Overview

The objective of this wet lab experiment is to synthesize the EGFR (Epidermal Growth Factor Receptor) cyclic peptide using the designed BioBrick parts. This synthesis will enable the subsequent bispecific antibody production.

Plasmid preparation

Plasmid synthesis was carried out by using Xbal and HindIII restriction sites. Initially, the pET-24d(+) vector and Ebp-intein vector were individually digested with restriction enzymes, and the resulting fragments were subsequently purified using gel electrophoresis. DNA fragments were extracted using a mini column. The desired product was obtained by ligating the two vectors together using T4 DNA Ligase. This stage involved the following experiments:

1.

Restriction digestion of pET-24d(+) vector and cloning vectors carrying synthesized inserts with XbaI and HindIII to generate inserts with compatible ends

2.

DNA gel electrophoresis of restriction digested inserts

3.

Gel purification of restriction digested insert

4.

Ligation of inserts and pET-24d(+)

Plasmid amplification and sequencing

Plasmid amplification was done using TOP10 competent cells. Initially, the intact plasmid was introduced into the TOP10 cells via heat shock method, followed by plating on agar plates to allow colony growth. Multiple colonies were randomly selected, and the obtained products were subjected to sequencing to verify the successful insertion of the target fragment and to identify any potential mutational events within the plasmid. This process aimed to confirm the availability and reliability of the plasmid. This stage involved the following experiments:

5.

Transformation of ligation product into TOP10 or BL21(DE3) competent cells

6.

Colony PCR

7.

Recombinant plasmid purification by miniprep

Peptide/protein expression

BL21(DE3) competent cells were chosen as the host for protein expression. Initially, the purified plasmid was transformed into BL21(DE3) cells and spread onto an agar plate. Selected colonies were subsequently transferred to LB broth and cultured overnight. Upon reaching an optical density (O.D.) of 0.6, protein expression was induced by adding 0.2 mM of IPTG (isopropyl β-D-1-thiogalactopyranoside). The target protein was subsequently extracted using a lysis buffer together with sonication. Protein expression levels were evaluated using SDS polyacrylamide gel electrophoresis analysis (SDS-PAGE).

8.

Protein expression

9.

Polyacrylamide Gel Electrophoresis analysis to check expression level

Peptide/protein purification

The expressed proteins were made up of CBD and intein, so a chitin column was used for purification. The purification intermediates (EGFR peptide with chitin) were extracted from cell lysates and subjected to purification using the chitin column. Through elution, the intein1 portion was detached after incubation overnight in B2 solution. Subsequently, after incubation overnight in B4 solution, the target cyclic peptide chain was selectively released from the chitin column and cyclized.

10.

Protein purification by Chitin column

11.

Liquid Chromatography Mass spectrometry analysis

Conjugation of chemically synthesized cyclic peptide to antibody

Bifunctional linker was used to cyclize and conjugate cyclic EGFR-targeting peptides via phthalaldehyde-amine capture reaction onto anti-c-MET monoclonal antibody to generate the c-MET x EGFR peptidic bispecific antibody, Polyneerab.

12.

Conjugation of chemically synthesized cyclic peptide to antibody

Functional assays of the BsAb

The Osimertinib resistance of HCC827 cells was confirmed by MTT cytotoxicity assay. The BsAb binding affinity against EGFR and c-MET targets was evaluated by ELISA assay, respectively. Finally, the response of Osimertinib resistant HCC827 towards the BsAb was determined by MTT cytotoxicity assay.

13.

Enzyme linked Immunosorbent assay (ELISA)

14.

MTT Assay

15.

Bliss Analysis

Restriction digestion of pET-24d(+) vector and cloning vectors carrying synthesized inserts with XbaI and HindIII to generate inserts with compatible ends

Materials

Restriction Enzyme - XbaI (10 U/µL) (Thermo Scientific #ER0681)

Restriction Enzyme - HindIII (10 U/µL) (Thermo Scientific #ER0505)

10X NEBuffer (New England BioLabs #B7002S)

Gene Synthesized Inserts

Procedures

1.

Set up the restriction digestion reaction as follow:

pET-24d(+) vector / Ebp-intein vector1 µg
XbaI (10 units/ µL)0.5 µL
HindIII (10 units/ µL0.5 µL
10 X NEBuffer5 µL
ddH2O34 µL
Total volume50 µL
Table 1
2.

