biacore:

*

* From subsequent mutagenesis and ribosome diplay

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1

2

3

4

5

6

Nature Biotechnology (2000) 18:1287

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Start here:Library in DNA form

Primer must carry T7 promoter

via an E. coli S30 extract

Insulin (target here)

T7 pol.

,which is stalled

Insulin (target here)

B B= biotin

Avidin bead

Selecting anti-insulin antibodies from a naïve library

16Ribosome display

Absence of a stop codon is necessary to prevent dissociation of the newly synthesized protein from the ribosome.

Affinity selection in rabbit reticulocyte ribosome display is antigen-dependent and requires the absence of a stop codon to keep the protein-ribosome-mRNA complex together

In the presence of a stop codon, the protein synthetic complex dissociates, as stop codon recruits release factors.

RT-PCRproduct of affinity captured material

From Hanes et al., FEBS Letter, 450, 105-110, 1999)

biotin

antigen

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DNAT7 promoter 3’ AA spacer

Prokaryotic ribosome binding site

5’ stem-loop

3’ stem-loopscFv

scFv: single chain Fragment of variable chain (VH + VL)

DNA construct assembled by ligation and PCR: no cloning in E. coliHere: 2 x 109 molecules. Stem-loops protect from degradation.Spacer allows for AAs in ribosome tunnel.Later conservative estimate of no. of molecules that can be screened:2.6 × 1011 per ml of reaction (Nature Methods 4:269 (2007))

mRNA

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CDR3 targeted here

VL VH

CDRs bkgds coloredFramework gray

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Specific insulin binding activity (gray-white bars) was evident over a smaller amount of non-specific (or different-specific) binding, as assayed by binding of 35S-labeled translated protein in the translational complex.

0

20

40

60

80

100

120

0 50 100 150 200

nM cold insulin

cou

nts

per

min

ute

Specific

Non-specific

50-50 mix

B

dish

BB BB B B B

35S-labeled anti-insulin“Cold” (unlabeled) anti-insulin

Radioimmune assay (RIA)

Insulin

wellwell

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Plenty of framework mutations introduced by the PCR (i.e., not in the HuCAL library)

Varied in the

HuCAL library (CD3)

Length variations

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Only a 8 of the 49 possible framework combinations were used. Three were used most often. So there may be a preference for particular frameworks for this antigen.

7x7 grid

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“Consensus WT” HuCAL sequence(without the PCR mutations)All selected clones exhibit tighter binding. That is, the addiitonal mutations introduced by PCR were selected.

Entirely cell-free: no bottlenecks due to relatively inefficient DNA transfer steps. One is always dealing here with populations of molecules.

Eventually clone in a plasmid in E. coli for expression and testing.

23DNA shuffling (Stemmer - Maxygen)

(by PCR)

PCR (no oligo primers)

Base changes can be purposely introduced prior to shuffling• by chemical mutagenesis • by error-prone PCR during the process• by “faithful” PCR during the process (lower level of mutation)• by using different members of a gene family (paralogs or homologs)

Analogous functionsin the same organism;e.g. families of proteases

Same functionin a different organism; e.g., interferons

“recombination” via PCR priming

Stemmer, W., Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10747-51.                       

Fragment DNA with DNase

Reassemble fragments

Iterate

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Why alter a natural protein?

For therapeutic proteins:• Tighter target binding (enabling lower doses, perhaps)• Enable desirable side reactions• Disable undesirable side reactions•Improve half-life in bloodstream• Improve production characteristics (e.g., secretion, stability)

For industrial enzymes:• Altered (even new) substrate specificity• Greater stabilty • More robust (e.g., function at extreme pH’s)• Faster

From:Advances in directed protein evolution by recursive geneticrecombination: applications to therapeutic proteinsAaron L Kurtzman et al. Current Opinion in Biotechnology 2001, 12:361–370

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Example 1:

Human interferon alpha (IFNa ): induces cellular antiviral functions

20 IFNa genes in a gene familyEach human protein is poor at inducing virus resistance in mouse cells

Shuffle the 20 family genes, only 2 rounds, mixing the non-conserved a.a. differences

Select for induction of viral resistance in mouse cells

Achieve 185X more activity in a shuffled protein.

