Assignment on Immunotherapy, Types of immunotherapeutics, humanisation antibody therapy, Immunotherapeutics in clinical practice

1. IMMUNOTHERAPY
Immunotherapy is the treatment of disease by influencing
the immune response given by the immune system.
TYPES
Active immunotherapy
Passive immunotherapy

2. IMMUNOTHERAPY
Active immunotherapy
It is the type of immunotherapy that attempts to stimulate the host intrinsic
immune response to a disease.
• Specific active immunotherapy
• Non specific active immunotherapy

3. CONTINUED….
Specific active immunotherapy
The generation of cell mediated and antibody immune responses focused on
specific antigen.
E.g. cancer vaccines
• Cellular therapies
• adjuvants

4. CONTINUED…
• Cancer vaccines
cancer vaccines are active immunotherapy because they meant to trigger the
patient immune system to respond.
cancer vaccines may contain cancer cells ,part of the cell , or purified tumor
specific antigen.

5. CONTINUED…
Two categories of cancer vaccine
• cell based
in which the patient cancer cell is cultured with patients own immune
system cells and derived back to the same patient.
• Vector based
in which the engineered virus or other vector is used to introduce cancer
specific proteins and other molecule in order to stimulate the patient immune
system to recognize the tumor cells to fight the cancer.

6. CONTINUED…
Examples
• Tumor cell vaccines-kidney , ovarian breast cancer
• Antigen vaccines-prostate , colorectal cancer.
• Dendritic cell vaccine
• DNA vaccines
• Vector based vaccines

7. CONTINUED…
• Cellular therapies
These are single type agent derived from the cancer patients which are
modified in the lab. to become more adapt at recognizing and killing the tumor
cells
this type of immunotherapy is designed to boost specific part of immune system
to cause tumor cell death
Vaccines in contrast attempts to get the body immune system react to specific
antigen
E.g. lymphocyte activated killer cell therapy

8. CONTINUED…
• Adjuvant immunotherapy
an adjuvant is an any material which when injected together with an antigenic
protein or other substance like monoclonal antibodies , cancer vaccines increases
or boost the immune response to the particular system.
E.g. BCG vaccine

9. CONTINUED…
Non specific immunotherapy
the generation of general immune system response using cytokines
Non specific active immunotherapy
• Cytokines
destruction of tumor cell by two mechanism
• direct antitumor
e.g. TNF alpha,IL-6
• Indirect enhancement of antitumor response
Eg;IL2 promote T-cell and NK cell growth

10. PASSIVE IMMUNOTHERAPY
Passive immunotherapy
This comprised of antibodies and other immune system component that are
made outside the body and administered to the patient to provide immunity
against the disease.
It do not stimulate a patient immune system to actively respond to a disease in
the way vaccines does.

11. CONTINUED…
• Monoclonal antibodies therapy
• Cytokine inhibitors
• Tolerance induction
• IV immunoglobulin
• Haemopoitic stem cell transplantation

12. CONTINUED…
• monoclonal antibody therapy
• Types
Naked monoclonal antibodies,
e.g. Cetuximab, trastuzumab
Conjugated monoclonal antibodies
antibodies contain immunotoxin eg.gemtuzumab
Radiolabelled antibodies eg;tositumomab
Chemolabelled antibodies eg;brentuximab

13. CONTINUED…
• CYTOKINES INHIBITOR
These are cytokine specific substance that inhibit the biological activities of
specific cytokines in a number of different ways
• Production can be blocked e.g. etanercept
• Intracellular process which produce the active protein can be inhibited
• Cytokines can be neutralized in the circulation e.g. infliximab
• Specific receptor can be blocked e .g. kineret

14. CONTINUED…

15. CONTINUED….
IV immunoglobulins
• It contains the pooled IgG extracted from the plasma of over one
thousand blood donors.
• IVIG's effects last between 2 weeks and 3 months.
It is mainly used as treatment in three major categories:
• Immune deficiencies .
• Autoimmune diseases e.g. Immune thrombocytopenia
• Acute infections.

16. CONTINUED…
• IVIG dose
• Dosage of IVIG is dependent on indication.
• For primary immune dysfunction 100 to 400 mg/kg of body weight
every 3 to 4 weeks is implemented.
• For neurological and autoimmune diseases 2 grams per kilogram of
body weight is implemented for three to six months over a five day
course once a month. Then maintenance therapy of 100 to
400 mg/kg of body weight every 3 to 4 weeks follows.