Incubate the reaction at 37°C for 3 hours.

3.

Inactivate the enzymes by heating at 65°C for 20 minutes.

Size of restriction digested fragments

Plasmid backbone:

●

pET-24d(+): 5249bp

Inserts:

●

Ebp-SspMxe: 1539bp

●

scrb-SspMxe: 1539bp

●

Ebp-NpuSsrA: 978bp

●

scrb-NpuSsrA: 978bp

●

eGFP-NpuSsrA: 1659bp

DNA gel electrophoresis of restriction digested vector

Materials

●

Agarose (Invitrogen #16500500)

●

1X TAE buffer (Thermo Scientfic #B49)

●

6X DNA loading dye (Thermo Scientfic #R0611)

●

GeneRuler 1 kb DNA Ladder (Thermo Scientific #SM0311)

●

10000X GelRed Nucleic Acid Stain (Millipore #SCT123)

Procedures

1.

Place the pre-prepared 0.8% (w/v) agarose gel containing GelRed stain (5 µL of GelRed stain to 50 mL of agarose gel) into the gel tank.

2.

Fill the gel running tank with the gel running buffer (1X TAE) until it just covers the gel surface.

3.

Spin down briefly (10 seconds) the double restriction digested cloning vector tube.

4.

Add 10 µL of loading dye (6X) to the digestion reaction. Mix gently and spin down.

5.

Carefully load ALL the reaction mixture into the wells of the gel.

6.

Load 5 µL of the 1 kb DNA size marker to a lane.

7.

Place the lid onto the gel tank. Connect the electrodes to the power supply.

8.

Perform electrophoresis at about 150V (constant voltage) for 30 minutes.

9.

Run until the dye front has moved about half of the way down the gel.

10.

Switch off powe. Carefully lift the gel tray and place the gel onto a UV light box.

11.

Make sure the long UV wavelength is selected. Make sure the UV shield is properly placed to block UV from your eyes and skin.

12.

Switch on the UV light box. Observe the gel pattern and then cut out the gel pieces that contain the DNA fragments you want.

Gel purification of restriction digested products

Materials

●

Agarose (Invitrogen #16500500)

●

Gel Extraction Kit (Promega #A9281)

Procedures

1.

Measure the weight of 1.5 mL microcentrifuge tubes. Label sample name and weight of tube clearly on each tube.

2.

With a pair of cover slip, cut out the slice(s) of agarose gel containing the DNA band(s) of interest (vector: ~5.3 kb, Inserts: ~1kb - 1.5kb) Warning: Wear UV protective goggles or UV face shield!

3.

Calculate the net weight of agarose.

4.

Follow the DNA extraction procedure below:

Dissolving the Gel Slice

1.

Add 10 μL Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65°C until the gel slice is completely dissolved.

Binding of DNA

1.

Insert SV Minicolumn into Collection Tube. Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.

2.

Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

Washing

1.

Add 700 μL Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

2.

Repeat Step 3 with 500 μL Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.

3.

Empty the Collection Tube and centrifuge the column assembly again for 1 minute.

Elution

1.

Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube. Do not let the Minicolumn touch the solution inside the Collection Tube.

2.

dd 30 μL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.

3.

Discard Minicolumn and store DNA at -20°C.

Ligation of Ebp-intein gene and pET-24d(+)

Materials

●

T4 DNA Ligase (New England BioLabs #M0202S)

●

10X T4 DNA Ligase Reaction Buffer (New England BioLabs #B0202S)

●

milli-Q H2O

●

PCR tubes

Procedure

1.

Before ligation, measure the concentrations of the gene insert and the cloning vector eluates by nanodrop.

2.

Setup the ligation reactions in a PCR tube as follow:

Tube number (molar ratio of vector to insert)1:31:1
Add milli-Q H2O to a final volume of 9.5 µLUp to 9.5 µLUp to 9.5 µL
10X Ligation buffer 1 µL1 µL
Double restriction digested cloning vector50 ng50 ng
Then adding each tube
T4 DNA Ligase0.5 µL0.5 µL
Table 2

*add all the components to the bottom of the tube in the following order: 1) Water 2) Buffer 3) DNA 4) Enzyme

3.

