MAbs Notes

7/27/2019 MAbs Notes

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MONOCLONAL ANTIBODIES

An antibody is a protein used by the immune system to identi-

fy and neutralize foreign objects like bacteria and viruses. Eachantibody 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 dif-

ferent cell lines.

DISCOVERY OF MONOCLONAL ANTIBODIES

The process of producing MAbs (hybridoma technology)was invented by Georges Kohler and Caeser Milstein in

1975, for which they were awarded Nobel Prize in 1984.

1990 Milstein produced the first monoclonal antibodies.PRODUCTION OF ANTIBODIES

The hybridoma technology:

Healthy antibody producing B lymphocytes from spleen ofimmunized mice are fused with the murine myeloma cells

(that have lost ability to secrete antibodies)

This results in clone of fused cells (Hybridomas) that re-tains the Ab-forming capacity of B-lymphocytes with an

ability of the myeloma tumour cell lines ability to prolifer-

ate endlessly in tissue culture


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The procedure yielded a cell line capable of producing one type

of antibody protein for a long period.

Methods of production:

1. In vivo: Production in animals

2. In vitro: Production in cell-culture

Batch tissue-culture methodsGrowing of hybridoma cultures in batches and purify the Mabs

from the culture medium(mostly fetal bovine serum is used as

culture medium)

Semi permeable membrane-based systemSemi permeable membrane with low molecular weight cut-

off(10,000-30,0000kDa)based system permits cells to grow at

high densities.

Depending on cell culture methods

1. Robottle cell culture process.

2. Membrane binded cell culture process.

3. Microcarrier cell culture process.

4. Suspended cell culture process.

CLASSIFICATION OF MABS:-

First generation MAbs

Murine MAbs Second generation MAbs

Chimeric MAbs Humanized MAbs Human MAbs Primatized MAbs Genetically engineered MAbs1.First generation MAbs:

Majority of the earlier MAbs available were murine,

rabbit or rat proteins purified following immuniza-

tion of the animal with an antigen preparation

Ex: Muromonab, Ibritumomab

Disadavantages:

a. Shorter t1/2b. Induce human anti-mouse(HAMA)/ human

anti-rabbit antibodies(HARA) allergic reac-

tions.

c. They are not able to recruit human effectorfunctions such as antibody-dependent cellu-

lar cytotoxicity (ADCC) and complement-

dependent cytotoxicity(CDC),which causes

destruction of a malignant cell.


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2. second generation Mabs:

Genetic engineering is used to construct hybrids composed of

human antibody regions linked with a murine or primate back-

bone.

i. Chimeric Abs: these are composite of Abs from 2 diff.species. The Abs combines the Ag binding

parts(variable region)of the mouse Ab with effector

parts(constant region) of a human Ab.

Ex: Infliximab, rituximab, abciximab

ii. Humanized Abs: The Ab combines only the amino ac-ids responsible for making antigen binding site(the hy-pervariable region) of a mouse Ab with rest of human

Ab molecule.

Ex: Daclizumab, herceptin, vitaxin

iii. Primatized Abs: it is composite of primate variable re-gions and human constant regions.

iv. Genetically engineered Abs:

. Fab portion from rodent

Abs is attatched to Fc portion of human Abs. Human

MAbs have been produced by immortalizing human B

lymphocytes with the Epstein-Barr virus and using spe-

cial immunodeficient mice immunologically reconsti-

tuted with immunocompetent cells of tissue.

v. Human Mabs: they can be produced by the followingtechniques:

(a) Recombinant DNA technology. By inserting random

human genes coding for variable Fab portion of human

Abs into the genome of filamentous bacteriophages. As

the bacteriophage replicate they display Fab portion Ab

on their surface. The bacteriophages are subsequently

mixed with an antigen to select those producing com-

plementary Fabs. Those bacteriophage genomes are

then converted into plasmids that can subsequently pro-

duce specific Fabs in bacteria.

(b) Transgenic mice . Transgenic technology has

been exploited to make a transgenic mice that have hu-

man Ab gene loci inserted into their bodies (using em-

bryo stem cell method) and their own genes for making

antibodies 'knocked out'. Therefore, mouse can be im-

munized with the desired antigen and produces human

Abs.

(c) Phage display . This is another technique for

making human MAbs. It is used when MAbs do not di-

rectly recognize antigen or when antigen is undetecta-

ble normally and expressed only in disease.

NOMENCLATURE:-

The United States adopted name (USAN) Council has outlined

specific guidelines for the nomenclature of MAbs. Theseguidelines provide a foundation of knowledge about a specific

MAb just by looking at the generic name.


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All MAbs end in the suffix - mab. It is used to iden-tify a class of medicines.

