This document describes a case study of a 4-year-old male patient presenting with generalized fatigue and abdominal protrusion for 2 weeks. On examination, enlarged lymph nodes were found and the liver and spleen were enlarged. Laboratory tests found anemia and abnormally high white blood cell count with many lymphocytes. Bone marrow aspiration revealed 98.5% lymphocytes. The patient was diagnosed with lymphoblastic leukemia and treated with iron injections over 33 days with little improvement. He later developed a rash typical of chickenpox and fever.

1. Case study
T.P 4 yrs. /M
Generalized Fatigue
Protuberant abdomen × 2 weeks
Feb 1951
LEO,USA

2. O/E
Vitals - stable
GE – palpable lymph nodes in the cervical,
epitrochlear, and inguinal areas
P/A – Hepatosplenomegaly

3. Relevant Lab results
Hb – 5.6 g /dL
TLC – 175000 cells /mm3
N3L97
BM –Bone-marrow aspiration revealed 98.5 per cent
lymphocytes (81% small, 14.5% intermediate, 2%
large, 1% blast)
Platelets – 150000/ mm3
Diagnosis – Lymphoblastic Leukemia

4. Treatment and follow up
Inj. Saccharated Iron oxide 4.8 gm. i.v. Over 33
day period
Discharged (without much improvement on May 2)
Readmitted on May 15th - developed scattered
watery vesicles on the face, scalp, and neck with
fever
typical of varicella, with subsequent crops of the
eruption until May 23.

6. Oncolytic Virotherapy
Dr.Gopisankar M G

7. Virus – A drug ??
A drug is defined as a chemical substance of known
structure, other than nutrient or an essential dietary
ingredient which when administered to a living
organism, produces a biological effect
“Slimy Liquid Poison “

8. “Drugs don’t work in patients who
don’t take them.”
General C. Everett Coop

9. General Properties of Viruses
Acellular Organisms
Obligate intracellular parasites
Nucleoprotein
Lipid overcoat
Uses host metabolic machinery and ribosomes
assemble into particles called VIRIONS

10. Viruses cannot make energy or proteins independent
of a host cell
Viral genome are RNA or DNA but not both
Viruses lack the enzymes necessary for protein and
nucleic acid synthesis
Viruses do not have the genetic capability to multiply
by division
Viruses occupy the twilight zone that separates the
‘living’ from the ‘nonliving’

11. 1800 1900 2000
1798 ‘85 ‘92‘84 ‘98
1915 1939 1948 -1955
1952
1952 1985 20051903
Virology milestones

12. Annual meeting of Academy of Medicine
September 18, 1903 ( Dr.George Dock )
Birth of oncolytic virotherapy
Cleveland , Ohio
Report of Influenza making leukemia better
WBC count reduced from 365000 to 7500 per µL
He cited a number of similar cases

13. Dr. George Dock (1860 - 1951)
Professor of Medicine
University of Michigan
“It might seem that the process could be imitated, and a
symptomatic, if not a causal, treatment be discovered”

14. Leviditi and Nicolou found that vaccinia virus and
herpes virus caused regression of mouse skin
tumors and sarcoma
Chickenpox improved the count and lymph node
status in case of lymphatic leukemia
Measles produces improvement in the case of
leukemia, Hodgkin’s, and Burkitt’s lymphoma
Rabies vaccination favorably modified the course
of carcinoma of the cervix

15. Observations Trials

18. How to study the anticancer effect?
Virus pharmacokinetics
Virus pharmacodynamics

19. Virus Pharmacokinetics
Absorption
Distribution
Metabolism
Excretion

20. Dosage and Administration
TCID50 - 50% Tissue Culture Infective Dose
Plaque forming units(pfu)
Focus forming assay
Haemagglutinin assay
Electron microscopic methods
Flow cytometry
TRPS(Tunable Resistive Pulse Sensing)

21.  Moraten dose ≥108 TCID50  prolonged survival
after administration of the strain in murine
intraperitoneal model of human ovarian cancer
1×107 pfu of the second generation HSV inoculated
into brain resulted in no adverse effects in mice
Virus JX-594 maximum tolerated dose was 1×109
pfu
ONYX 015 even when administered up to 1011 pfu
did not cause any dose limiting toxicity.

22. Dose –dependent increase in levels of the
inflammatory cytokines
Optimal dose for bystander response also
Maximal dosage

23. How to find the adequate dose ?
Conducting dose range studies and doing tumor
biopsies to recover the virus
JX594 an oncolytic vaccinia virus is under trial for
HCC appeared in tissue biopsies only when the
threshold dose was more than 109 IU

24. Controlling the availability
Controlled release viral preparations are available for
Adenovirus
Polymer coat
Lipid layer
Polyethylene glycol

25. Routes of administration
Intra tumoral ( Most common )
Oral, rectal, inhalation, intra-arterial, intraperitoneal,
intramuscular or intravenous are also tried
Convection Enhanced Delivery

27. Distribution
Details like Bioavailability , half life of the virus
Pre dose titers  community exposure
The virus levels may spike several times over a
period of time due to replication

28. Viral titer over time or viral RNA/DNA/proteins in blood can be
calculated over a time period

29. Surrogate markers

31. Visual analysis - fluorescent labeling of the vector
Transcription units coding for soluble marker
peptides are inserted by genetic engineering - β-
hCG, CEA
 BBB – parvo virus

32. Gamma camera images of MV-NIS treated mice
in the s.c. xenograft model

33. Histochemical detection of β- galactosidase

34. Metabolism and excretion
Peak activity of virus will increase due to replication
Non replicative vectors are developed - limited
success
Major route of elimination  Immunological
mechanism

