The document discusses immunotherapy and gene therapy in relation to cancer treatment, focusing on the tumor microenvironment and its interactions with the immune system. It highlights the mechanisms by which tumors evade immune detection and outlines gene therapy's application in genetic diseases like cystic fibrosis, as well as its challenges and potential vectors. The text also reviews novel therapies and the role of immune checkpoint inhibitors in managing advanced head and neck cancer.

1. IMMUNOTHERAPY AND
MODALITIES OF GENE
THERAPY
ASHISH K.GUPTA
SMS medical college
Jaipur

2. IMMUNOTHERAPY

3.  It is important to explore the fundamentals of the complex interactions
within the tumour microenvironment and how malignancies effectively
‘escape’ immune detection.
 Changes that cause acquisition of cancer related phenotype-
• limitless replicative potential of tumours
• self-suffciency in growth signals and insensitivity to anti-growth signals
• ability to evade apoptosis
• increased angiogenesis
• invasion and metastasis

4. TUMOUR MICROENVIRONMENT
 The cancer microenvironment, describes the
non-cancerous cells present in the tumour.
 Tumours not only contain malignant cells but
surrounding stroma, which comprises various
types of mesenchymal cells, extracellular
matrix and immune cells, also includes the
proteins produced by all of the cells present
in the tumour that support the growth of the
cancer cells.

5.  As tumours progress, the surrounding microenvironment co-evolves into an
activated state through continuous tumour–host interactions .
 Several studies have shown that tumour stroma contains growth factors and
cytokines that can promote angiogenesis, tissue invasion and metastasis.
 Stroma also provides a chemoresistant capability to the tumour, most likely
due to the presence and function of Carcinoma associated fibroblasts and
tumour associated macrophages (TAM) that promote resistance to oncological
therapies.

6. CELLULAR COMPONENT OF TME
 1.CD8+cells
Malignant cell+ MHC-1
Recognised by CD8+ T cells
TUMOR LYSIS &/APOPTOSIS

7.  2.CD4+ T Cells
 TME can become dominated by CD4+ Treg cells that suppress anti-tumour
effector T cells by the production of suppressive cytokines - TGF-β, IL-10,
(PGE2) and adenosine .
 Treg cells also suppress cytotoxic T cells.
 Higher CD8+/Treg cell ratios have been shown to be associated with a
favourable prognosis.
CD4+ T CELLS- (Th-1,Th-2,Th-17)
Help to enhance the magnitude and duration of anti-tumor response

8.  3.Other Cells-
 Tumour infiltrating lymphocytes (TIL) have been shown to promote
angiogenesis and therefore tumour progression and metastasis.
 Stromal cells have been shown to secrete cytokines and growth factors
including VEGF, PDGF and IL-8, which are strong inducers of angiogenesis and
tumour progression.
 Carcinoma-associated fibroblasts are the major cell population in the tumour
stroma
 When compared with normal oral cavity fibroblasts, CAF were found to
• exhibit rapid growth
• increased proliferation

9. CYTOKINES AND CHEMOKINES IN TME
 Cytokines and chemokines are key in controlling communication
within and between individual cells in the TME.
 Tumours produce cytokines in a dysregulated manner that contributes
to an ineffective host immune response.
 The release of such cytokines induces ECM remodelling, basement
membrane degradation,tumour cell proliferation and angiogenesis,
and hence favours tumour progression and metastasis

10. INFLAMMATION IN TME
 A chronic inflammatory environment is associated with neoplastic transformation
and progression in many malignancies including HNSCC.

 Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) pathways, which both produce
derivatives from arachidonic acid, are activated during HNSCC development.

 The COX-2 enzymes specifically catalyze the production of prostaglandins.
 Raised PGE2 in HNSCC is known to promote tumour growth and inhibit apoptosis by
upregulating Bcl-2 expression.
 PGE2 is also known to increase the production of angiogenic factors such as VEGF
resulting in the promotion of invasion and tumour metastasis.

11. TUMOUR ESCAPE MECHANISMS
 Tumour- specific antigens -antigens are solely presented by tumour cells and never by normal cells.
 Tumour-associated antigens -Other antigens are presented by tumour cells but at raised levels when
compared with normal cells.
 Immunoediting -encounter between the immune system and tumours initiates a process.
 Three outcomes: Elimination of the tumour;
Tumour equilibrium
Tumour escape, the growth of tumour variants that resist immune destruction.