17. IMMUNOMODULATOR
Immunomodulatory are drugs that either suppress or stimulate the
immune system.
• Immunostimulants
that stimulates the immune system.
• Immunosuppressant
that suppress the immune system

19. Mechanisms of immunomodulation
Drugs may modulate immune mechanism by either suppressing or by
stimulating one or more of the following steps:
• Antigen recognition & phagocytosis
• Lymphocyte proliferation/differentiation
• Synthesis of antibodies
• Ag –Ab interaction
• Release of mediators due to immune response
• Modification of target tissue response

20. CONTINUED…
• The importance of immune system in protecting the body against
harmful molecules is well recognized
• However, in some instances, this protection can result in serious
problems
• E.g, the introduction of allograft can elicit a damaging immune
response causing rejection of the transplanted tissue

21. CONTINUED..

22. CONTINUED..

23. Characteristics of an ideal immunomodulator
Should be
• Stimulate both specific and non specific immune response
• Act as an adjuvant along with vaccine
• Active through oral route
• Compatible with other drugs
• Short withdrawal period with low tissue residues
• Defined chemical composition and biological activity
• Inexpensive

24. IMMUNOMODULATORS
……Should not be
• Toxic
• Antigenic
• Pyrogenic
• Long side effects in the body

25. IMMUNOSTIMULANTS
• Immunostimulants are substances that stimulate the
immune system by inducing activation and increasing
activity of any of its components.
• They are used in disorders includes immunodeficiency
diseases, cancer, viral, fungal and certain autoimmune
disorders

26. CONTINUED..

27. BACILLUS CALMETTE GEURIN(BCG)
Live, attenuated culture of BCG strain of Mycobacterium Bovis
Mechanism of action:
BCG and its methanol extracted residue contain muramyl dipeptide as an active
immunostimulant ingredient.
It causes activation of macrophages to make them more effective killer cells.
It induces the production of lymphocyte activity factor resulting of phase 1 of
immune response.
Therapeutic uses:
Treatment and prophylaxis of Bladder Carcinoma and acute lympholytic
leukaemia
Adverse Reactions:
Hypersensitivity
shock
chills

28. LEVAMISOLE
• Levamisole was synthesized originally as an anthelmintic.
• It is orally active levo isomer of tetramisole,restores depressed T-cell
function
• Mainly act by raising c-GMP levels through interaction with thymopoietin
receptor sites
• Lead to decrease in metabolic inactivation of c-GMP accompanied with increased
breakdown of c-AMP
• Increase in c-GMP level induces lymphocyte proliferation & augmentation of
chemotactic responses
• This reflects into increased antibody production lymphocyte production,
increased phagocytosis.

29. CONTINUED..
Therapeutic uses:
• Adjuvant therapy with 5- fluorouracil colon
cancer, Used to treat
immunodeficiency associated with Hodgkin
disease,Rheumatoid arthritis
Adverse Reactions:
• Flu like symptoms, allergic manifestation,
• nausea and muscle pain

30. Recombinant cytokines
These are now use by rDNA technology
Application in treatment of viral infection, autoimmune and neoplastic diseases.
INTERFERONS: Interferon alpha-2b,Interleukin 2
Mechanism of action:
Low molecular weight glycoprotein cytokines produced by host cells in response to viral infections &
suppresses cell proliferation; although the exact mechanism of action is not known
Bind to cell surface receptors and initiates intracellular events like
• Enzyme induction
• Inhibition of cell proliferation
• Enhancement of immune activities
• Increased phagocytosis
• Affect viral replication

31. CONTINUED…
Therapeutic uses
Hairy cell leukemia
Malignant melanoma
Hepatitis B
Adverse Rections:
Flu like symptoms – Fever, chills, headache
CVS – Hypotension, Arrhythmia
CNS- Depression, Confusion

32. INTERLEUKIN
It is a protein that regulates the activities of white blood cells (leukocytes,
often lymphocytes) that are responsible for immunity. IL-2 is part of the
body's natural response to microbial infection, and in discriminating
between foreign ("non-self") and "self". IL-2 mediates its effects by
binding to IL-2 receptors, which are expressed by lymphocytes.
Therapeutic uses:
Metastatic renal cell carcinoma
Melanoma
Toxicity
Hypotension

33. ISOPRINOSINE
Leads the production of cytokines such as IL-1, IL-2, and IFN- 𝝲𝝲,
increase proliferation of lymphocytes in response to mitogenic or
antigenic stimuli
Therapeutic uses:
Herpes simplex infection, Measles viruses
Adverse reactions:
Rise in uric acid in serum and urine, Nausea

