Immunology and immunotherapeutics

Immunology and immunotherapeutics

1. Immunology and I mmunotherapeutics 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)

9. 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 IFN α/β, IFN γ, TNF α, IL-12 or contact dependent signals such as NKG2D

10. 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

11. Phagocytosis

12. Principle Cells of the Immune System

13. 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

14. 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

15. 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

16. Immunological Memory

17. 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

18. 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)

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. The Immune Synapse

26. 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

27. 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)

28. 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

29. 435 immune-based clinical trials currently open in oncology (27/10/15 NCI database search term ‘immunotherapy and cancer’)

30. 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

31. 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

32. 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

33. 415 patients assessed by IHC for CD3 infiltration Tumour infiltration by CD3+ T cells predicts OS

34. Milestones in Tumour Immunology

35. Immunoediting Model of Cancer Development

36. Immunoediting Model of Cancer Development - Evidence for Elimination

37. Immunoediting Model of Cancer Development - Evidence for Equilibrium

38. 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!

39. M2 macrophage: Express TGF β, 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

40. 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 >TNF α, IL-1 β – 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

41. 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

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

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

44. 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. α CTLA-4, α PD-1 / α PD-L1

45. 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

46. 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

47. 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)

48. 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

49. • 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

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

51. 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

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

53. The Immunogenicity of Radiotherapy

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

55. The Immunogenicity of Radiotherapy

56. 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)

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

58. 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

59. 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

60. 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

61. Thank You!!!