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i l f i i f h iPrecise Control of Diversity for SyntheticPrecise Control of Diversity for SyntheticPrecise Control of Diversity for Synthetic A tib d Lib D i & C t tiAntibody Library Design & ConstructionAntibody Library Design & Constructiony y gChris Hebel1 Christoph Eicken1 Qi Zhu1 Xiaochuan Zhou1,2 Xiaolian Gao3Chris Hebel1, Christoph Eicken1 Qi Zhu1, Xiaochuan Zhou1,2, Xiaolian Gao3, 1LC Sciences and 2Atactic Technologies, Houston, TX 77054 , 3Dept of Biology and Biochemistry, University of Houston, Houston, TX 77004LC Sciences and Atactic Technologies, Houston, TX 77054 , Dept of Biology and Biochemistry, University of Houston, Houston, TX 77004
Electron microscope Synthetic antibody libraries have proven to be effective tools for drug discovery and development through the generation of functional, high-affinity antibodies against a wide varietyphoto of the Paraflo® Synthetic antibody libraries have proven to be effective tools for drug discovery and development through the generation of functional, high affinity antibodies against a wide variety
of antigens They are an expanding alternative to standard hybridoma technology especially for application to particularly difficult therapeutic challenges that cannot be addressedchip. of antigens. They are an expanding alternative to standard hybridoma technology especially for application to particularly difficult therapeutic challenges that cannot be addressed with antibodies from the natural repertoire The performance of a synthetic antibody library depends in large part on the diversity of the library which must be designed based onIt features: with antibodies from the natural repertoire. The performance of a synthetic antibody library depends in large part on the diversity of the library which must be designed based on th h d t di f th tib d t t d f ti F d di it id ffi i t th t tib d did t d i d f ti l f i• 10 µl total volume thorough understanding of the antibody structure and function. Focused diversity can provide an efficient path to antibody candidates designed for exceptional performance in • 10 µl total volume
4000 rxn chambersspecialized applications if precise control over design and construction is exercised. The use of degenerate oligos and other standard methods of diversity introduction lack this precise
• 4000 rxn chambersp pp p g g g y p
control and can introduce unwanted or useless codons into the library, thus limiting its performance. Fully designed library diversity is enabled through parallel in-situ (on-chip)• 270 pl/rxn chamber
control and can introduce unwanted or useless codons into the library, thus limiting its performance. Fully designed library diversity is enabled through parallel in situ (on chip) synthesis of tens of thousands of specific (non-degenerate) oligonucleotide sequences We demonstrate the bioinformatics-based design and high-throughput synthesis of a mutant
• uniform flow rate synthesis of tens of thousands of specific (non-degenerate) oligonucleotide sequences. We demonstrate the bioinformatics-based design and high-throughput synthesis of a mutant phage display library to improve affinity of anti ErbB2 single chain monoclonal antibody A21across the chip phage display library to improve affinity of anti-ErbB2 single chain monoclonal antibody A21.p
bl h l h h fl dfl ® fl d l h l f Programmable DLP Photolithography Microfluidic Reaction DevicesµParaflo® Microfluidics Oligo Synthesis Platform g g p yµ g y
Digital Light Projection drives light directed chemical reactions at specific sites in an array Three components are at the core of the µParaflo® Technology:
Mi fl idi hi (4K 30K
g a g oj o d g d d a a o a p a a ayformat and eliminates the need for expensive, inconvenient microfabricated photomasks. A
p µ gy
FluidReaction ChamberMicrofluidic chip (4K-30K
ti h b ) id
o a a d a d o p , o o ab a d p o o acomputer generates the digital mask and a Digital Light Projector (DLP) projects the light beam 1. Photogenerated reagent chemistry (PGR-chemistry). Cover GlassFluid
ChannelChamber
reaction chambers) provides l d t th t
o pu g a d g a a a d a g a g oj o ( ) p oj g b avery accurately into the micro reaction chambers where a photogenerated reagent is produced.2 Digital photolithography directed (programmable) synthesis
Channel
an enclosed system that f ilit t th f th
y a u a y o o a o a b a p o og a d ag p odu d2. Digital photolithography directed (programmable) synthesis.Distributionfacilitates the use of the
