1.
Professor Rama S Verma M.Phil., Ph.D., FMSF, FAMI
Stem Cell and Molecular biology Lab.
Department of Biotechnology
Indian Institute of Technology Madras
Chennai 600 036
vermars@iitm.ac.in

2.
Immunotoxins are used to treat cancer – Leukemia and
solid tumors
Ligand-toxin
conjugate
Immunotoxin – A molecule carrying target
antibody or ligand moiety and killing toxin moiety
Target moiety Linker toxin moiety
Ligand
Antibody
Antibody-toxin
conjugate

3.
Used to treat all types of cancers specifically
It targets cancer cells or tissues specifically by binding via
target moiety and destroys them using the drug or toxin moiety

4.
Target and toxin molecules being used
Target Molecules Toxin Molecules
1. Cytokines- IL-2, IFN, TNF
targeting respective Receptors (IL-2 for
CTCL, renal cell cancer, melanoma)
2. Monoclonal antibodies- for
tumor specific antigens-EpCAM
3. Immunoglobulins – single chain
variable fragmens (ScFv).
4. Growth factors – VEGF, GnRH
1. Bacterial toxins- Diphtheria Toxin,
Pseudomonas Exotoxin, Shiga,
cholera, anthrax toxins
2. Fungal toxins- a-sarcin, restrictocin
3. Plant toxins- ricin, abrin, saporin
(SAP), pokeweed antiviral protein (PAP),
gelonin etc
3. Humanized toxins-
Pro-apoptotic proteins-TRAIL, DFF,
Bcl-2, FASL and RNases.

5.
Immunotoxins developed in the laboratory of Prof. Rama
Shanker Verma
1. DT-HN-1 for Head and Neck Carcinoma- Potala &
Verma, Mol Biol Rep, 2010.
2. DT-SCF for solid tumors - Potala & Verma, Appl Biochem
Biotechnol, 2009.
3. DT-IL2 for Leukemia- Sirisha Potala, Rama S. Verma,
Journal of Biotechnology 2010.

6.
DT-HN-1 Immunotoxin DT-SCF Immunotoxin
Immunotoxins developed in the laboratory

7.
DT-IL2 Immunotoxin

8.
Other related Publications
1. Potala, Sahoo and Verma, Drug Discovery Today, 13, 2008
2. Mathew & Verma, Cancer sci, 2009, 100, (8), 1359-1365
3. Swati Choudhary, Mrudula Mathew and Rama S. Verma, 2011, 16, 495-503
4. Madhumathi & Verma, Current Opinion in Microbiology 2012, 15:300–309
Ongoing Immunotoxins
DT-SCF and SCF-ETA: Targets C-kit positive cells like Colorectal
cancer, Neuroblastoma and Mast cell leukemia.
Work in progress in the Laboratory

9.
Limitations of Bacterial/Fungal/Plant based Immunotoxins
1. Immunogenicity: immune system reacts to both the murine antibody fragment and
to the protein toxin, by generating HAMA and human anti-toxin antibodies (HATA).
2. Tissue toxicity:
 VLS- Immunotoxins are usually given intravenously and cause damage to
endothelial cells surrounding the blood vessels causing Vascular Leak Syndrome
(VLS) that can be severe and even cause death. VLS is characterized by
increased vascular permeability accompanied by extravasation of fluids and
proteins resulting in interstitial edema and organ failure. It results in fluid
retention, Hypoalbuminemia, increase in body weight, peripheral edema, pleural
and pericardial effusions, ascites, anasarca and, in severe form, signs of
pulmonary and cardiovascular failure.
 Hepatotoxicity- Nausea, Vomiting, Abdominal pain, Loss of appetite, Diarrhea
 Other side effects- encephalopathy, central nervous toxicity.
3. Off-target toxicity: Toxicity due to non-specific killing of normal cells by bacterial
toxins

10.
Humanized Immunotoxins
Cytotoxic moiety is either a protein involved in the apoptotic pathway or
human RNase.
1. Proapoptotic proteins:
• TNF-related apoptosis inducing ligand (TRAIL)
• Granzyme B
• DNA fragmentation factor (DFF40)
• Bax, Bik, Bak etc
2. RNases:
• Human pancreatic RNase 1
• Eosinophil derived neurotoxin or RNase 2
• Eosinophil cationic protein or RNase 3
• Angiogenin or RNase 5

11.
IISc
presentation
Mathew and Verma,Cancer Sci | 2009
15-02-13
Humanized Immunotoxins Reported

12.
1. GMCSF -DFF40 for Leukemia- Mathew, Zaineb, Verma, Apoptosis, 2013
2. IL2-TRAIL for leukemia- Madhumathi, Sridevi, Verma, 2015
3. SDF-TRAIL for breast/prostrate/cervical/ovarian cancer
Humanized Immunotoxins developed in Prof.R.S
Verma’s Laboratory

13.
GMCSF -DFF40

14.
DNA Fragmentation Factor 40
(Caspase Activated DNase /CAD)
• N terminal residues for DNase activity and C terminal residues for nuclear
transport.
• Exists as an inactive complex with ICAD ( Inhibitor of CAD/ DFF45) in
proliferating cells. ICAD needed for proper folding.
• Apoptotic signals such as binding of death factors to their receptors, growth
factor deprivation and genotoxic agents activate the caspase cascade.
• Caspase 3 cleaves away ICAD activating DFF40 leading to chromosomal
DNA fragmentation.
GMCSF (granulocyte monocyte colony stimulating
factor)
• A hematopoietic cytokine.
• GM-CSF receptor over expressed in AML and on solid tumors, including
lung, breast, and gastrointestinal carcinomas.
GMCSF -DFF40

15.
In vitro Cytotoxicity Assay
Fig. In vitro cytotoxicity of GM-CSF-DFF40. Dose response curves after 48 hrs of
treatment. The values represent mean ± SE. The error bars represent SE of the
mean.