Mix briefly and gently.

4.

Spin down all contents to the bottom of the tube.

5.

Incubate at room temperature (25°C) for 3 hr.

6.

Store at -20°C until perform transformation next time.

Transformation of ligation product into TOP10 OR BL21(DE3) competent cells

Materials

●

TOP10 Competent Cells (Invitrogen #C404010)

●

Ice bucket

●

42C water bath

●

LB medium

●

Kanamycin antibiotics

●

LB agar plates (Kan)

Procedure

1.

Set up 3 transformation tubes as follow:

Tube numberAmount
1Ligation reaction tube number 1:310 µL
2Ligation reaction tube number 1:110 µL
VecVector plasmid (+ve control)1 µL
Table 3
2.

Thaw the competent cells on ice. Keep them on ice whenever its possible before the heat shock.

3.

Add 10 mL of the ligation mixture and 1uL vector to each 100 µL of the competent cell suspension.

4.

Mix the reaction by stirring gently with the pipette tips and store on ice for at least 10 minutes. Do not pipette up and down!

5.

Transfer the tube to a 42C water bath for exactly 45 seconds (very important!).Warning: Do not shake the tubes.

6.

Immediately transfer the tube to the ice bath.

7.

Allow the cells to chill for 2 minutes.

8.

Add 900 mL of LB medium (antibiotic-free) to the tube.

9.

Use tape to secure the tube cap and incubate at 37°C while shaking for 45 minutes.

10.

Label three LB agar plates (Kan) with sample name clearly.

11.

Take out 100 mL of the bacterial culture from each tube and add onto the agar plates, respectively.

12.

Use a sterile spreader to spread them out evenly on the agar plate.

13.

Let it absorb into the agar for about 5 minutes.

14.

Turn the plate over, pack your 3 plates together with tape and incubate the plates at 37°C overnight.

Colony PCR

Materials

●

2X PCR master mix (Thermo Scientific #K0171)

●

10 µM T7 universal forward primer- 5’-GCTAGTTATTGCTCAGCGG-3’ (BGI Genomics)

●

10 µM T7 universal reverse primer - 5’-TAATACGACTCACTATAGGG-3’ (BGI Genomics)

Procedure

1.

Setup the PCR reaction in a PCR tube as follow (1 reaction for 1 colony):

Tube number (molar ratio of vector to insert)
Add milli-Q H2O to a final volume of 10 µLUp to 9.5 µL
PCR Master Mix (2X)5 µL
Forward primer (1 µM)1 µL
Reverse primer (1 µM)1 µL
Table 5
2.

Pick a single colony with a sterile pipette tip and swirl in a small amount of LB medium with antibiotics. Pick 5-10 colonies in total to test.

3.

Swirl the pipette tip in the PCR reaction.

4.

Briefly centrifuge the PCR tube and Perform the PCR using the following conditions:

StepTemperature (°C)TimeNumber of cycles
Initial denaturation952 min1
29530s35
Annealing5030 s35
Extension721 min 30 s35
Annealing7210 min1
Table 6
5.

Run the samples following the Gel Electrophoresis protocol. Calculate the size of the DNA bands.

Recombinant plasmid purification by mini-Prep

Materials

●

TIANprep Mini Plasmid Kit (TIANGEN #4992423)

Procedure

Purify the recombinant plasmids from the liquid cultures according to the following:

1.

Harvest the bacterial cells by centrifugation at 7,000 rpm in a bench-top centrifuge for 5 min (or 4,000 rpm for 15 min) at room temperature, remove supernatant.

2.

Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μL Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube.

3.

Re-suspend the bacterial pellet in 250 μL Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.

4.

Transfer the resuspended cell to a new 1.5mL microcentrifuge tube. Add 250 μL Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times. Note: Do not vortex!! If necessary, continue inverting the tube until the solution becomes viscous and slightly clear, but less than 5 min.

5.

Add 350 μL Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.

6.

Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.

7.

Wash the Spin Column CP3 by adding 600 μL Buffer PW (ethanol (96%-100%) has been added) and centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube. Repeat Step 7 one more time.

8.

Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.

9.

Place the Spin Column CP3 in a clean labeled 1.5 mL microcentrifuge tube.To elute DNA, add 30 μL Buffer EB to the center of the Spin Column CP3,incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).

