MONOCLONAL ANTIBODIES AND THEIR ROLE IN PHARMACOLOGY

Dr.Harmanjit Singh PG Resident (pharmacology)GMC, Patiala

Monoclonal Antibodies

Monoclonal antibodies are Monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell.

Class of highly specific antibodies Produced by clone of single hybrid cells or

hybridomaFusing B lymphocytes with a tumour cell

The Structure of an Antibody

2 identical light chains (~220 amino acids long)

Variable domain: VL

Constant domain: CL

2 identical heavy chains (~440 amino acids long)

Variable domain: VH

3 Constant domains: CH1, CH2, CH3Covalent, disulfide bonds between cysteine residuesFlexible “hinge region”

Antibodies have two major functions: • Recognize and bind antigen • Induce immune responses after

binding

The variable region mediates binding• Affinity for a given antigen is

determined by the variable region• The variable region confers absolute

specificity for an antigen

The constant region mediates immune response after binding• Different classes of constant regions

generate different isotypes• Different isotypes of antibody have

differing properties

Antibody Function

Constant region

Variable region

History

1975 :Hybridoma Technology

George Kohler and Cesar Milstein devised a method to obtain large amounts of a mAb

1984 :

The Nobel Prize for Medicine

- In 1988, Greg Winter et al pioneered the techniques to humanize monoclonal antibodies

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Hybridoma production

Healthy B lymphocytes

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Fusion of mouse myeloma cells

Murine Mabs

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HGPRT – HYPOXANTHINE GUANINE PHOSPHORIBOSYL TRANFERASEHAT – HYPOXANTHINE, AMINOPTERIN, THYMIDINE

Transgenic mice

Transgenic mammalian cells (e.g. Chinese hamster ovary cells ) grown in culture are the industry standard for producing full-length mAb

Why should we be interested ?The outlook for monoclonal antibody therapeutics is

healthy

mAbs drive the development of multibillion dollar biotechnology industry.

US$26 billion by the end of the decade.

Quicker and less costly development higher success rates premium pricing and potentially reduced threat from generics

100 mAb were Expected By 2010

Origin

First generation

Murine, rabbit or rat proteins purified

after immunisation with antigen

Abs to these proteins (Ag) generated in

patients human antimouse antibody

(HAMA)

Block effectiveness of therapy

Adverse events serum sickness or anaphylaxis

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Origin

Second generation

DNA technology or genetic engineering

used to construct hybrids composed of

human Abs regions with murine

Chimeric Abs

Humanized

Human

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EVOLUTION OF MONOCLONAL ANTIBODY

1. TRANSGENIC

2. LIBRARIES

a.BACTERIOPHAGE

b. mRNA

c. Cell Surface

Ist generation mab

2nd generation mab

daclizumab

Types of mAbs

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Purple denotes human component orange murine component

Murine

Derived from mice

Patients treated with murine mAbs develop

a human antimouse antibody (HAMA)

response

Rapid clearance of the mAb

Poor tumour penetration

Hypersensitivity reactions

90Y-ibritumomab 131I -Tositumomab

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Chimeric Abs

Antigen binding parts (variable region) of

mouse

Ab with effector parts (constant region) of

human

Infliximab

Abciximab

Rituximab15

Humanized

Human Ab with complimentary determining

region

(CDR) or hypervariable region from non

human source

Daclizumab

Trastuzumab

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Human Abs

Recombinant DNA technology:

Genes for variable Fab portion of human Abs

is inserted in genome of bacteriophages &

replicated

Mixed with Ag & complementary Ab

producing

phages selected

e.g. Adalimumab

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Nomenclature of Monoclonal AntibodiesPrefix Target Source Suffix

variable

-o(s)- bone -u- human

-mab

-vi(r)- viral -o- mouse

-ba(c)- bacterial -a- rat

-li(m)- immune -e- hamster

-le(s)- infectious lesions -i- primate

-ci(r)- cardiovascular -xi- chimeric

-mu(l)- musculoskeletal -zu- humanized

-ki(n)- interleukin -axo- rat/murine hybrid

-co(l)- colonic tumor

-me(l)- melanoma

-ma(r)- mammary tumor

-go(t)- testicular tumor

-go(v)- ovarian tumor

-pr(o)- prostate tumor

-tu(m)- miscellaneous tumor

-neu(r)- nervous system

-tox(a)- toxin as target

Nomenclature

Suffix

Human: -umab

Humanized: -zumab

Murine: -momab

Chimeric: -ximab

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Examples ab- + -ci- + -xi- + -mab:

chimeric monoclonal antibody used on the cardiovascular system.

tras- + -tu- + -zu- + -mab: humanized monoclonal antibody used against a tumor.