Best was even 3.5 X more active than the best mouse IFN.

And still equally effective vs. human cells.

So an example of a dramatic change in biological specificity (binding?)

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Shuffling an engineered human gene with a mouse gene

Boxed nts are not conserved. So lots of differences here. Low temp PCR was required due to the poor homology (Klenow instead of Taq)

Stemmer, W., Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10747-51.                       

27Example 2: Antibodies: breaking the natural limit on affinity selection

Natural affinity ceiling of 10-10 M (100 pM):Kd = off time / on time

Endocytosis rate ~~ 10 min to several hoursSo no selection for off –rate longer than ~<3 h (104 sec)Diffusion-limited on-rate ~ 10-6 M/s Selection limit of affinity of natural antibody evolution:Off rate 1/off time; on rate 1/on time Kd = off rate / on rate = (1/10+4) / (1/10-6)= 10-10 = Kd

---------------------------------------------------------------------------------------------------

J Foote and HN EisenKinetic and Affinity Limits on Antibodies Produced During Immune ResponsesPNAS 1995; 92: 1254-1256

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2x10-6 per second = 5 dayshalf-life

Iterations

Off

tim

e (t

1/2)

Antibodies from yeast scFv selection (Boder et al. and Wittrup, PNAS 2000)

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Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Eric T. Boder, Katarina S. Midelfort, and K. Dane Wittrup. Proc Natl Acad Sci U S A. 2000; 97(20): 10701–10705.

Used the FACS to selected single chain anti-fluorescein antibodies displayed on the surface of yeast cells. Competed with free fluorescein. DNA shuffled. 4 cycles. Selected for slow off times.

Achieved 50 fM affinities. That’s femtomolar, 50 x 10-15 M = 5 x 10-14 M = 0.05 pM (compare 100 pM above)Slower off-rate than even biotin-avidin (>5d).

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Selections other than Ag binding:

scMAb + DNA shuffling + ribosome display + selection in DTT (reducing agent). Ab folding without need for disulfide bond (-SH oxidation to –S-S- not needed), as well as high ligand affinity.

Lutz Jermutus, Annemarie Honegger, Falk Schwesinger, Jozef Hanes, and Andreas Pluckthun, PNAS 98:75-80 (2001)

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Example 3: Enzyme stabilization:

PAI-1, a protease inhibitor (TPA inhibitor)

Error prone DNA shuffling 245X increase in stability (days)Assay = tPA bindingFound 11 aa changes, presumably affecting protein folding

Back-cross to remove non-contributory mutations:DNA shuffle best clone with original WT DNA.Maintain selective pressure.Analyze “progeny”: see 2 of the 11 aa changes lost, not needed, replaced by WT sequence.

J Mol Biol. 2001 Jan 26;305(4):773-83.Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during

latency transition and proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries.

Stoop AA, Eldering E, Dafforn TR, Read RJ, Pannekoek H.

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Example 4: Viral tropism: murine leukemia virus, a retrovirus

MLV, 6 strains, all poor infection of CHO cells.

DNA shuffled envelope gene of 6 strains chimeric virus that can infect CHO cells

And selected incidentally for resistance to inactivation under conditions of laboratory manipulation (100X centrifugation resistant)

Nat Genet. 2000 Aug;25(4):436-9.Molecular breeding of viruses.

Soong NW, Nomura L, Pekrun K, Reed M, Sheppard L, Dawes G, Stemmer WP.

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Zhang et al., Nature 415, 644 (2002)Can mix Streptomyces genomes by protoplast fusion. effective diploid bacteria

The fused cells will generate recombinant haploid spores.

Target: tylosin production (an antibiotic)

Mutagenize a culture, collect 22,000 survivors.

Screen them all for tylosin synthesis, pick the top 11.

Protoplast fuse all 11 with each other. Collect 1000 progeny.