10.

Send the plasmids to sequencing company and check the sequencing result.

Protein expression

Materials

●

LB medium

●

Kanamycin antibiotics

●

Spectrophotometer

●

100 mM IPDG

Conditions:

Temp4h6hOvernight
25°C
30°C
1.

Inoculate 0.3 mL of starter culture into a 10 mL sterile LB medium containing antibiotics.

2.

Incubate at 37°C with shaking (~250 rpm).

3.

Setting the BLANK of the spectrophotometer using the LB medium. Every 30 min, take out the broth culture from the shaker and aseptically transfer 0.1ml to a clean cuvette containing 0.9 mL LB. Pipette up and down to mix well. Avoid bubbles. Determine its O.D.. Put the 10ml culture back to the shaker at 37°C and keep shaking until O.D.600 reaches 0.06 (after dilution).

4.

Separate the culture into two falcon tubes (each one has 5 mL now). Name one tube as “uninduced (UI)” and another tube as “induced (I)”.

5.

Induced with 0.2 mM IPTG (stock concentration: 100mM) in the “(I)” tube.

6.

Express protein for overnight at 25°C or 30°C. Aliquot samples at different times (4h, 6h, overnight) for gel electrophoresis.

7.

Centrifuge the cells at 7000 rpm for 15 min at 4°C. Discard supernatant. Store the cell pellets at -80°C overnight.

Polyacrylamide Gel Electrophoresis analysis to check expression level

Materials

●

TGX Stain-Free™ FastCast™ Acrylamide Kit (Biorad # 1610180)

●

Precision Plus Protein Unstained Protein Standards (Biorad #1610363)

●

4X SDS Sample Buffer (Milipore #70607)

●

10X SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) (Homemade)

●

10% ammonium persulfate (Homemade)

●

Tetramethylethylenediamine (Thermo Scientific #17919)

●

Staining solution (0.025% Coomassie dye in 10% acetic acid) (Homemade)

●

De-stain solution (10% acetic acid) (Homemade)

1.

Prepare resolving gel acrylamide solution by combining equal volumes of resolver A and B solutions in a 50 mL tube

1 GelStacker tubeResolver tube
Resolver A/2 ml
Resolver B/2ml
Stacker A1 ml/
Stacker B1 ml/
TEMED2 µL2 µL
10% APS10 µL20 µL
2.

Add the required volume of TEMED and freshly made 10% APS to the combined resolver solution and mix well. Use an appropriate pipet to steadily dispense the solution into the cassette. Do not let bubbles form or solution mix with air. Fill the cassette to 1 cm below the bottom of the teeth on the comb. Immediately prepare and pour the stacking solution as directed in the next steps.

3.

Prepare stacking gel acrylamide solution by combining equal volumes of stacker A and B solutions

4.

Add required volume of TEMED and freshly made 10% APS to the combined stacker solution and mix well. Pipet solution down the middle of the cassette, filling to the top of the short plates. Apply slowly and steadily to prevent mixing with the resolving solution. Align and insert the comb in the cassette carefully to prevent air from being trapped under the comb teeth. Allow the gel to polymerize for 30-45 min before electrophoresis.

5.

The cast gels can be covered with a paper towel wetted with deionized water and stored in a zipper sealed plastic bag at 4°C for up to 4 weeks.

Prepare 1X Buffer

Add 100 mL 10X SDS Running Buffer to 900 mL deionized water to prepare 1X Buffer.

Load Sample

Add 4 µL of SDS Sample Buffer to 12 µL of sample. Boil at 95°C for 10 min. Load 16 µL of protein sample on the gel.

Load Buffer

Fill the Buffer Chamber with 800 mL of 1X Running Buffer.

Run Condition

Set 60V on the power supply and run the electrophoresis for stacking gel, then increase to 100-120V for separating gel until the dye front reaches the bottom of the gel.

Gel Staining

1.

Stain the gel with 0.025% Coomassie dye in 10% acetic acid for 15 min. Gels can be maintained in a staining solution for several days.

2.

De-stain the gel twice in 10% acetic acid. Each incubation should last 15 min.

3.