12. Immune-suppressive tumour-escape mechanisms in HNSCC
• Spontaneous apoptosis/anergy of tumour infiltrating and circulatory T cells
• Imbalanced and decreased absolute counts of T cell subsets
• Signalling defects in effector cells
• Cytokine imbalance favouring immunosuppression
• Poor expression of co-stimulatory molecules and high expression of co-inhibitory
receptors on tumour cells
• Deficient antigen-presenting and processing mechanisms
• Loss of HLA class I molecules by HNSCC cells
• Inhibition of NK cell activity
• Overexpression of Treg cells both in the circulation and intra-tumourally

13. TUMOUR BIOMARKERS
 Tumour biomarkers can be divided into either disease- related ones (i.e. at
the time of diagnosis prior to any therapy) or therapy-related ones (i.e. prior
to, during and after therapy)
 T cell infiltrates emerged as the strongest independent prognostic factor,
more significant than the conventional clinicopathological criteria such as
tumour size, depth of infiltration, differentiation, or the nodal status .
 key role of the B cell signature in HNSCC where intra-tumoural CD20+ B cells
were significantly more frequent in metastatic lesions than in primary
tumours

14. NOVEL THERAPIES FOR THE FUTURE
 Despite the introduction of new cytotoxic drugs, the management of
advanced head and neck cancer remains challenging.
 agents with more targeted mechanisms of action or agents that are
able to manipulate the immune system to provide tumour control
(immunotherapy).

15. Monoclonal antibodies
A desirable target for MAbs would have the following properties:
 Wide distribution on tumour cells
 High level of expression
 Bound to tumour, allowing cell lysis
 Absent from normal tissues
 Trigger activation of complement on MAb binding

16.  Early clinical trials of immunotherapy in the majority of cancers were
troubled by systemic toxicity or difficulties in local administration.
 The aim of these therapies is to cause the tumour to be targeted in a specific
fashion by the host immune defences.
 Targeting specific elements of the immune system such as tumour antigens
with antibodies and therapeutic cancer vaccines have all shown promising
results

17.  Cetuximab (Anti-EGFR) is the only approved antibody by FDA for use in
patients with locally advanced HNSCC .
 Bevacizumab (Anti-VEGF) first anti-angiogenic drug to have induced a survival
advantage in cancer therapy.
 A variety of other anti-EGFR agents such as the small molecule tyrosine kinase
inhibitors (TKI) lapatinib, dacomitinib and afatinib and the anti-EGFR
nimotuzumab and panitumumab, are currently under investigation .

21.  Mainly there were 2 trials –
 second-line phase III trial CheckMate-141 (for
approval of Nivolumab which is a monoclonal
antibodies against programmed cell death
protein-1 (PD-1), an ‘immune checkpoint’
receptor. )
 non-randomised, multicohort phase Ib study
KEYNOTE-012 (for approval by FDA for
pembrolizumab )

24.  Patients enrolled between June 2014 and August 2015 were randomised in a
2:1 ratio to receive either nivolumab (236 of 240 assigned) or a single-agent
of the investigator’s choice (111 of 121 assigned). In the intention-to-treat
population (n = 361), median age was 60 years with 113 (31%) patients being
‘elderly’ (aged 65 or over).
 A/E occurred at similar rates in the two arms (59% with nivolumab versus
78%), but grade 3–4 toxicities were less frequent with the experimental drug
(13%) than the drug of the investigator’s choice (35%).
 In the nivolumab-treated group, fatigue (14%), nausea (9%), rash (8%),
decreased appetite (7%), pruritus (7%), and diarrhoea (7%) were the most
common side effects of any grade, while other toxicities did not exceed 6%.

25.  There were two treatment-related deaths in the nivolumab cohort (caused by
pulmonary embolism and hypercalcemia) and one in the standard therapy arm
(lung infection)

27.  Using a schedule of 10 mg/kg pembrolizumab, again an anti-PD-1 antibody,
administered I/V every 2 weeks, the investigators demonstrated efficacy and
toxicity outcomes similar to nivolumab in the CheckMate-141 study.
 no deaths were attributed to pembrolizumab.

