1. Creation of anti-CD33
antibody targeting Acute Myeloid
Leukemia
Subarnarekha Chatterji1, Mariya Dyka2, Abdullah Elsayed3, Abhishek Goyal4, Emine Taytaş5
1School of Life Sciences, Manipal University, Manipal, Karnataka, India
2 Technical University of Munich, Munich, Germany
3 Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
4 Jaypee Institute of Information Technology, Noida Sector 62, Uttar Pradesh, India
5 Erasmus University College, Rotterdam, The Netherlands
Objectives
Methods
Results Results (contd.)
Conclusion
Acute Myeloid Leukemia is a type of blood cancer that develops in the
bone marrow. CD33 is a surface glycoprotein expressed on leukemic
cells but not on hematopoietic stem cells. This makes it a popular target
for new immunotherapeutic approaches with the intention of
conjugating to radioisotope for therapeutic use. The gene coding for
CD33 has been identified which allowed us to produce potential
antibodies against this glycoprotein.
1. We have used the Pichia Pastoris in BMMY cultures to produce
potential anti-CD33 scFv. Involved vectors and restriction enzymes:
• Vectors: pBluescript and pPICZα
• RE’s: Xho1 and Not1
2. PCR: Amplification of the gene of interest
3. Ligation: After restriction digest, anti-CD33-scFv insert and pPICZα vector were
ligated together using T4 DNA ligase
4. Transformation of the desired vector + insert to competent E.coli occurred using
heat pulse in 42℃ and then left to grow overnight on LB-Zeocin cultures
5. Electroporation: transformation of the desired vector + insert into yeast cultures
A: 100 bp DNA Ladder; B: 1 kb DNA ladder; C-E: MiniPrep
Sample 1-3; F: Restriction Digest 1; G: Restriction Digest 2;
H: Restriction Digest 3
The red box in F, G and H shows successful
transformation of vector into E. coli after
restriction digest.
A: 100 bp DNA Ladder; B: pPICZα; C: pBluescript+insert;
D: 1 kb DNA Ladder; E: PCR based cloning product
The bands marked with red indicate that the
PCR product and the restriction digest are a
match due to their similar sizes.
A: 100 bp DNA Ladder; B: 1 kb DNA Ladder; C-E: PCR 1-3;
F: Negative Control.
The clear bands in lanes C, D, and E suggest
the presence of the anti-CD33-scFv insert
Fig. 1: Anti-CD33 antibody production
Fig. 2: Gel analysis of restriction digest and PCR
product
Fig. 3A: Gel analysis showing successful
transformation after mini-preps and restriction digest
Fig. 3B: Gel analysis showing successful
transformation after PCR
6. Time course analysis of anti-CD33-scFv expression was done after 0, 24, 48, and
72 hours.
7. Agarose Gel Electrophoresis & SDS-PAGE: Analysis of both DNA and proteins
after each step which showed successful bands of the desired products
Acknowledgment
A: Protein marker; B: 0h protein; C: 24h protein; D: 48h
protein; E: 72h protein; F: Flowthrough after NPI-10; G:
Flowthrough after NPI-20; H: Elution after NPI-500; I:
Second protein elution; J: protein marker.
The arrows indicate the presence of the
antibody
Fig. 4: SDS-PAGE of time course yeast cultures and
post-protein purification
Our results suggest that we successfully produced the scFv of the anti-CD33 antibody
using the yeast expression system. While figure 2 shows that we have successful starting
templates (insert + vector), figure 3 shows successful ligation and transformation. Finally,
figures 4 and 5 indicate successful protein product (anti-CD33 ScFv). Further studies are
required (eg., combining the antibody of interest with a potential radioisotope) to
determine their therapeutic efficacy in patients suffering from Acute Myeloid Leukemia.
References
We would like to thank our course supervisors
Dr. Dan Lloyd and Dr. Rosalyn Masterton along
with their whole team at the School of
Biosciences, University of Kent, for their
invaluable guidance and support that has made
this project a success.
1. L.M. Emberson et al./Journal of Immunological Methods 305 (2005) 135-151
1000 bp
750 bp
Fig. 5: Mass spectrum before and after deconvolution
showing the protein mass of anti-CD33 ScFv as 19048 Da