Transfer the gel to water

Protein purification by Chitin column

Materials

●

Lysis buffer (20mM Tris-HCl, 500 mM NaCl, 1mM EDTA, 0.1% Tween-20, 20 μM PMSF, pH 8.5) (Homemade)

●

Chitin Resin (New England Biolabs #S6651S)

●

2-mercaptoethanesulfonic acid (Sigma Aldrich #63707)

●

EDTA (Sigma Aldrich #03609)

●

Tris (Cytiva #GE17-1321-01)

●

NaCl ((Sigma Aldrich #S9888)

●

Pierce centrifuge columns (Thermo Scientific #PI89898)

Buffer preparation

Buffer B1

●

20 mM Tris-HCl, pH 8.5

●

500 mM NaCl (or 50-1000 mM NaCl)

●

1 mM EDTA

Buffer B2

●

20 mM Tris-HCl, pH 6.5

●

500 mM NaCl (or 50-1000 mM NaCl)

●

1 mM EDTA

Buffer B4

●

20 mM Tris-HCl, pH 8.5

●

500 mM NaCl (or 50-1000 mM NaCl)

●

50 mM 2-mercaptoethanesulfonic acid

●

1 mM EDTA

Cell lysis by sonication

1.

Thaw the cell pellets from the previous expression experiment. Cell pellets from a one-liter culture should be resuspended in at least 100 ml of ice-cold cell lysis buffer.

2.

Transfer the cell suspension to 50 mL falcon tubes.

3.

Sonicate the cell suspension at 70% amplitude with 30s ON and 30s OFF pulse, for 20 minutes at 4°C water bath. Note: An increase in viscosity indicates when the cells are broken. If the mixture becomes extremely viscous, it may be necessary to dilute the cell lysate and/or add 10 μg/ml of protease-free DNase plus MgCl2 (to 5 mM final concentration) to reduce viscosity.

4.

Centrifuge the lysate at 7500 rpm for 20 minutes to separate soluble proteins from insoluble proteins.

Preparation of Chitin Column

The chitin-binding domain(s) (CBD) present in the intein-tag(s) allows affinity purification of the fusion protein. 1 mL of chitin resin was loaded to a 10 mL Pierce centrifuge column. 10 mL of Buffer B1 was added to equilibrate the column. Centrifuge at 100 x g for 1 minute. The flowthrough was discarded.

Loading the Clarified Cell Extract

10 mL of soluble fraction of cell lysate was added to the column. The column was incubated at 4°C with gentle shaking for 1 hr. It was then centrifuged at 100 x g for 1 minute.

Washing unbound proteins

10 mL of Buffer B1 was added to the column. The tube was inverted 5 times for mixing. It was then centrifuged at 100 x g, 4°C for 1 minute. This step was repeated 2 more times.

Induction of On-column Cleavage of Intein 1n

10 mL of Buffer B2 was added to the column. The column was incubated at room temperature with gentle shaking overnight. It was then centrifuged at 100 x g for 1 minute.

Washing unbound proteins

10 mL of Buffer B1 was added to the column. The tube was inverted 5 times for mixing. It was then centrifuged at 100 x g, 4°C for 1 minute. This step was repeated 2 more times.

Induction of On-column Cleavage of Intein 2

2 mL of Buffer B4 was added to the column. The column was incubated at room temperature with gentle shaking overnight.

Elution of the Target Protein

The target protein was eluted by centrifugation at 100 x g, 4°C for 1 minute.

Liquid Chromatography-Electrospray Ionization-Mass spectrometry analysis

Materials

●

C4 LC column (Thermo Scientific #25502-052130)

●

4% formic acid (Sigma Aldrich #27001)

●

Acetonitrile (Acetonitrile Sigma Aldrich #34851)

●

MilliQ water

Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS) experiments were performed with an Agilent 6540 QTOF mass spectrometer coupled with an Agilent 1290 Infinity UHPLC system. Elution fraction from chitin column was injected into a C4 LC column and eluted with a linear gradient from 98% solvent A: 2% solvent B to 2% solvent A: 98% solvent B, where solvent A was milliQ water and solvent B was acetonitrile, each with 0.1% formic acid. The mass spectrometer was operated in a positive ion mode. ESI-MS data was acquired with a m/z range of 600-1600, from which multiply-charged mass spectra were obtained. The multiply charged mass spectra were deconvoluted by the MassHunter BioConfirm program to obtain the molecular mass of proteins. The calculated molecular mass of cyclic eGFP was 18 Da less than that of linear eGFP (26941 Da), resulting in a mass of 26923 Da.