l ti h t h i t3. Microfluidic devices containing high density 3D-reaction chambers of pico-liter volumes.Distribution Channelsolution photochemistry,
t t if it
3. Microfluidic devices containing high density 3D reaction chambers of pico liter volumes. Channel
Ph t G t d A id (PGA) D t ti Ch i tpromotes spot uniformity
d d ibilit dPhoto Generated Acid (PGA) Deprotection Chemistry and reproducibility and h ki ti f Light Beam
( ) p yenhances kinetics for h i l th i ll
Light Beam
A novel solution photochemical approach employing chemical synthesis as well i bi di / t
Photogenerated R
p pp p y glight directed parallel synthesis and deprotection as various binding/capture Reagent
l kg p y p
with a photogenerated reagent enables high-yield assays. Uniform Flow Sequence input &
Digital mask Programmable lightp g g g y
parallel synthesis using standard DMT protected Si Substrate
Distribution Across all R ti Ch b
Sequence input & mask generation
projection Programmable light driven reactionsp y g p
phosphoramidites. Si Substrate Reaction Chambersmask generation driven reactionsp p
Other Methods Have Limitations TooLimits of Conventional Codon Randomization MethodsDesigned Library Diversity with Specific Oligo Sequences Other Methods Have Limitations TooLimits of Conventional Codon Randomization MethodsDesigned Library Diversity with Specific Oligo Sequences
Library Design & Diversity Amino Acid Codon Representations Using Different Randemization Synthesis Methods • TRIM – Trinucleotide Mutagenesis - The codon-based Library Design & DiversitySynthesis Units (3) NNG/T (4) NNC/G/T(1) NNN (2) NNC/T
uc eot de utage es s e codo basedtrinucleotide synthesis for building oligonucleotides provides
Diversity - the variability carried by the amino acid # of CodonAmino Acid 32 4864 32
t uc eot de sy t es s o bu d g o go uc eot des p o desspecific codon for each amino acid, but it is expensive to be of y y y
sequences of a synthetic antibody libraryAmino Acid
Codon Presence# Presence% Presence# Presence% Presence# Presence% Presence# Presence%
32 4864 32 spec c codo o eac a o ac d, but t s e pe s e to be oroutine use and suffers from biased or low efficiency coupling q y y y
Alanine(A) GCX 4 6.25 2 6.25 2 6.25 3 6.25Arginine(R) CGX, AGA/G 6 9.38 2 6.25 3 9.38 4 8.33
out e use a d su e s o b ased o o e c e cy coup gissues.
• Synthetic libraries are diversified by design – design is in-silico using one of many g ( ) ,
Asparagine(N) AAC/T 2 3.13 2 6.25 1 3.13 2 4.17Aspartic Acid(D) GAC/T 2 3 13 2 6 25 1 3 13 2 4 17
ssues
• S lit Mi S lit M th d Off b tt t l f i idy y g g g y
published algorithms. Naturally occurring antibodies provide guidance on how to limit Aspartic Acid(D) GAC/T 2 3.13 2 6.25 1 3.13 2 4.17
Cysteine(C) UGC/T 2 3.13 2 6.25 1 3.13 2 4.17• Split-Mix-Split Method – Offers better control of amino acid
di t ib ti d iti f d i d hp g y g p gsynthetic diversity. Glutamine(Q) CAA/G 2 3.13 0 0.00 1 3.13 1 2.08
Glutamic acid(E) GAA/G 2 3.13 0 0.00 1 3.13 1 2.08distribution and composition of randomized sequences, however, th i i ifi t l it d t ti l f h
y y• Diversity designs can be created in one or more of the light and/or heavy chain
Glutamic acid(E) GAA/G 2 3.13 0 0.00 1 3.13 1 2.08Glycine(G) GGX 4 6.25 2 6.25 2 6.25 3 6.25
Hi tidi (H) CAC/T 2 3 13 2 6 25 1 3 13 2 4 17
there is significant complexity and potential for human error as th i i i d lti l tiDiversity designs can be created in one or more of the light and/or heavy chain
variable domains (VH and VL) and by varying the length of the hypervariable loopsHistidine(H) CAC/T 2 3.13 2 6.25 1 3.13 2 4.17Isoleucine(I) ATA/C/T 3 4.69 2 6.25 1 3.13 2 4.17
synthesis resins are mixed multiple times.variable domains (VH and VL) and by varying the length of the hypervariable loops present in the variable domain (length diversity) to allow diverse structural
Leucine(L) CTX,TTA/G 6 9.38 2 6.25 3 9.38 4 8.33Lysine(K) AAA/G 2 3 13 0 0 00 1 3 13 1 2 08 • XYZ or Custom Mixtures of building blocks can combat codon present in the variable domain (length diversity) to allow diverse structural
conformations to form in this region and further increase the diversity of the libraryLysine(K) AAA/G 2 3.13 0 0.00 1 3.13 1 2.08
Methionine(M) ATG 1 1.56 0 0.00 1 3.13 1 2.08Ph l l i (F) TTC/T 2 3 13 2 6 25 1 3 13 2 4 17
o Custo tu es o bu d g b oc s ca co bat codobiases but also are limited by the potential for imprecise conformations to form in this region and further increase the diversity of the library.