28.  Unlike other targeted drugs (e.g. EGFR-inhibitors) and cytotoxic
chemotherapy, checkpoint inhibitors can elicit delayed clinical effects and
may also lead to long- term off-treatment survival .
 Taken together, in patients with R/M-SCCHN, the PD-1-directed immune
checkpoint inhibitors nivolumab and pembrolizumab are well tolerated novel
anticancer agents producing a modest overall response rate of about 15% in
second-line treatment, but the induced tumour regression is usually durable,
even in platinum-resistant/ refractory cases.
 cetuximab as an adjunct to the platinum/5-fluorouracil combination in the
first-line EXTREME trial.

29. OTHER ONGOING TRIALS
 RTOG 3504 examines the efficacy and safety of nivolumab in the definitive
and adjuvant settings .
 Citoplurikin, a primary human cell-derived biologic with multiple active
cytokine components, is being tested in a randomised phase II trial of
neoadjuvant and adjuvant therapy in pa- tients with newly diagnosed curative
resectable stages II, III, or IVA oral cavity cancer .

30.  Those affected by Epstein–Barr virus (EBV)-positive nasopharyngeal carcin-
oma. In these cases, vaccines and adoptive T-cell transfer have demonstrated
biological activity in boosting the anti-cancer properties of T-cells.

32. GENE
THERAPY

33. GENE THERAPY
 Gene therapy is the means of delivering
exogenous genetic material for therapeutic
purposes into the host cell target using
vectors.
 As of 2012, some 1800 human clinical gene
therapy trials (including phase I to IV) exist
worldwide.

34.  The disease processes involved can be broadly divided into four groups as listed in descending
order of number of clinical trials:
 cancer
 monogenetic diseases such as cystic fibrosis (CF)
 haemophilia B, cardiovascular disease
 infectious disease trials, which entirely comprise human immunodeficiency virus (HIV)
 The treatment aims to replace or repair the defective gene causing a given disease or to provide
a new or altered function in a cell.
 The most common vectors are a variety of replication-deficient viruses although non-viral
vectors, such as liposomes, are also used.

35. DELIVERY
 The common goal of all gene therapy is to
achieve expression of the gene of interest in
the targeted cell.
 (i) Targeting – ideally only cells which require
the gene would be affected
 (ii) Binding and internalization (transfection) –
once a gene reaches the cells it must bind and
become internalized
 (iii) Cellular trafficking to the nucleus
(transduction) – most methods of
internalization require the gene to escape from
endosomal degradation and traffic through the
cell to the nucleus
 (iv) Nuclear expression – once in the nucleus
the quantity of gene expression and stability of
expression for a given strategy also need to be
determined.

36. VECTORS OF GENE THERAPY

37. ADENOVIRUS
 Non-encapsulated, DNA virus.
 Adenovirus efficiently infects both dividing and non-dividing cells by binding
to the cox-adenovirus receptor.
 Once a cell binds the viral vector, adenovirus is internalized, escapes from the
endosome and is trafficked to the nucleus very efficiently.
 Even if a specific cell is lacking viral receptors, the virus can be combined
with complexes to be internalized via non- receptor-mediated mechanisms.

38. DRAWBACKS
 It is immunogenic that which limits its ability to re-infect.
 Both cell- and humoral-mediated immune systems are activated after viral
delivery, especially if >10 particles are delivered.
 Besides limiting redelivery, this immune response also results in the clearance
of cells expressing the transgene.
 Immune modulation at the time of delivery –may decrease inflammation

39. ADENO-ASSOCIATED VIRUS
 AAV -- single- stranded encapsulated virus belongs to the group of human
parvoviruses.
 Advantages-
 It infects human cells this virus has not been associated with a pathologic
human disease.
 Recombinant AAV vectors do not encode viral proteins, delivery of this vector
results in very little immunogenicity.
 AAV also persists and infects dividing and non- dividing cells.
 AAV viral vector harbouring human lipoprotein lipase AAV, alipogene
tiparvovec (Glybera®) became the first commercially available gene therapy
drug in Europe to treat familial lipoprotein lipase deficiency disease in 2012 .

40. DISADVANTAGES
 limited loading capacity (up to 4.2kb)
 Inefficient expression and delayed onset of expression
 however, newer generations of recombinant AAV are now becoming available
and are proven to have shorter turnover of transcription and enhanced gene
expression potencies.