Liquid Chromatography-Electrospray Ionization-Mass spectrometry analysis

Materials

●

Resin Preparation: Swell the resin in an appropriate solvent in DCM) for 30 min.

●

Peptide Chain Assembly: For each cycle of peptide elongation, repeat the following steps:

a. Fmoc Deprotection:

Treat the resin with a solution of piperidine in DMF to remove the Fmoc group.

Wash the resin thoroughly with DMF.

b. Amino Acid Coupling:

Prepare the activated amino acid solution: Dissolve the Fmoc-protected amino acid, activation reagent, and an organic base in DMF.

Add the activated amino acid solution to the resin and allow the coupling to proceed.

Wash the resin thoroughly with DMF.

Repeat steps a and b until the desired peptide sequence is obtained.

Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS) experiments were performed with an Agilent 6540 QTOF mass spectrometer coupled with an Agilent 1290 Infinity UHPLC system. Elution fraction from chitin column was injected into a C4 LC column and eluted with a linear gradient from 98% solvent A: 2% solvent B to 2% solvent A: 98% solvent B, where solvent A was milliQ water and solvent B was acetonitrile, each with 0.1% formic acid. The mass spectrometer was operated in a positive ion mode. ESI-MS data was acquired with a m/z range of 600-1600, from which multiply-charged mass spectra were obtained. The multiply charged mass spectra were deconvoluted by the MassHunter BioConfirm program to obtain the molecular mass of proteins. The calculated molecular mass of cyclic eGFP was 18 Da less than that of linear eGFP (26941 Da), resulting in a mass of 26923 Da.

3.

Peptide Cleavage and Deprotection:

Treat the resin-bound peptide with a cleavage cocktail to liberate the peptide and remove side-chain protecting groups.

Precipitate and wash the peptide in cold ether or another suitable solvent.

Optionally, purify the peptide using reverse-phase high-performance liquid chromatography (RP-HPLC).

4.

Peptide Analysis: Analyze the peptide using amino acid analysis, mass spectrometry (MS), and/or HPLC to confirm its purity and identity.

Conjugation of chemically synthesized cyclic peptide to antibody

Materials

●

Anti-cMet monoclonal antibody

●

Cyclic Peptide-OPA Conjugate (cEBP-OPA)

●

Bifunctional Linker 1

●

Water

●

Borate Buffer, pH 8.5

●

Phosphate-Buffered Saline (PBS), pH 7.4

●

Zeba Spin Desalting Column, 40K MWCO (Thermo Scientific, USA)

●

MALDI-TOF Mass Spectrometer

●

EGFR binding peptide

Steps

1.

Preparation of Antibody Stock Solution: Dissolve anti-cMet monoclonal antibody in PBS (pH 7.4) to create a 1 µg/µL stock solution.

2.

Peptide cyclization: Treat the bifunctional linker 1mM react with EBP (1 mM) in borate buffer (pH 8.5) for 1 h at room temperature.

3.

Characterization of the Product: Use MALDI-TOF mass spectrometry to confirm the cyclization process.

4.

Antibody Conjugation with cEBP-OPA:

Without further purification, add the cEBP-OPA to the prepared antibody solution in PBS.

Use different mole ratios for the antibody:cEBP-OPA (e.g., 1:5, 1:10, 1:20, 1:50).

Stir the mixture at room temperature for 30 min.

5.

APurification and Formation of the cEBP-IgG or EGFR x cMet pBsAb:

Filter the mixture through a Zeba Spin Desalting Column (40K MWCO) at 1500 g for 2 min, following the manual instructions, to remove excess unconjugated cEBP-OPA.

Lyophilize the product to obtain a powder form for storage and further use.

Enzyme linked Immunosorbent assay (ELISA)

Materials

●

ELISA Plates, 96 Well (Wuxi NEST Biotechnology Co., Ltd.)

●

V-bottom Plates, 96 Well (Bibby Sterilin Ltd.)