• Often randomization or tailored randomization of oligonucleotide sequences isPhenylalnine(F) TTC/T 2 3.13 2 6.25 1 3.13 2 4.17
Proline(P) CCX 4 6.25 2 6.25 2 6.25 3 6.25
b ases but a so a e ted by t e pote t a o p ec semixing/handling.• Often randomization or tailored randomization of oligonucleotide sequences is
employed to introduce diversity
( )Serine(S) UCX,AGC/T 6 9.38 4 12.50 3 9.38 5 10.42
Threonine(T) ACX 4 6 25 2 6 25 2 6 25 3 6 25
g/ a d g
employed to introduce diversity.h bili i l d fi h fi l di i f lib f ili h
Threonine(T) ACX 4 6.25 2 6.25 2 6.25 3 6.25Tryptophan(W) UGG 1 1.56 0 0.00 1 3.13 1 2.08
• The ability to precisely define the final diversity of a library facilitates the f i l i h i i d i i i ib d l d
Tyrosine(Y) UAC/T 2 3.13 2 6.25 1 3.13 2 4.17Valine(V) GTX 4 6.25 2 6.25 2 6.25 3 6.25
Synthetic Antibody Library Construction Processprocess of isolating, characterizing, and optimizing an antibody lead.( )
Stop codon UAA/G,UGA 3 4.69 0 0.00 1 3.13 1 2.08 Synthetic Antibody Library Construction ProcessMax. 9.38 12.50 9.38 10.42Min 1 56 0 00 3 13 2 08Min. 1.56 0.00 3.13 2.08
in excess 6.0 3.0 5.0
Recombinant Monoclonal Antibody LibraryRecombinant Monoclonal Antibody Library
Li kL1 L2 L3N • NNN dLinkerL1 L2 L3N • NNN codon
• has six fold difference in amino acid representation. H3 H2 H1C
p
• the amount of the expressed full length sequences drops quickly due to stop codonH3 H2 H1C • the amount of the expressed full-length sequences drops quickly due to stop codon termination and codon-dependent low expressionAntigenic
ligand binding
termination and codon-dependent low expression.
CDRH1Sequence
ligand binding to surfaces of
CDRs• generates an impractically large library where almost all variants are inactive, reducing
th lik lih d f fi di i t ith d i bl tiSequence Variations
CDRs the likelihood of finding a variant with desirable properties.
• NNC/T codon is incomplete in representing amino acids (missing 5 of the 20 amino acids)./ p p g ( g )
• NNG/T and NNC/G/T codons overcome some limitations but still suffer from the difficulties• NNG/T and NNC/G/T codons overcome some limitations but still suffer from the difficulties of being inflexible in codon choices and sequence biasof being inflexible in codon choices and sequence bias.
• None of the above methods would allow deletion or insertions.
• None offer the flexibility in choosing specific codons for protein expression optimization.None offer the flexibility in choosing specific codons for protein expression optimization.