Pali- + -vi- + -zu- + -mab humanized mab used against a

virus (RSV)

Pharmacokinetics: mAbs

Routes of administration: Subcutaneously (Rituximab, Trastuzumab,

Adalimumab) Intramuscularly (Palivizumab) Intravenously

IV route: preferred because of 100% bioavailability

Route for elimination of antibodies Via uptake & catabolism by reticuloendothelial

system & target tissue.21

P/K: mAbs

Half-life Chimeric : 4 –15 daysHumanized: 3 - 24 daysRecombinant human: 11– 24 days

Human antimouse antibody (HAMA) response develops 7–10 days following exposure to murine antibody

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List of some important Mabs

Therapeutic agent Indication

Alemtuzumab B cell CLL

Bevacizumab Met. Colon cancer

Ranibizumab Neovas. Macular degenration

Cetuximab Met. Colon cancer

Gemtuzumab AML

Panitumumab Colorectal cancer

Rituximab Low grade NHL

Trastuzumab Met. Breast cancer

Therapeutic agent Indication

Abatacept Severe RA

Basiliximab, Renal, heart transplant

Daclizumab Renal transplant, M.Sclerosis

Efalizumab Psoriasis

Adalimumab RA

Abciximab ACS

Omalizumab Allergic asthma

Palivizumab RSV infection

Natalizumab M.Sclerosis

Therapeutic uses

Immunosuppression

Autoimmune diseases

Malignancies

Antiplatelet therapy

Infectious diseases

Asthma

Osteoporosis

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1.Immunosuppression

Muromonab CD-3: Murine mAb

 1st mAb  approved for clinical use in humans

Act on CD3 receptors

Apoptosis of the T cells

Prevents graft rejection in renal transplant

ADRs: skin reactions, fatigue,

fever, chills, myalgia, headaches, nausea & 

diarrhea 26

Autoimmune diseases

• Therapeutic uses of Infliximab: • Blocks TNF-α

Crohn’s disease & Ulcerative colitisRA & ASPsoriatic arthritis

• ADR- Reactivation of TB, Hepatitis BLeukopenia, Neutropenia,

Thrombocytopenia & Pancytopenia 

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Autoimmune diseases

Epratuzumab:

Humanized Ab

Binds to the glycoprotein CD 22 of mature

and malignant B-cells

Therapeutic use:

NHL

SLE

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Autoimmune diseasesEfalizumab: Humanized Ab MoA

  binds wth CD11, a subunit of lymphocyte function-associated antigen 1

Therapeutic uses Plaque psoriasisADRsBacterial sepsis, viral meningitis, invasive

fungal disease and progressive multifocal leukoencephalopathy (PML)

Withdrawn from market 29

2.MalignanciesRituximab:

• First chimeric IgG-I mAb

• MoA:

Directed against CD 20 on B cells

• P/K

Bioavailability: 100% (IV)

Half life: 30 to 40 hours

Excretion: Phagocytosis and catabolism in RES30

Malignancies

• Therapeutic uses of Rituximab:

Non hodgkins lymphoma

Multiple myeloma

ADRs:

Severe infusion reactions

Cardiac arrest

Tumor lysis syndrome

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MalignanciesAlemtuzumab Humanized IgG1 MoA:

Binds to CD52 B & T cells Therapeutic uses

 Chronic lymphocytic leukemia (CLL) in  failed fludarabine therapy

Cutaneous T-cell lymphoma(CTCL)

ADRs: Hypotension, rigors, fever, shortness of breath, bronchospasm, chills, Lymphopenia, Anaemia, Thrombocytopenia

