Immunology and immunotherapeutics

1. Immunology and
Immunotherapeutics
Dr Chandana Sanjee
DNB Resident
HCG Hospitals

2. • Innate immune system is absolutely
indispensible for survival
• Comprised of both cellular and acellular
components
• CO-ORDINATES the ADAPTIVE SYSTEM
• Direct effects on pathogens: Phagocytosis,
lysis

3. Innate Immune System
• First line defense;
response is the same
each and every time (no
memory)
• Phagocytes:
Neutrophils, monocytes
& macrophages
• Natural killer (NK) cells

4. System / Organ Active component Effector mechanism
Skin Squamous cells , Sweat Flushing , organic acids
Serum Lactoferrin and Transferrin Iron binding
Serum IFN
TNF-alfa
Lysozyme
Fibronectin
Complement
Anti-viral proteins
Anti-viral phagocyte activation
Peptidoglycan hydrolysis
Opsonisation and phagocytosis
Opsonisation, phagocytosis and
inflammation

5. Key players in immune system

6. Innate Immune System
• First line defence; response is the same each
and every time (no memory)
• Non-specific: although can detect the
difference between self and non-self!
• Innate immune cells recognise
PathogenAssociated Molecular Patterns via
pattern recognition receptors
• TOLL-like receptor family considered to be the
primary sensors of pathogens

7. Pathogen recognition: TOLL-like
receptors (TLRs)

8. Monocyte/ macrophage
• Play a major role tissue repair, clearance of
apoptotic cells and necrotic debris
• Potent APCs (antigen presenting cells)

10. Natural Killer cell
• Activation of NK cells
depends on the balance
of stimulatory and
inhibitory signals. The
Killer Inhibitory
Receptor (KIR) searches
for MHC-I (present on
virtually all nucleated
cells) – ligation of the
KIR with MHC-I
prevents cell death
• NK cells generally
require signals from
accessory cells for
activation – these can
be soluble factors i.e.
proinflammatory
cytokines IFNα/β, IFNγ,
TNFα, IL-12 or contact
dependent signals such
as NKG2D

11. Phagocytosis
• Innate cells such as neutrophils, macrophages
and dendritic cells are highly phagocytic and
can engulf free bacteria/virus and whole cells
that are either infected or
• The formation of a phagylysosome acidifies
the contents and leads to proteolytic
degradation of the pathogen

12. Phagocytosis

13. Principle Cells of the Immune System

14. Adaptive Immune System
• Comprised of T and B lymphocytes: both
derived from the bone marrow
• Cells do not rely on recognition of pathogen
associated molecular patterns like the innate
system
• Express receptors that can differentiate
between self and non-self by sequence-
specific recognition of antigen

16. T cells
• Cytotoxic T cells (TC , CD8+ ): effector immune cells
that can identify and kill infected / neoplastic cells
• Helper T cells (TH , CD4+ ): produce cytokines that
can promote CD8+ (e.g. IL-12) or B cell activation
and function (e.g. IL-4)
• Regulatory T cells (Treg, CD4+CD25+FOXP3+ ):
mediate peripheral tolerance of CD8+ T cells
through production of both soluble factors e.g.
TGFβ or cell-cell contact e.g. CTLA-4

17. Adaptive Immune System
• Comprised of T and B
lymphocytes: both derived
from the bone marrow
• Specific and can differentiate
between infections agents
e.g. HSV/HPV
• Adaptive Immune System
Adaptive: ability to ‘learn’
and respond more efficiently
during subsequent infections
>> Immunological Memory

18. Immunological Memory

19. Key concept - Clonality
• Each lymphocyte is specific for a restricted
number of antigens
• Specificity is developed randomly - many
lymphocytes never encounter an antigen
• During a primary response few cells are
specific (perhaps ~1/50,000-1/100,000)
• Antigen specific cells divide and produce
effector and memory populations
• ~50% of all T cells in adults memory, the result
of previous antigen exposure

20. What are T cell antigens?
• T cell antigens are peptides typically 9 - 12 amino
acids in length
• Recognition is sequence specific
• Bound in the groove of an antigen-presenting
Major HistoCompatibility antigen
• Antigens are presented by specialised antigen
presenting cells (APC)
• T cells require the capacity to respond to non-self
but must be tolerant of self antigens (or will
produce autoimmunity)

21. T-Cell Receptor-MHC Interaction
• TCR does not recognize
MHC antigens alone
• The TCR sees both MHC
and the peptide complexed
with it
• The whole complex defines
a TCRs specificity
• If the combined affinity
(1+2+3) is above a certain
value the T-cell is activated
and can perform its
effector functions

22. The Major Histocompatibility Complex
• Region on the short-arm of
chromosome 6
• Contains the genes for many
proteins of importance for
the immune response
• Of primary relevance for T
cell immunity are the human
leukocyte antigens (HLA);
these are sometimes called
MHC antigens Class I: HLA-A,
HLA-B, HLA-C Class II: HLA-
DR, HLA-DP, HLA-DQ
• Each person expresses 2
variants of each antigen; HLA-
type