Example: Recombinant l l tib d libDesigned (Non-Random) Sequence Variations monoclonal antibody libraryDesigned (Non Random) Sequence Variations
Application Ex Directed Evolution for Increased Affinity Bioinformatics Based Library Design ResultsApplication Ex – Directed Evolution for Increased Affinity Bioinformatics Based Library Design Results
Bi i f ti b d d i d hi h th h t i fl idi th i f N t ll i tib di id id h t li it th ti di it ABioinformatics-based design and high-throughput microfluidic synthesis of Naturally occurring antibodies provide guidance on how to limit synthetic diversity. An l ith h l t d t i d t d P d d i t d d h
Several rounds of screening process
mutant phage display library to improve affinity of single chain antibody A21 algorithm helps us to determine advantageous codons. Pseudo-codons are introduced such th t th t t lib t i i bl bi ti f t ti (d t i d Scanning Liquid biotin antigen screening more than 103 clonesuta t p age d sp ay b a y to p o e a ty o s g e c a a t body that the mutant library contains every conceivable combination of mutations (determined t b d t ) t t h ti ll f f hil i i i i th
Scanning - Liquid biotin antigen screening, more than 103 clones
Anti ERBB2 Antibody A21to be advantageous) at a stochastically even frequency of occurrence while minimizing the i t d ti f d i d id d th i f tl b d i t l tib di
Greater than 200 clonesAnti-ERBB2 Antibody A21 introduction of undesired residues and those infrequently observed in natural antibodies.The next
( l ll d / d 8 ) 8 b d l h A21 CDR region Kabat classificationScreening - Screening to clone the soluble antibody and
phage antibodies by ELISAround of screeningERBB2 (also called HER2/neu and p185) is a 185 KDa trans-membrane epidermal growth
f ( ) f l h k
A21 CDR region Kabat classificationCDRs Sequence Class Single Mutation Double
MutationTotal phage antibodies by ELISA screening
factor receptor (EGFR) family protein with intrinsic tyrosine kinase activity. ERBB2 contains l l bl h h l d bl f h h
Positions Peptide Seq
Positions Peptide Seq
Positions Peptide Seq
~30-50 clonesSPR determination of wild-type and mutant single chain antibody affinity C2C10Bseveral plausible tyrosine phosphorylation sites and is capable of associating with other
h d f k l d h
q q q
L1 KSSQTLLYSNNQKNYLA Class 3 13 272 84 2372 97 2644A21scFv Western-Blotting or SPR Preliminary single-chain antibody affinity C2C10B
EGFR proteins to enhance a cascade of kinase activities in signal transduction pathways k b ll l f d/ ll h h
L2 WAFTRKS Class 1 7 42 24 5461 31 5503
g ycomparison of affinity
known to be active in cell proliferation and/or cell apoptosis. ERBB2 is shown to have up-l d l l h h b d
L3 QQYSNYPWT Class 1 8 72 28 6176 36 6248 ~20 clonesregulated expression in several malignant human tumors such as breast and ovarian
d l ll k h l dH1 GYSFTGYFIN Class 1 9 90 36 3219 45 3309
cancer compared to normal cells, making the protein an intensively pursued target as h d b k f ll d
H2 HISSSYATSTYNQKFKN Class 2 17 272 136 19362 153 19634CDR, L3 Sequence analysis
cancer therapeutics and as a biomarker for tumor cell detection. H3 SGNYEEYAMDY Length specified
11 110 55 20425 66 20535
5 10 clones
b d d h h b f ll hQ Q Y S N Y P W T
specified
Total 65 858 363 57015 428 57873~5-10 clones
Antibody A21 is a predominant growth inhibitor of cells over expressing the ERBB2 gene h f k d d d h h ff
Q QDetermination of the ka (1/Ms) kd (1/s) KD
with significant in vivo work done toward producing higher affinity mutants. However, b d ff f f l d b d d f h d Lib C t ti
relative affinity of eukaryotic expression
( ) ( )
SKN3 1.72e5 8.99e-5 5.21e-10antibody-affinity maturation in vivo often fails to produce antibody drugs of the targeted
k f h ff b l d d d dLibrary Construction eukaryotic expression
C2C10B 2.56e5 3.73e-6 1.45e-11
potency making further in vitro affinity maturation by computational design and directed l
yIt k i i !evolution necessary.
Oligonucleotide SynthesisIt works in vivo!It works in vitro!Oligonucleotide Synthesis It works in vitro!
Non-random
C l
Non random mutagenic oligos are synthesized on
Complex t l
are synthesized on a microfluidic chip
crystal
Synthesized oligos are cleaved from the Eukaryotic expression vector: chip and recovered in solution
y pmutant antibodies with human IgG Fc fusion protein
chA21 chA21 Epitopes
g p
chA21 chA21 Epitopes
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