34. ACTIVE IMMUNIZATION
• Vaccines
•  Administration of antigen as a whole, killed
• organism, or a specific protein or peptide
• constituent of an organism
•  Booster doses
•  Anticancer vaccines

35. IMMUNOSUPRESSANT MECHANISM-
OVERVIEW
• 1.Inhibition of gene expression
• 2.Selective attack on clonally expanding
lymphocytes
• 3.Inhibition of intracellular signalling
• 4.Neutralisation of Cytokines & receptors
required for T-cell stimulation
• 5.Selective depression of T-cells (or others)
• 6.Inhibition of co-stimulation by APC
• 7.Inhibition of Lymphocyte-target cell
interactions
• 8.Supressionof innate immune cells &
complement activation (not shown here)

36. CLASSIFICATION-IMMUNOSUPRESSANT
• Immunosupressants
• Glucocorticoids
• Calcineurin Inhibitors
• Antiproliferative/Antimetabolites
• Biologicals(Antibodies)

37. GLUCOCORTICOIDS
Glucocorticoids have broad anti-inflammatory effects on multiple components of cellular
immunity e.g-cortisone,betamethasone
MOA –
Binds with glucocorticoid receptor
Glucocorticoid glucocorticoid receptor complex translocate to nucleus
Binds to GREs in specific gene
Regulate the gene expression
Down regulate the inflammatory mediators

38. GLUCOCORTICOIDS-uses
• combined with other immunosuppressive agents to prevent and treat
transplant rejection.
• graft-versus-host disease in bone-marrow transplantation.
• Glucocorticoids are routinely used to treat auto-immune disorders such as
RA, psoriasis
Asthma and other allergic disorders,
inflammatory bowel disease
acute exacerbations of MS (see "Multiple Sclerosis").
• to block first-dose cytokine storm caused by treatment with muromonab-
CD3 and to a lesser extent ATG

39. GLUCOCORTICOIDS-toxicities
• Growth retardation in children,
• Avascular necrosis of bone, osteopenia
• Increased risk of infection
• Poor wound healing
• Cataracts
• Hyperglycemia
• Hypertension

40. CALCENEURIN INHIBITORS
• The most effective immunosuppressive drugs in routine use
• They target intracellular signaling pathways induced as a consequence of T cell–
receptor activation
DRUGS-Tacrolimus,Cyclosporine
Both drugs are functionally same but cyclosporine binds to cyclophilin-binding
protein&tacrolimus binds to FKBP-FK binding protein
Activated T-cells produces IL2 via dephosphorylation of NAFT(nuclear factor
activated T cell)
Calceneurin is needed for dephosphorylation
Translocate to nucleus
Transcription of IL2 gene(helps in further proliferation of T-cells)

41. CONTINUED..
USE -
• prophylaxis of solid-organ allograft rejection
• Clinical indications for cyclosporine are kidney, liver, heart, and other organ
transplantation
• Rheumatoid arthritis and psoriasis.
ADR-
• Nephrotoxicity,
• GI complaints,
• hypertension,
• tremor,
• hirsutism,
• hyperlipidemia,

42. SIROLIMUS
• Sirolimus is a macrocycliclactone produced by Streptomyces
hygroscopicus
PK-
•Systemic availability is 15%
•A loading dose of three times the maintenance dose will provide nearly
steady-state
•CYP3A4
USE –
•Prophylaxis of organ transplant rejection usually in combination
•At risk of calcineurin inhibitor–associated nephrotoxicity

43. MOA of Sirolimus

44. AZATHIOPRINE
• It is an imidazolylderivative of 6-mercaptopurine
• MOA –
• cleaved to 6-mercaptopurine (via Glutathione),
• A fraudulent nucleotide, 6-thio-IMP, is converted to 6-thio-GMPand finally to 6-
thio-GTP, which is incorporated into DNA.
• converted to additional metabolites that inhibit de novopurine synthesis
• Cell proliferation thereby is inhibited, impairing a variety of lymphocyte
functions.

