Increased Fab thermoresistance via VH-targeted directed evolution

Presented by Shadman tariq sadiq

(91150000630) PhD study

Supervised by Proff.dr. serap EVRAN

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

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

AbstractAntibody 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

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 andshown 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.

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

- 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 reducedaggregation upon heating compared with wild-type Fab.

Materials and methods

1-To construct derivative Fabs containingthe 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.

3-Protein purification :the proteins purified by size exclusion chromatography (SEC) using an AKTA FPLC system (GE Healthcare) and a 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) .

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.

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).

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 .

- 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

-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.

-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.

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