Screen 100 for tylosin, collect the best 7.

Protoplast fuse again. Collect 1000 again. Screen for tylosin again.

Characterize the best 2: Tylosin production is up 9-fold.

So productivity is up 9-fold, without a tremendous amount of work (22000 screen max)

A more global version of DNA -- genome shuffling: A different (unnatural) kind of genetic mixing using whole genomes

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Strategy: Create therapeutic proteins by combining hundreds of known binding domains from receptor proteins in new mutated random combinations and selecting for binding to a specific target by phage display.

A more supervised version of DNA shuffling

“Multivalent avimer proteins evolved by “exon” shuffling of a family of human receptor domains”

Nature Biotechnology 23: 1556 (2005)

Joshua Silverman, et al & Willem Pim C Stemmer

Avidia, Inc

Avimers = ligand binding proteins other than antibodies, based on recptor domains.

A misnomer; really domain shuffling)

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Typical receptor structures

of 217 A-domains

A-domains:(~35-40 AA’s/domain):determine binding specificity of many receptors

as a spacer between domains

(~metaphorically?)

Organization of binding domains in typical mammalian receptors

Degenerate oligos synthesized to coding for 35-40 AAs of the A domainsOnly AA’s naturally found at each position were coded for.Conserved structural AAs were kept constant (6 cys and 4 Ca binders + 2 others).Complexity = 1023 . Actually realized = 1010 as phage display particlesSelect one domain at a time, serially, by panning:

LRP = LDL receptor related protein; VLDLR =very low density lipoprotein receptor

2 domains cooperating

Bipartite domain

Dual specificity domain

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Monomer displayedon phage coat

Monomer protein Screened for binding

Build 20 phage dimer poolsfrom 20 best monomers

M13 phage

Isolation of a high affinity binding protein to IL6 ( interleukin 6 ) by iterative selection (IL6 is a target for cancer and inflammation).Phage display (M13).IL6 immobilized on plates.

Recovered phage from first cycle, cloned and tested for IL6 binding; 20 top binders pursued.

Added the domain library to each of the 20 first round winning domains. Again pick best 20 overall. After a third cycle pick the very best binder: = “C326”

IL6 = interleukin 6

One domain

Two domains

Three domains

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Add an IgG-binding domain at the end to prevent rapid clearance(measured half-life of 89 hours in monkeys)

Model structure

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     Activity of the anti-IL-6 tetramer C326. (a) AlphaScreen competition analysis comparing ability of C326 relative to IL-6 itself in inhibiting the interaction of IL-6 with its receptors. An avimer that does not bind to IL-6 is included as a negative control.

LaserReactive Oxygen

Luminescence

Binding measured by a competition assay (“AlphaScreen”)

Reactive oxygen species can react only over a short distance with and “acceptor” bead

IL6IL6 receptor

Avidin bead:biotinylated IL6 + gp130-Fc:Protein A beadCompetition: IL6 (non-biotinylated) or C326 avimer (10X tighter)

Laser

gp130 = natural IL6 receptor

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More AlphaScreens: effect of combining the 3 domains

20 pM

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Physical assay: Biacore surface plasmon resonance to measure binding kinetics

253 pM

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Biological assay:Stimulation of proliferation of TF-1 cells (erythroleukemia line)16 h of 3H-TdR incorporation to measure promotion of DNA synthesis

Commercial anti-IL6 antibodies

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C426 cMet 2 <0.1b 0.170c Actived

C65 CD40L 2 <0.1e 0.06c 0.1f

C326 IL-6 3 <0.2e 0.05c 0.0008g

C2810 CD28 3 0.1e n.d.h 0.6f

C2 BAFF 3 n.d.h 0.1c 0.4f

Table 1 Selected avimer affinities and activitiesa

Avimer TargetNo. of

domainsAffinity (nM)

IC50

(Biochemical) (nM)

IC50 (Biological) (nM)

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Acute phase inflammatory response induced by IL6 is reversed by avimer C326(in mice)

Specific for IL6-induced inflammation