45. AZATHIOPRINE
USE-
• adjunct for prevention of organ transplant rejection
• Severe RA
ADR –
• The major side effect of azathioprine is bone marrow suppression
• increased susceptibility to infections (HSV),
• hepatotoxicity, alopecia, GI toxicity, pancreatitis,

46. MYCOPHENOLATE MOFTEIL
• It is an ester of mycophenolic acid (MPA).
MOA –
• MMF is a prodrug that is rapidly hydrolyzed to the active drug, MPA
• MPA -a selective, noncompetitive, reversible inhibitor of inosine monophosphate
dehydrogenase (IMPDH),
• IMPDH is an enzyme @ de novo pathway of guanine nucleotide synthesis
• B and T lymphocytes are highly dependent on this pathway for cell proliferation
(others-salvage pathway)

47. MYCOPHENOLATE MOFETIL

48. CONTINUED..
USE-
• MMF is indicated for prophylaxis of transplant rejection, and it typically is used in
combination (Glucocorticoid/Calcineurin)
• off label -SLE
ADR –
• Gut -
• Hematologic –PRCA, leucopenia
• Congenital anomaly

49. ANTIBODIES

50. Mab Hybridoma Technique

53. ANTI IgE MONOCLONAL ANTIBODIES
• Drug- omalizumab
• It inhibits the binding of IgE to mast cell&basophils
• It inhibits the activation of IgE also that already bound to mast cells
&thus prevent their degranulation.
• Down regulates FCeR-1 receptor present on mast cells & basophils.
• That decrease allergic inflammation & asthma symptoms &
exacerbations.

54. HUMANISED ANTIBODY THERAPY
• What are antibodies?
An antibody is a protein used by the immune system to identify and neutralize
foreign objects like bacteria and viruses. Each antibody recognizes a specific
antigen unique to its target.
Monoclonal antibodies (mAb) are antibodies that are identical because they were
produced by one type of immune cell, all clones of a single parent cell.
• Polyclonal antibodies are antibodies that are derived from different cell lines.
They differ in amino acid sequence.

56. Practical steps in monoclonal antibody production:
1) Immunize animal
2) Isolate spleen cells (containing antibody-producing B cells)
3) Fuse spleen cells with myeloma cells (e.g. using PEG -
polyethylene glycol)
4) Allow unfused B cells to die
5) Add aminopterin to culture to kill unfused myeloma cells
6) Clone remaining cells (place 1 cell/well and allow each cell to
grow into a clone of cells)
7) Screen supernatant of each clone for presence of the desired
antibody.
8) Grow the chosen clone of cells in tissue culture indefinitely.
9) Harvest antibody from the culture supernatant.
10) (If you’re a biotech company) charge about $1,000-$2,000
per mg.

57. ANTIBODY MOLECULE

58. TYPES OF mAbs

59. WHY mAbs
Proven effective for a broad range of indications
Immunosuppression, inflammatory disease, infections and cancer
Have excellent safety profiles: highly specific
• Early decision by tissue cross-reactivity test (fast & clear
development path)
 Relatively short development cycle: 7 years vs 10-12 years
• Remarkably free of adverse effects and synergistic therapeutic
responses
Commercially successful
• High success rates and phase transition probabilities

60. HUMANIZEDANTIBODY THERAPY & IN
CLINICAL PRACTICE
Omalizumab is a humanized
antibody given in asthma
• Omalizumab is a recombinant
humanized antibody of IgG1k
subclass targeted against IgE.
• Approved by FDA on June 2003
for moderate to severe asthma

62. Omalizumab
• MOA
• 1) inhibits the binding of IgE to mast cells and basophils.
• 2) inhibits the activation of IgE already bound to mast cells and thus
prevent their degranulation.
• 3) down-regulates Fc€R-1 receptor present on mast cells and
basophils.

63. CONTINUED…
• Slowly absorbed, Bioavailability = 60 %
• Peak serum concentration is reached in 7 to 8 days
• Volume of Distribution = 78 +/- 32ml/kg
• Elimination of Omalizumab - IgE complexes occur in liver and reticuloendothelial
system and primarily excreted via hepatic degradation.
• Elimination half-life is 26 days
• A/E – Injection site reactions, headache, URTI, parasitosis,anaphylaxis, malignancy

64. OTHER ANTIBODIES IN TRIAL FOR ASTHMA
• MEPHOLIZUMAB-Phase 3 trial
• Reduced eosinophil entry in airways
• Dec. asthma exacerbation
• RESLIZUMAB-Phase 2 trial
• Pronuced in a subgroup of patients with highest blood and septum
eosinophils
• LEBRIKIZUMAB- Phase 3 trial
• Improvement of lung function in patients with moderate to severe
asthma

