1. Increased Fab thermoresistance via
VH-targeted directed evolution
Presented by
Shadman tariq sadiq
(91150000630)
PhD study
Supervised by
Proff.dr. serap EVRAN

2. Structure of Immunoglobulins
Antibody (or immunoglobulin) molecules are
glycoproteins composed of one or more units,
each containing four polypeptide chains: two
identical heavy chains (H) and two identical light
chains (L). The amino terminal ends of the
polypeptide chains show considerable variation in
amino acid composition and are referred to as
the variable (V) regions to distinguish them from
the relatively constant (C) regions. Each L chain
consists of one variable domain VL and one
constant domain CL. The H chains consist of a
variable domain, VH, and three constant domains
CH1, CH2, CH3

4. Directed evolution is a mimic of the
natural evolution cycle in a laboratory
setting.

5. Abstract
Antibody aggregation is frequently mediated
by the complementarity determining regions
within the variable domains and can
significantly decrease purification yields,
shorten shelf-life and increase the risk of
anti-drug immune responses. Aggregation-
resistant antibodies could offset these risks;
accordingly, we have developed a directed
evolution strategy to improve Fab stability

6. the process of work
1- A Fab-phage display vector was constructed
and the VH domain targeted for mutagenesis by
error-prone PCR.
2-Five unique variants were identified, each
possessing one to three amino acid substitutions.
Select mutations were combined and
shown to confer additive improvements to these
biophysical characteristics.
3-Finally, the wild-type and most stable triple
variant Fab variant were converted into a human
IgG1 and expressed in mammalian cells.

7. Introduction
The therapeutic monoclonal antibody market is
estimated to grow to nearly $58 billion in 2016.
multiple approved antibody therapeutics have been
developed using the same scaffold, One approach used
to confer antibody resistance to aggregation employed
heat denaturation of a single-domain antibody when
fused to the surface of the M13 phage, followed by
selection of correctly folded variants using a
conformation-specific binding partner .
This approach was subsequently used to improve the
thermoresistance of both human

8. - Next, a VH-targeted library was created by
error-prone PCR, displayed on phage and
transiently heated followed by selection for
binding to a CL-binding antibody. After four
rounds of panning,
five unique variants were identified and
characterized in phage display and soluble
formats.
- All exhibited higher activity levels and
reduced
aggregation upon heating compared
with wild-type Fab.

9. Materials and methods
1-To construct derivative Fabs containing
the thermoresistance mutations, Kunkel
mutagenesis was performed using a modified
annealing step and three 26 bp oligonucleotides
targeting each region for mutagenesis.
2-To create the Fab phage display vector, a
multiple cloning site was first added at the C-
terminus of the CH domain using standard
QuikChange mutagenesis.

10. 3-Protein purification :the proteins purified by size
exclusion chromatography (SEC) using an
Superdex S75 (GE Healthcare) column with a
phosphate-buffered saline.
4-Soluble Fab biophysical measurements and
ELISA Solubility Fabs was measured by
concentration using a single 10 kDa MWCO
amicon filter to 5–26 mg/ml, incubation for 4
days at 4°C, centrifugation for 10 min at high
speed (16 000 rcf) to pellet insoluble
aggregates and measurement of the remaining
soluble protein using a BCA assay
( bicinchoninic acid assay) .

11. Thermal stability was measured using a fluorescent
assay as described previously . Briefly, 0.1–0.5 mg
protein or a PBS buffer blank was assayed using the
Protein Thermal Shift Dye Kit (Applied Biosystems)
according to the manufacturer’s instructions.
Also Fabs tested for peptide binding by ELISA.

12. Results
- Resistance to thermally induced inactivation has been
shown to correlate with improved expression level and
increased solubility.
-To measure thermoresistance of the Fab, we next
performed an activity ELISA. Purified αEE Fab was
incubated at room temperature or heated to a single
temperature, ranging from 48 to 65°C for 1 h, cooled and
the remaining EE peptide-binding activity measured by
ELISA (fig 1).

13. Activity remaining after thermal stress. Purified Fab protein was heated for 10 min
at the indicated temperature, ranging from 25 to 65°C, cooled and binding to the
EE peptide assessed by ELISA. Incubation at 55°C results in 50% activity
remaining after cooling .

14. - To perform directed evolution using a VH-
targeted library, we first constructed a
vector for phage-based selection. A C-
terminal FLAG (is a polypeptide
protein tag that can be added to a
protein using recombinant DNA
technology) peptide was removed to
prevent interference with use in other
applications resulting in the vector pFabF ,
which was used for soluble expression

15. -To identify thermoresistant clones, monoclonal
phage were produced from 364 randomly
selected colonies from the output population for
rounds 3 and 4. Clones were screened by
monoclonal phage ELISA under three conditions:
replicate phage were incubated for 10 min at 25,
58 or 68°C, cooled and assessed for binding to
coated or uncoated control plate to assess
nonspecific binding.

16. -Results of Biophysical and antigen binding
properties of selected variants
We next wanted to determine whether increased
stability was also present in soluble Fabs containing
these amino acid changes. Phage Fab clones E1–E5
were transferred to the soluble Fab expression vector
and purified using the optimized procedure detailed
above. All engineered variants expressed better than
wild-type, an additional increase of up to 1.2 mg/l
culture.

17. Thanks for listening