41. RETROVIRUS/LENTIVIRUS
 Substantial advantage of persistent gene expression.
 Once internalized the RNA genome is reverse-transcribed and transported to the nucleus
where it integrates as a pro- virus into the host chromosome.
 In all retroviral gene therapy vectors, the genome of the virus has been made replication-
incompetent by removing the components of the viral gene required for packaging.
Limitation- only infect dividing cells
lentivirus genus of retroviruses-- advantage of infecting
dividing and non- dividing cells .

42. NON-VIRAL VECTORS
 Naked DNA is one of the most extensively studied methods of gene therapy.
 ADVANTAGES-
 Simplicity
 Ease of large scale production
 Minimal immune response and safety
 THE MAJOR OBSTACLE for plasmid gene therapy is efficiency. When DNA is
placed in an organism, most of the DNA is not internalized. Even if
internalization does occur, endosomal degradation destroys nearly all of the
remaining plasmid .
 Plasmid uptake is not receptor-mediated, targeting of the plasmid to a speci c
cell also remains a major obstacle.
 plasmid-mediated transfer results in transient expression because the plasmid
is lost with cell division.

43. APPLICATIONS OF GENE
THERAPY

44. CYSTIC FIBROSIS
 It is inherited with an autosomal recessive pattern .
 CF is caused by mutations in a cystic fibrosis transmembrane conductance
regulator (CFTR) gene on the long arm of chromosome 7.
 This gene encodes a CFTR protein, which has been shown to be a cAMP-
dependent chloride channel on the apical surface of the epithelium.
Abnormalities of CFTR impairs the chloride channel and dysregulates salt and
water transport across a variety of epithelia.
 This produces the increase of salt in sweat, obstructive pathology of the lung,
pancreatic insufficiency, malabsorption of the GI tract and male infertility
due to the lack or stenosis of vas deferens.

45. THE NASAL MODEL FOR GENE
TRANSFER IN CYSTIC FIBROSIS
 While the eventual goal of CF gene therapy will clearly be intra-pulmonary
adminis- tration, there are clearly some dif culties using the lungs for routine
experimentation.
 In a dose-escalating trial reported by Crystal et al.40 in 1994 of intra-
pulmonary adenoviral vector administra- tion, the patient who received the
largest dose developed a signi cant adverse reaction with opacities on chest
X-ray.
 The risk of a signi cant adverse reaction in a localized area of the nose or
sinuses is clearly less.

46.  The nasal potential in CF provides a measurable end- point for gene transfer
experimentation.
 In CF, the nasal mucosa, like the lung mucosa, exhibits the characteristic
chloride transport abnormality.
 This potential can be readily and reproducibly measured, in contrast to a
number of bronchopulmonary indices and has been a target of gene therapy
strategies in the nose.

47. THE HUMAN CF GENE THERAPY
TRIALS: ADENOVIRUS VECTOR
 Recombinant viruses are produced by replacing the DNA sequence responsible
for replication with CFTR cDNA. The viruses are thus replication-deficient,
but still remain sufficiently active to transport genetic material into the
target cell.
 Adenovirus vectors, while efficient delivery vehicles in the laboratory setting,
clearly have difficulties in human trials.
 The human respiratory tract has evolved to efficiently repel a variety of
microbial attacks, including of course adenoviruses.
 Adenovirus does not integrate into the genome and expression is transient
thus repeat administration is required. With repeat administration come
concerns of inflammation and immunogenicity.

48. ADENO-ASSOCIATED VIRUS VECTOR
 Serotypes 5 and 6 of the adeno-associated virus (AAV) enter airway cells from the
apical surface.
 AAV are thought to have the least cell toxicity and minimal immunoreaction
compared to other viral vector counter-parts
 AAV are also associ- ated with limited loading capacity, inef cient expression and
delayed onset of expression, thereby may not be an ideal vector in treating CF.

49. NON-VIRAL VECTORS
 These have included puri ed or naked DNA in plasmid form or ballistic gene
delivery, the so-called gene gun.
 Only exposed surfaces accessible to a microcarrier coated with DNA are candi-
dates for the gene gun. Although successful in mice lungs this approach is dif
cult to incorporate in animals with larger lungs due to the lack of equipment
design that would match the lung size.
 Most interest in non-viral gene delivery has centred on liposomes. Liposomes
bind to DNA, spontaneously forming complexes that have high affinity for
plasma cell membranes.
 They are non-immunogenic and there is no poten- tial for insertion
mutagenesis. The main dif culties with non-viral vectors relate to transient
expression and less ef cient gene transfer as compared to viral vectors.