65. RHEUMATOID ARTHRITIS
• Systemic autoimmune inflammatory disease
• Chronic inflammation of synovial tissue lining of joint capsule
• Signs
• Tenderness with warmth and swelling over joints
• Symmetrical joint involvement
• Rheumatoid nodules
• Symptoms
• Joint pain/stiffness
• Muscle pain, fatigue, fever, loss of appetite
• Joint deformity

66. TOCILIZUMAB
First interleukin-6 receptor inhibitor FDA approved to treat adults with:
Moderate to severe active RA who have had an inadequate response to one or more TNF
antagonist therapies
4 mg/kg IV infusion over 1 hour q 4 weeks
Increase to 8 mg/kg based on clinical response
MOA
Human monoclonal antibody
Binds specifically to both soluble and membrane bound IL-6 receptors
Elevated IL-6 is an important mediator of articular inflammation:
Proinflammatory cytokine
Involved in physiological processes

67. TOCILIZUMAB
• Humanized mAb IgG1
(MW ~150 kd)
• Key Features:
• Binds soluble and membrane bound IL-6R
• Weak/no CDC* or
ADCC** effector
functions (in vitro
Heavy chain
Light chain
CDR region
CDC: complement-dependent cytotoxicity
**ADCC: antibody-dependent cellular cytotoxicity

68. IL-6 is Produced by Multiple Cell Types and Is
Associated with Numerous Biologic Activities1,2
Endothelial cellsMonocytes/
macrophages
T-cell activation
Hepatocytes
Acute-phase response
Hepcidin, CRP
↓ CYP450
Maturation of
megakaryocytes
Thrombocytosis
Osteoclast activation
Bone resorption
B-cells
Hyper-γ-globulinemiaAuto-antibodies (RF)
IL-6
Mesenchymal cells,
fibroblasts/
synoviocytes

69. IL-6 Has Numerous Articular Effects in RA
Synoviocytes
Osteoclast activation
bone resorption
Endothelial cells
VEGF
Pannus formation
Joint destruction
Mediation of chronic
inflammation
IL-6Macrophage
T-cell
B-cell
Neutrophil
Antibody
production

70. TOCILIZUMAB-side effects
• severe stomach pain with constipation,
• bloody or tarry stools,
• coughing up blood or vomit that looks like coffee grounds,
• painful blistering skin rash with burning/itching/tingly feeling,
• upper stomach pain,
• vomiting,
• runny or stuffy nose,
• sinus pain,
• sore throat,

71. IMMUNOTHERAPY IN NSCLC
• Immunotherapy targets in NSCLC:
CTLA-4 and PD-1 pathways (1 of 2)
CTLA-4 and PD-1 pathways are immune checkpoint pathways
that play critical roles in controlling T-cell immune responses1
Deactivated
CD8+ T-cell
Tumor
antigen
presentation
TCR
MHC
Tumor cell
PD1: PD-L1
binding
PD-L1
PD-1
Tumor cell growth
and proliferation
CTLA-4 pathway PD-1 pathway
T-cells can become unresponsive after CTLA-4 binds B7 molecules on APC,
or when PD-1 binds PD-L1 or PDL-2 on target cells

72. CONTINUED….
• Anti-CTLA-4, PD-1, or PD-L1 antibodies can restore
T-cell activation and killing of tumor cells1
Activated
CD8+ T-cell
Anti-CTLA-4 antibody
CTLA-4
CD28
B7
Tumor cell death
PD-L1
Cytolytic molecules
Activated
CD8+ T-cell
PD-1
Anti-PD-1 antibody
• Anti-CTLA and -PD-1 antibodies are associated with
unconventional response patterns and immune-related
adverse events1
APC, antigen-presenting cell; CTLA-4, cytotoxic T-
lymphocyte-associated antigen-4;
NSCLC, non-small cell lung cancer; PD-1, programmed
cell death protein-1;
PD-L1, programmed cell death ligand-1

73. REFERENCES
• Golan D.E, Tajshjian A.H, Armstrong E.J, Armstrong A.W, Principles of
pharmacology The pathophysiologic basis of drug therapy,3rd Edition, Lippincott
Wlliams & Wilkins,2012,790-806.
Brunton LL, Lazo JS, Parker LK, Goodman & Gilman's The Pharmacological Basis
Of Therapeutics, 11th Ed., McGraw-Hill, 2006,1291-1306
• Bousquet J,et al. Expert Opin Biol Ther 2012;8(12):1921- 8
• Patil U.S, Jaydeokar A.V, Bandawane D.D, immunomodulators : a
pharmacological review, international journal of pharmacy & pharmaceutical
sciences, vol 4, suppl 1, 2012