23. MHC Polymorphism
• Class I MHC Molecules -
HLA-A
n = 893
• HLA-B
n = 1,431
• HLA-C
n = 569
• Σ= 3,007 alleles
• Class II MHC Molecules
HLA-DR (A + B chain)
n = 817
• HLA-DP (A + B chain)
n = 164
• HLA-DQ (A + B chain)
n = 141
• Σ= 1,154 alleles

24. MHC class I binds intracellular peptide
antigens
• MHC-I presents viral /
mycobacterial / self & mutated
peptides
• MHC class I (MHC-I) is expressed by
virtually all nucleated cells
• Antigens are processed into
peptides in the
immunoproteosome and shuttled
into the ER via the transporter
associated with Ag processing (TAP)
• Peptides are loaded onto MHC-I
and can present antigen only to
CD8+ T cells

25. MHC class II binds extracellular peptide
antigens
• MHC class II (MHC-II) presents
exogenous antigens following
phagocytosis
• Expression of MHC-II is restricted
to professional Antigen Presenting
Cells (APC)
• Antigens are processed in through
the endolysosomal pathway and
loaded onto MHC-II
• Peptides bound to MHC-II can
present antigen only to CD4+ T
cells

26. MHC class II binds extracellular
peptide antigens
• MHC class II (MHC-II) presents
exogenous antigens following
phagocytosis
• Expression of MHC-II is restricted to
professional Antigen Presenting Cells
(APC)
• Antigens are processed in through the
endolysosomal pathway and loaded
onto MHC-II
• Peptides bound to MHC-II can present
antigen only to CD4+ T cells
• Professional APC can also load
exogenous antigen onto MHC-I for
presentation to CD8+ T cells

27. The Immune Synapse

28. Review of Antigen Presentation
• MHC-I is present on virtually all nucleated cells
permitting CD8+ T cells to scan intracellular
antigens and identify / kill infected cells and those
expressing altered self peptides
• T cell activation requires 2 simultaneous signals TCR
- MHC Co-stimulation e.g. CD28 (T cell) – CD80/86
(APC)
• Not all cells can license T cell activation - Most
efficient 3. APC is the Dendritic Cell
• Once activated, T cells can mediate cytotoxicity
through a variety of mechanisms e.g.
perforin/granzyme

29. The Dendritic Cell (DC)
• Immature DC Non Stimulated
DC (Day 10 of Culture x40)
• Mature DC Stimulated with
1.25x105 cfu/ml of BCG (Day 10
of Culture x40)

31. • In addition to being APCs, Dendritic Cells express a
diverse array of cytokines/chemokines that
influence the intensity and phenotype of the
nascent immune response

32. 435 immune-based clinical trials currently open in oncology
(27/10/15 NCI database search term ‘immunotherapy and
cancer’)

33. Immunotherapeutics- Objectives
• To consider the role of the immune system
during cancer initiation and progression
• Consider the processes that lead to immune
failure in the control of neoplastic disease
• To review some of the different
immunotherapeutic approaches being
developed in oncology

34. Immune system has 3 primary roles in
tumour prevention
1. Elimination of virus’s that drive neoplasia
2. Resolution of acute inflammation (i.e. to pathogens) to prevent
a chronic inflammatory environment that can directly influence
neoplastic transformation
3. Identification and elimination of transformed cells

35. This study in colorectal cancer characterised the
tumour-infiltrating immune cells in large cohorts
of patients by gene expression and in situ
immunohistochemistry. Data paired to long-
term outcome

36. • 415 patients assessed by IHC for CD3 infiltration
Tumour infiltration by CD3+ T cells predicts OS

38. Milestones in Tumour Immunology

39. Immunoediting Model of Cancer
Development

40. Immunoediting Model of Cancer Development - Evidence for
Elimination

41. Immunoediting Model of Cancer Development -
Evidence for Equilibrium

42. Immunoediting Model of Cancer
Development - Evidence for Escape
• Tumours may evolve by a Darwinian-selection
mechanism to circumvent the immune response or
may induce local immuno-suppression…. or both?
• When a immune cell recognises a foreign antigen
(i.e. viral/bacterial peptide) the result is normally
activation
• But when the immune system ‘sees’ a tumour cell
the result is often anergy or tolerance
• To launch an anti-tumour immune response we
must first break tolerance - Without eliciting an
auto immune disease!