16.
ImmunofluorescencestainingofGM-CSF-DFF40treatedTHP-1cells
Untreated Treated
Colocalization of Bax in mitochondria seen in treated cells
Detection of Bax colocalisation in mitochondria

17.
Patient
GM-CSF-DFF40
(Percent viability)
1 46.23
2 70.00
3 61.54
4 46.67
Mean (±SE) 56.11 ± 6.8
Comparison of the cytotoxic effect on different
patient samples

18.
IL2-TRAIL

19.
IL2-TRAIL
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-
Induces apoptosis by Extrinsic Pathway
TRAIL- member of TNF superfamily- 40 kDa type II
transmembrane protein
Expressed by normal immune cells upon stimulation- T cells, NK cells,
Macrophages, Dendritic cells, B cells, monocytes and in prostate and spleen
tissues.
Role in normal cells:
Tumor suppressor, suppresses metastasis, prevents auto-immunity
TRAIL

20.
Binds to-
Death receptor 4 (DR4/TRAIL-R1),
Death receptor 5 (DR5/TRAIL-R2),
Decoy receptors DcR1 and DcR2 and
Soluble receptor osteoprotegrin
TRAIL receptors
Receptors- R1 and R2 (DR4/DR5)-
expressed in cancer cells not normal cells -
Selectively kills cancer cells but not most
normal cells
(Yagita, 2001)

21.
 Selective toxicity for tumour cells- (Yagita et al., 2004, Koschny et al., 2007, Carlo-Stella et
al., 2007).
 No apparent systemic toxicity of rTRAIL in non-human primates
 Shows bystander effect (therapeutic effect toward neighboring tumor cells that
lack expression of the target antigen).
 Induces apoptosis in premalignant cells-(Lu et al., 2004)
 Increased ability to induce apoptosis when combined with chemotherapy
(Liu et al.,2001)
 scFv54–sTRAIL : colorectal & breast carcinomas
 scFv425–sTRAIL :Squamous cell carcinomas
 scFvCD7–sTRAIL: Leukemias
TRAIL based immunotoxins
Advantages of TRAIL
Mathew and Verma, 2009
Bremer et al., 2004 a, b, 2005
The TRAIL based fusion constructs reported for solid tumors:

22.
HL60
Hoechst Anti-DR5-FITC Overlay
MOLT-4
K562
KG-1
Localization of TRAIL receptors

23.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
0
20
40
60
80
100
120
TRAIL
IL2 TRAIL
Concentration (M)
Percentage
viability
(%)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
0
20
40
60
80
100
120
TRAIL
IL2 TRAIL
Concentration (M)
Percentage
viability
(%)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
0
20
40
60
80
100
120
TRAIL
IL2 TRAIL
Concentration (M)
Percentage
viability
(%)
24 hrs 48 hrs 72 hrs
TRAIL IL2-TRAIL
0
20
40
60
80
100
120
140
**
Percentage
viability
(%)
TRAIL IL2-TRAIL
0
50
100
150
*
Percentage
viability
(%)
TRAIL IL2-TRAIL
0
50
100
150
Percentage
viability
(%)
ALL CLL Normal PBMC
Cytotoxicity in Leukemic Cell line KG-1
Cytotoxicity in Leukemic Patients

24.
Cytotoxicity of IL2-TRAIL
Cell Lines Patient Samples

25.
Control
IL2-TRAIL
Apoptosis induced by IL2-TRAIL

26.
Guzman ML, Jordan CT. Cancer Control. 2004
Tannishtha Reya et al, Nature, 414, 2001
Targeting Cancer Stem Cells
Targeting both CSCs and the dividing cells would be required for complete
tumor eradication
Immunotoxins targeting specific surface proteins of cancer stem cells
could be the solution

27.
 Highly resistant to chemo- and radio-therapy
 Inhibition of apoptosis- long-term survival is primarily by deregulation of apoptosis.
 Self-renewal capability
 Quiescence
Characteristics of CSC’s
Resistance of CSC’s
CSC’s are typically resistant to cancer drugs and are
responsible for recurrence of all cancers.
1. Evasion of apoptosis is one of the major mechanisms of
immortality in human cancers (Hanahan and Weinberg,
2000).
2.Chemoresistance – due to efflux of anticancer drugs
through multidrug resistance transporter 1 (MDR1)

28.
Percentage viability of leukemia cells with different
concentrations of Methotrexate
Percentage viability of leukemia cells with different
concentrations of 5- FluroUracil
PRELIMINARY RESULTS

29.
MOLT-4
Control Methotrexate Treated
HL60
KG-1
Efflux of Hoescht dye by the Side population (SP)

30.
Madhumathi J and Rama S Verma, Therapeutic targets and recent advances in protein immunotoxins,
Current Opinion in Microbiology 2012, 15:1–10,
IL2-TRAIL
GM-CSF-DFF40
Mechanisms of different Immunotoxins

31.
Dr.Sirisha Potala, Ph.D Dr.Mridula Mathew, Ph.D
Miss. Swati Chowdary, Ph.D Dr.Madhumathi, Post doctoral fellow
Students

32.
15-02-13
IISc
presentation
33

33.
Acknowledgments
 Department of Biotechnology, Govt. of India
 Department of Science and Technology, Govt. of India
Funding Source for the projects