●

LEDETECT 96 Microplate Reader (Labexim Products)

●

Coating Buffer (0.05 M Carbonate-Bicarbonate, pH 9.6)

●

PBST Buffer (PBS + 0.05% Tween 20)

●

PBS Buffer without Potassium (Salty PBS Buffer)

●

PBS Buffer

●

Albumin Bovine V (BSA) (Roche #738328)

●

Human c-MET Protein (Sino Biological Inc., 10692-H08H)

●

Human EGFR Protein (Sino Biological Inc., 10001-H08S)

●

c-MET Antibody, Rabbit mAb (Sino Biological Inc., 10692-R243)

●

HRP Donkey anti-rabbit IgG Antibody (Biogend®, 406401)

●

TMB Two-Component Substrate solution (freshly prepared) (Phygene Scientific, PH1595).

●

2M HCl

Antigen Coating and Blocking

1.

Dilute the c-MET antigen to 0.25 mg/mL with ddH2O to prepare a stock solution.

2.

Dilute the “ELISA binding on c-MET” stock solution to 0.5 μg/mL with a coating buffer.

3.

Dilute the EGFR antigen to 0.25 mg/mL with ddH2O to prepare a stock solution.

4.

Dilute the “ELISA binding on EGFR” stock solution to 1 μg/mL with a coating buffer.

5.

Add 100 μL/well antigen solution from (step 2) and (step 4) solution for coating and incubate at 4°C and overnight.

6.

Discard the solution from (step5), then wash the plate once with a PBST buffer (150 μL/well).

7.

Block the plate with 1% BSA (100 μL/well) and incubate for 1h at room temperature.

8.

Wash 3 times with a PBST buffer (150 μL/well) before adding antibodies.

Preparation and binding of primary antibody (pBsAb, EGFR and c-MET)

1.

Dilute the c-MET antigen to 0.25 mg/mL with ddH2O to prepare a stock solution.

2.

Dilute the “ELISA binding on c-MET” stock solution to 0.5 μg/mL with a coating buffer.

3.

Dilute the EGFR antigen to 0.25 mg/mL with ddH2O to prepare a stock solution.

4.

Dilute the “ELISA binding on EGFR” stock solution to 1 μg/mL with a coating buffer.

5.

Add 100 μL/well antigen solution from (step 2) and (step 4) solution for coating and incubate at 4°C and overnight.

6.

Discard the solution from (step5), then wash the plate once with a PBST buffer (150 μL/well).

7.

Block the plate with 1% BSA (100 μL/well) and incubate for 1h at room temperature.

8.

Wash 3 times with a PBST buffer (150 μL/well) before adding antibodies.

Preparation and binding of primary antibody (pBsAb, EGFR and c-MET)

1.

The concentration of each component:

2.

For “ELISA on c-MET”: Top working concentration is 20 nM

3.

c-MET Antibody : 1:500

4.

For “ELISA on EGFR”: Top working concentration is 200 nM

5.

c-MET Antibody : = 1:500

6.

Prepare top work concentrations of c-MET and EGFR with Salty PBS Buffer.

7.

Gently mix well then incubate at RT in the dark for 30 mins.

8.

Using V-bottom plate to dilute top work concentration 5-fold 8-dose for “ELISA on c-MET” and 2-fold 8-dose for “ELISA on EGFR” with PBS Buffer.

9.

To a V-bottom plate, dilute the antibodies top work solutions with PBS buffer to the following concentrations.

10.

Transfer the primary antibody solution to ELISA 96-well plate (100 μL/well) and add Blank Control (PBS buffer) and Negative Control (PBS Buffer).

11.

Incubate at RT for 2 h.

12.

Discard the solution from (step 5), then wash the plate 3 times with PBST buffer (150 μL/well) before adding a secondary antibody.

Preparation and binding of secondary antibody

1.

Dilute the secondary antibody to 5000-fold with PBS Buffer.

2.

Transfer the diluted secondary antibody to each well EXCEPT Blank control (100μL/well).

3.

Add PBS Buffer (100μL/well) to the Blank Control.

4.

Incubate at RT for 1 h in dark.

5.

Discard the solution from (step 2) and (step 3), then wash the plate 6 times with PBST buffer (150 μL/well) before adding the TMB solution.

Adding TMB Substrate Solution

1.

Mix the TMB solution buffer (TMB Solution A : TMB Solution B = 1:1).