50. HEAD AND NECK CANCER
 The approaches to target specific cancer cells fall into four basic categories:
 (i) chemosensitization;
 (ii) cytokine gene transfer;
 (iii) inactivation of protooncogene production; and
 (iv) selective oncolytic viruses.

51. Chemosensitization
 Selective sensitization of cancer cells using gene therapy would be an ideal
way to kill cancer cells. Using this approach, the expected gene is delivered
only to cancer cells and then a second therapy (e.g. radiotherapy or che-
motherapy) is used to induce killing in the cells that express the transgene.
 The best example of this model is delivery of herpes simplex thymidine
kinase (HSV-TK).
 In this strategy, HSV-TK is delivered to cancer cells. Once expressed, this
enzyme changes the prodrug gancyclovir to its toxic nucleoside analogue,
which induces cell death.
 In the bystander effect, the infected cell spreads the expressed genes to the
cells surrounding it via cell–cell contacts.

52.  Transfer of p53 is another example of chemosensitization
 Methods to increase cancer targeting may make these strategies more
amenable for human trials in the future.
 These strategies include redesigning the adenovirus binding site to increase
cancer cell selectivity.

53. Cytokine gene transfer
 Immune suppression increases the risk of cancer development.
 It has been shown that individuals with head and neck cancer lack an
effective local immune response even early in the disease .
 This dysfunction occurs as a result of the normal immune system not
recognizing the tumour cells.
 Causes of this include-
 immunological ignorance
 downregulation of major histocompatibility complexes
 loss of costimulatory receptor and pathways.

54.  One method to break this immune dysregulation is to overexpress the
downregulated cytokines.
 When IL-2 is administered systemically, significant toxicity develops,
including capillary leak syndrome.
 Gene therapy offers the ability to increase local expression and possibly
improve tumour response while limiting systemic toxicity.

55.  Even if such single agent cytokines do not prove useful, it may be possible to
deliver combinations, which activate the immune pathway in a synergistic
manner.

56. Inactivation of protooncogene production
 Restoring the function of a key cellular gene whose dysfunction has resulted
in cancer progression can be a major goal of gene therapy.
 The most common mutations of these genes in squamous cell cancer of the
head and neck are p53 and p16.
 This gene plays a role in triggering cell death in many different path- ways
involving apoptosis.

57. Adenoviral vector placed intralesionally along with mutated gene
33 patients with locally recurrent SCC in phase-1 trial
SURGICAL NON SURGICAL
Pts. Received treatment pre-
op/intraop/and post-op
At end of 1yr
4 alive 9 died of ds 2 died of
unrelated
causes
At end of 1 yr
2 showed
partial
response
6-stable ds
9-progressive
ds

58.  Gendicine, a drug with modified adenovirus harbouring p53 gene was
approved by the Chinese State Food and Drug Administration to treat head
and neck cancer in China in 2004, becoming the first gene therapy approved
for clinical use in humans.
 However, the western version of Ad-p53 (AdvexinR) for the treatment of head
and neck cancer was refused for FDA approval in the USA in 2008.
 The overall efficacy of Ad-p53 therapy in head and neck cancer continues to
be closely evaluated.

59. SELECTIVE ONCOLYTIC VIRUS
 Using replication-selective viruses to treat cancer is not entirely new.
 The ONYX-015 is an adenovirus therapy design to accomplish this goal.
 ONYX-015 is missing the adenovirus E1B gene that normally inhibits the
cellular p53 genes.
 p53 cellular expression allows only minimal viral expression.
 Approximately 60% of squamous cell carcinoma (SCCA) do not express
functional p53. The ONYX-015 virus will selectively replicate only in SCCA
cancers with p53 mutations.

60. Conclusion
 Our increased understanding of cellular oncogenesis will lead to the development of novel
cancer therapies in the years to come.
 Gene therapy is likely to be a part of these therapies.
 A head and neck oncologist will need to understand both the viral vectors and their
strategies of implementation to offer a full range of treatment to the cancer patient.