37. Poor correlation between viral titers and increasing
antibody titer
PEGylation of virus reduces immunogenicity
Virus will concentrate in the RES and from there it is
slowly removed by immune mediated mechanisms
Pre conditioning of the MPS receptors - polyinosinic
acid , clodronate-loaded liposomes , gamma
globulins , gadolinium chloride
MPS follows saturable kinetics

38. Cell carriers and stem cells
Cells can function as vehicles for therapeutic
virus
They escape immune attack
Natural habitat
Stress situation – low PH , hypoxia
Stem cells have tumor homing properties
Due to chemokines produced during
carcinogenesis

39. Virus Pharmacodynamics
Mechanism of action of virus
Aberrant pathways
Nonfunctioning of certain normal pathways
Certain receptor over expression in the cancer cells

40. Signaling Pathways
Interferons have anti viral and anti proliferative
actions
Their antiviral mechanism involves activation of PKR
pathway, 2-5A synthases and activation of some
specific proteins
PKR gets activated if comes in contact with ds RNA
derived from virus
It phosphorylates itself and other substrates like eIF2
which is a translation initiation factor of viral
translation
Phosphorylation leads to eIF2 inactivation and thus
inhibition of virus and host protein translation

42. CD46 (membrane co-factor protein) is
overexpressed in cancer cells compared to normal
cells to protect them from complement mediated
destruction.
Measles virus have a tropism to CD46 and they uses
this receptors to enter into the cells
The oncolytic activity of CVB3 (Coxsackie B3 virus)
is found to be proportional to the percentage of CAR
expressing cells times the percentage of DAF
expressing cells

43. Use of a CD20-targeted recombinant measles virus
can be used a substitute to Rituximab in the FCR
Fludarabine, Cyclophosphamide, and Rituximab
regimen for lymphoma management

44. MOI
Multiplicity of infection (MOI) ratio
It is the ratio of infectious agents to the targets
As the MOI increases the percentage of cells
infected with at least one viral particle also increases
It is used for quantifying the affinity of the cells and
viruses

45. Augmenting the immune response against
cancer
Release of tumor associated antigens (TAA)
Augusto et al used Adeno viral vector with HSV-TK
protein which functions as a super antigen and
stimulates T cells and secretion of cytokines like IL-2
Local intratumoral injection of viruses will have a
global effect

46. Combination virotherapy
 Virus and chemotherapy
 Virus and Radiotherapy
 Virus and other treatments
 Virus with virus

47. Virus and chemotherapy
G207 and cisplatin
1726 and mitomycin C
ONYX-015 and cisplatin/5FU
ONCOS-102 + GM-CSF
Cyclophosphamide

48. Virus and Radiotherapy (Radiovirotherapy)
Lytic activity of NV1020 was enhanced with radiation
in HuH7 xenografts in athymic mice
G207 and radiation in cervical cancer is found to
have increased efficacy

49. Virus with other treatments
ONCOS-102 is a chimeric Ad5/knob3 vector added
with GM-CSF production
Hyaluronidase enzyme along with Ad5/35GFP fiber-
chimeric adenovirus which enhanced virus
transduction and spread within prostate cancer
Losartan enhances the intratumoral spread of
oncolytic HSV as it disturbs the transforming growth
factor beta1 signaling

50. Virus with virus
 Interferon sensitive VSV which is a RNA virus is
combined with pox virus encoding a secreted
interferon antagonist had better oncolytic effect due
to VSV and better intratumoral spread due to pox

51. Advantages
Safer drugs
Less chance of transmission
Bystander response
Virus drugs can be deposited in resection cavity
which will track the remaining neoplastic cells thus
reducing the risk of recurrence

52. Obstacles
Immune attack
Metastatic cases
Adequate animal models
Some metastatic lesions won’t respond
Repeated histopathology confirmation

53. Risks and Adverse effects
New regulations
Immunocompromised
Massive cytokine release
Drug interactions –
Reo virus when trialed for solid tumors it raised the
ALT/AST levels in patients who were taking
acetaminophen and thus should be avoided
The same drug if used with cyclosporine will lose its
therapeutic benefit in tumor models

54. Approved Drugs
1. Talimogene laherparepvec (Oncovex)
T-VEC was approved by US FDA in 2015, with the
brand name ‘Imlygic’ for the treatment of malignant
melanoma in patients with inoperable tumors
Oncolytic herpes simplex virus type 1.
The neurovirulence factor ICP34.5 is inactivated by
recombinant DNA technology to prevent neuronal
involvement and is replaced with GM CSF encoding
genes
It is administered intralesionally as 0.4mL of 106 to
108 pfu/mL.

55. In OpTIM study, T-VEC was compared with G-CSF
alone. Durable response rate lasting ≥ 6 months
(16.2 vs2.1%, p<0.001) was superior in the T-VEC
arm
Median overall survival was 23.3 months compared
to other arm which was 18.9 months. T-VEC
compared with ipilimumab (anti CTLA4) showed 56%
response rate.

56. The Future
0
50
100
150
200
250
300
1945 1955 1965 1975 1985 1995 2005 2015
NUMBER OF REGISTERED ONCOLYTIC VIRUS STUDIES
(1952 - 2015)

57. 36
12
6 5 5
3 3 3 3 3 3 2 2 2 2 2 2 2 1 1 1 1 1
0
5
10
15
20
25
30
35
40
Trials registered in clinicaltrials.gov.in
(2004 - 2016)

58. Thank you