2.

Add TMB Substrate Solution (100μL/well) at RT and immediately start recording the time: “ELISA on c-MET”: 5mins; “ELISA on EGFR”: 15mins.

3.

After specific waiting time, add 2M HCl (100μL/well) immediately to terminate the reaction.

Measurement of OD value and Data Treatment

1.

Measure the ODs with the LEDETECT 96 Microplate Reader at 450 nm.

2.

Using Prism GraphPad 9 to interpret the data and obtain the EC50.

MTT Cytotoxicity Assay

Materials

●

1X MTT reagent (5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide in PBS) & 1X Solubilization buffer (isopropanol with hydrochloric acid)

●

96 Well Plates (Wuxi NEST Biotechnology Co., Ltd.)

●

Cell Line: Mock & Osimertinib Resistant HCC827

●

DMSO

●

Osimertinib (MedchemExpress #HY-15722)

●

PBS Buffer without Potassium (Salty PBS Buffer)

●

c-MET Antibody MAb (Medchemexpress, HY-P99391)

●

Culture Medium: Roswell Park Memorial Institute (RPMI) w/t 10% (FBS) & 1% Penicillin-Streptomycin (PS)

Cell Culture

1.

Osimertinib resistant HCC827 cell line was cultured continuously using RPMI supplemented with 10% FBS and 1% PS in a 37°C incubator with 5% CO2.

2.

Fed with fresh medium every 3 days, and prepare to sub-culture every 3 to 4 days when confluence gets to 70% to 80%.

3.

Washed the cells by 1X PBS to wash off the waste and cell debris.

4.

Treated the cells with 2 ml 0.0625% Trypsin with 0.05% EDTA in PBS for 5 minutes in a 37°C incubator.

5.

Terminate digestion with 8 mL of PBS.

6.

Centrifuged at 1500 rpm for 3 minutes.

7.

Split in 1:3 to 1:5 ratio adding to new culture dishes with RPMI.

Seed Cell

1.

When the cell confluence reaches 70% to 80%, repeat the process in step 3 & 4 in the cell culture part. Then disperse the cells in 3 ml of fresh RPMI medium.

2.

Calculate cell concentration

3.

Dilute the cell concentration to 4 × 104 cells/mL containing 10% FBS.

4.

Seed cells at a concentration of 8 × 103 cells/well in 96 Well Plates and each well with 200 μL RPMI culture medium).

5.

Observe the cell under a microscope and multiple layers of cells should be observed.

6.

Incubate cell culture for 24 h at 37 °C and 5% CO2.

Preparation Polyneerab and Osimertinib

1.

The concentration of each component:

a.

Osimertinib = 0-6.3 μM

b.

c-MET Antibody = 0-200 μg/mL

c.

EGFR peptide = 1:500

2.

Prepare top work concentrations of c-MET and EGFR with Salty PBS Buffer.

3.

Gently mix well then incubate at RT in the dark for 30 mins.

4.

Dilute the Polyneerab and Osimertinib top work solutions to the above range of concentrations.

MTT application and solubilization

1.

Mix MTT with 5% FBS medium (Solution volume MTT : 5% FBS medium = 1:10)

2.

Add 110 μL of the MTT solution (final concentration 0.5 mg/mL) to each well.

3.

Incubate the 96 Well Plates for 3 hours in a humidified atmosphere (37 °C, 5% CO2).

4.

Mix the dissolving solution (50 nm HCl) by isopropanol and HCl

5.

Aspire all solution on the 96 Well Plates

6.

Add 200 μL of the dissolving solution into each well.

7.

Place the plate on the shaker for 10 minutes

Bliss Analysis

Data analysis

1.

Calculate the individual and combination drug effects on cell viability based on the assay results. This may involve comparing the drug-treated groups to the control group and determining the percentage of cell viability.

2.

Apply the Bliss independence model to analyze the combination drug effects. This model predicts the expected combined effect based on the assumption of non-interaction between the drugs. The Bliss score can be calculated using the formula: Bliss score = (A + B) - (A x B), where A and B represent the effects of the individual drugs.

3.

Compare the observed combination effects with the predicted Bliss score. Positive deviations from the Bliss score indicate synergy, while negative deviations indicate antagonism.