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Malignancies

Cetuximab

Chimeric Ab

Directed against EGFR

Inhibits tumor growth

Therapeutic use

Metastatic colorectal cancer (EGFR over

expressive)

Head and neck cancer

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Malignancies

ADRs of Cetuximab:

Acne-like rash

Fevers, chills, rigors, urticaria, pruritis,

rash, hypotension, bronchospasm,

dyspnea, wheezing, angioedema,

dizziness, anaphylaxis

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Malignancies

Trastuzumab

• Humanized Ab

• MoA: Binds to HER-2/neu,

Downregulation of receptor

• Therapeutic use Metastatic breast ca (over express

HER-2/neu)

ADR : Cardiomyopathy35

Malignancies

BevacizumabBevacizumab Humanized mAb MoA:

Directed against VEGF• Blocks VEGF in neovascularisation• Therapeutic uses

Metastatic colorectal ca, Pancreatic ca, Breast ca

Prostate ca

ADRsRisk of bleeding, Bowel perforation, Nasal

septum perforation36

3.Antiplatelet therapyAbciximab

Chimeric mAb MoA

Glycoprotein IIb/ IIIa receptor antagonistPrevents platelet aggregation Inhibits fibrinogen, von Willebrand factor

Therapeutic usesAcute coronary syndromePCI

ADRs: ↑ risk of bleeding, Thrombocytopenia

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5.AsthmaOmalizumab Humanized Ab MoA

Binds IgE # interaction with basophils & mast cells

Therapeutic use B. Asthma in adults & adolescents

refractory to inhaled corticosteroid therapy

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Osteoporosis Denosumab - Human mab - Act against RANK Ligand -  Approved by FDA for use in

postmenopausal women with risk of osteoporos

- ADRs UTI  and RTI Cataract , constipation, rashes and

joint pain

Future aspects Zanolimumab

CD4 specific mAb Cutaneous T cell lymphoma Phase III clinical trial

Daclizumab: Humanized mAb (Phase II

clinical trials )

Binds to IL – 2

Therapeutic use : Prophylaxis of acute organ

rejection in renal transplant

Multiple Sclerosis 40

Future aspects

Ipilimumab Human monoclonal antibody  Undergoing clinical trials treatment

of melanoma

Tremelimumab Human IgG2 monoclonal antibody Phase III clinical trial for

advanced melanoma 

Ruplizumab  Humanized monoclonal antibody  Systemic lupus erythematosus  Lupus nephritis 41

Future aspects Efungumab : for Invasive Candida Infection Panobacumab : for Pseudomonas Infection Solanezumab : for Alzheimer’s Disease Natalizumab : for Multiple Sclerosis

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OTHER USES IMAGING : Arcitumomab : colorectal carcinoma Nofetumomab : small cell lung cancer DIAGNOSTICS : HIV Diagnostic kit A monoclonal antibody can be used to

detect pregnancy only 14 days after conception.

Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal, and Chlamydia infections.

Ethical issues: Freund’s complete adjuvant (FCA) (to enhance the immune

response): painful lesions at the injection site. Pristane as a "priming" agent - granulomatous

reactions Respiratory distress: due to ascites. Shock - rapid fluid loss. FCA and pristane should not be used where it is

possible to use no adjuvant or less irritant adjuvants. FCA should not be used more than once in individual

mice. The volume of FCA and pristane used should not

exceed 0.1ml and 0.2ml respectively. Individual mice should not be inoculated with

adjuvant more than 3 times. A priming agent should not be used in individual

mice more than once. Ascites fluid should only be harvested once at the

time of euthanasia.

TO SUMMARISE : MAbs are highly specific Abs produced by

a clone of single hybrid cells formed by fusion of B cell with the tumor cell.

The hybridoma formed yields higher amount of MAbs.

MAbs can be produced in vitro and in vivo . Animals are utilized to produce MAbs, but

these antibodies are associated with immunogenicity and ethical problems.

Recombinant DNA technology, genetic engineering and transgenic animals are used to produce humanized MAbs or pure human MAbs, with fewer ADRs

Used for treatment of cancer, autoimmune disorders, graft rejections, infections, asthma etc.

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