43. • M2 macrophage: Express TGFβ, PGE2 , IL-10, IL-4
contribute to suppression of effector T cell
function and support Treg / tolerogenic DC
Differentiation. Express Indoleamine deoxygenase
(IDO) and Arginase (Arg)
• As for Tumour Associated Macrophage but also
expresses high levels of reactive oxygen species
that can modify the TCR
• Express TGFβ. Also express high levels of IL-2
receptor and can sequester available IL-2
depriving effector T cells of survival signal. Can
express CTLA-4 and LAG-3 which are negative
regulators of DC function.
• Express TGFβ and other suppressive cytokines.
Present antigens on class I and II MHC but in the
ABSENCE of co-stimulation

44. Tumour cells can drive immunological
tolerance
• Tumour cells actively induce tolerance – Loss of
MHC expression
• Up-regulation of inhibitory molecules e.g. PD-L1,
CTLA-4 >TNFα, IL-1β –normally associated with
infection and cellular stress, ischaemia > Low level
of damage-associated molecular patterns (DAMPs)
• Sterile environment i.e. no pathogen associated
molecular patterns (PAMPs).
• Also lack of ‘danger’ signals e.g. Heat shock
proteins, Inflammatory factors

45. Tumour cells can drive immunological
tolerance
• Tumour cells can
produce
immunosuppressive
factors e.g. TGF-b, IL-10,
PGE2 , IDO
(indoleamine
deoxygenase), arginase
• Many of these are
targets for
immunotherapy

46. Generation of anti-tumour T-cell responses can be
modulated at multiple levels

47. Generation of anti-tumour T-cell responses can
be modulated at multiple levels

48. Immunotherapeutic Strategies
1.Therapeutic vaccination
• Various strategies employed e.g. Autologous DC-
based vaccines, Allogeneic tumour cell vaccines,
recombinant viral vectors delivering Tumour
associated antigens (TAAs +/- costimulatory
molecules), oncolytic virus’s
2.Targeting co-stimulatory / co-inhibitory pathways
– Targeting immune checkpoints
• Specific agonists i.e. TLR-ligands
• Antagonists i.e. αCTLA-4, αPD-1 / αPD-L1

49. Dendritic cell-based cancer vaccine –
Provenge
• First ever cancer vaccine to achieve FDA approval (April
2010) for metastatic castration resistant prostate cancer
• Tumour antigen: Prostatic acid phosphatase, expressed in
the prostate and elevated in cancer. This is linked to
Granulocyte Macrophage Colony Stimulating Factor (GM-
CSF) stimulating DC maturation

50. Dendritic cell-based cancer vaccine –
Provenge
• Phase III IMPACT trial in asymptomatic or minimally
symptomatic metastatic Castration-resistant
prostate cancer – Patients received either
Sipuleucel-T q2w x 3 or placebo

51. Targeting immunological checkpoints
• Numerous options for
investigation!! These are
the various ligand/receptor
interactions between an
APC and a T cell
• CTLA-4
• B7-H1 (PD-L1)
• PD-1
• TLRs (TOLL-like receptors)

52. Cytotoxic T-Lymphocyte Associated
Protein-4 (CTLA-4)
• Ipilimumab (Bristol-Myers
Squibb) ~16 trials in
melanoma, prostate,
pancreatic Tremelimumab
(CP-675,206) MedImmune
~17 trials Phase I-III in
melanoma, pancreatic,
colorectal, prostate,
bladder, renal, NSCLC

55. • 62% 1 year survival, 43% 2 year
survival
• Median response duration in patients
with objective tumour regressions (31%)
was 2 years
• 71% of patients maintained responses
following treatment discontinuation

60. Anti-cancer immune responses are modulated at multiple levels
– NEED FOR COMBINATION

61. • Phase 2 study in first line
• Response rates were 61%
for ipi/nivo combination vs.
11% for ipi alone
• CR in 22% of combined
group and 0% for ipi alone
• Grade 3 or 4 AE’s reported
in 54% in combined
therapy vs 24% for ipi
alone
• This led to 38% and 13%
patients with grade 3 or 4
AEs discontinuing therapy

62. Anti-cancer immune responses are modulated at multiple levels
– NEED FOR COMBINATION

63. The Immunogenicity of Radiotherapy

64. Solid tumours generate an environment that
suppresses anti-tumour immune responses

65. The Immunogenicity of Radiotherapy

66. Preclinical tool compound
• R848: selective murine
TLR7 agonist (also hits
TLR8 in man) - a more
potent analogue of
Imiquimod (which is
approved for topical
treatment of BCC and
genital warts)

68. Systemic TLR7 therapy improves survival in a mouse model of T cell
lymphoma

69. • Patients had low-grade B-
cell lymphoma (relapsed)
n=15 • Low-dose RTx was
administered to a solitary
tumour site (2x2Gy
fractions) • CpG (PF-
3512676; Pfizer) 6mg
intra-tumoural
immediately before the
first radiation dose, after
the second dose and q1w
x 8

70. Intratumoural vaccination induces objective clinical
responses
A) Complete response in
patient 3, treated site:
occipital; visualized site:
bilateral axillae.
B) Partial response in
patient 10, treated site:
suprasternal cutaneous;
visualized site: supra-
orbital cutaneous

71. Conclusion
• Targeting one molecule / pathway is not enough for
therapy due to redundancy in pathways
• Combination may be key to tackle overlapping layers of
immunosuppression
• »i.e. with adoptive T cell therapy, multiple checkpoint
blockade and/or non-myeloablative therapies i.e.
Radiotherapy
• Need for a personalized approach
• –Biomarker assays that can predict responders/non-
responders
• –Patient selection criteria

72. Thank You!!!