The infix preceding - mabdenotes the source of theproduct.

e.g., u = human i = primate ,o = mouse, e = hamster ,a =

rat, xi = chimera ,zu = humanized

The infix preceeding the source of the Ab refers tomedicine target.

e.g., vi(r) - viral ci(r) - cardiovascular

ba(c) - bacterial co(l) - colonic tumor

li(m) - immune le(s) - infectious lesions

me(l) - melanoma ma(r) - mammary tumor

go(t) - testicular tumor go(v) - ovarian tumor

pr(o) - prostate tumor tu(m) - miscellaneous tumor

When combining a target or disease infix stem with the

source stem for chimeric or humanized MAb the last

consonant of this target syllable is often dropped to

make the name more pronounceable (e.g., -tum-

changed to -tu- ).

TARGET SOURCE MAb STEM

e.g., ci(r) -xi -mab Abciximab

tu(m) -zu -mab Trastuzumab

The starting prefix is distinct syllable carrying nospecial meaning & unique for each drug.

TYPES OF MAbs THAT ARE USED IN TREAT-

MENT:

(a)Naked Mabs:-

These are those without any drug or radioactive materi-al attached to them.

They attach themselves to specific Ag on cells, e.g.cancer cells. They mark the cancer cells for the immune

system to destroy it.

Others attach to certain Ag sites called receptors whereother molecules that stimulate the cancer cell growth

might otherwise attach. By blocking other molecules

from attaching there, MAbs prevent cancer cells from

growing rapidly. Some examples of naked MAbs avail-

able for use are:

1. Trastuzumab - For advanced breast cancer2. Rituximab - For B cell non-Hodgkin's lymphoma3. Cetuximab - For advanced colorectal cancer4. Bevacizumab - For metastatic colorectal cancer5. Alemtuzumab - For B cell chronic lymphocytic leuke-

mia


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(b)Conjugated MAbs:-

MAbs, because of their inherent specificity, areideal targeting agents

They can be used to deliver radionuclides, tox-ins, or cytotoxic drugs to a specific tissue or ma-

lignant cell population. They are also referred to

'tagged' 'labeled' or 'loaded' antibodies.

MAbs with radioactive particles attached are re-ferred to as radiolabeled and this type of therapy

is known as radioimmunotherapy.

MAbs attached to toxins are known as immuno-toxins.It delivers the toxic substance to where it

is needed most, minimizing damage to normal

cell. Examples are:

1. Ibritumomab tiuxetan- Radiolabeled MAb to treat B cell

non-Hodgkin's lymphoma.

2.Tositumomab- Radiolabeled MAb for non-Hodgkin's

lymphoma

3.Gemtuzumab ozogamicin - Immunotoxin MAb for

AML.

MECHANISM OF ACTION OF MAbs:-

The mechanism by which MAbs achieve therapeutic effect is

not very clear. Potential mechanisms include:

1. Blocking or steric hindrance of the function of targetantigen i.e., T-lymphocytes, B lymphocytes, tumour

necrosis factor-a (TNFa) and interleukin (IL) which are

capable of transducing intracellular signals.

2. Cytotoxicity to the cell expressing target AG by ADCCor CDC.

3. Inhibition of growth factors: Epidermal growth factorreceptor (EGFR) is a cell surface receptor involved inregulation of cell proliferation, survival&angiogenesis

Theses factors can be inhibited to arrest growth of can-

cer cells e.g., cetuximab act as EGFR inhibitor.

PHARMACOKINETICS:-

MAbs are used by parenteral route and remain es-sentially intravascular.

They have small volume of distribution and limitedtissue penetration.

They remain in circulation for 2 days to 2 weeks. T1/2- Chimeric : 415 days, Humanized: 3 - 24

days, Recombinant human: 1124 days.


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Antibodies can have exquisite specificity of targetrecognition and thus generate highly selective out-

comes following their systemic administration.

While antibodies can have high specificity, the dos-es required to treat patients, particularly for a chron-

ic condition, are typically large

ADVERSE EFFECTS:-

Adverse effects with MAbs are related to one of three mecha-

nisms:

1.

Xenogenetic nature of MAb used.2. Suppression of physiological function.3. Activation of inflammatory cells or mediators after

binding of MAb to its target.

APPLICATIONS OF MAbs:-

1. Diagnostic Application:

a. They are used in Diagnostic tests- for example:western blot test, immuno dot blot test,. immunohistochemis-

try. immunofluorescence test, biosensors, microarrays,

blood grouping.

b. Identification & characterization of tumour specific an-tigens.

c. Localization of cancer.d. Diagnostic imaging of cancer (99mTc-mAb)e. Pregnancy tests- HCG levels.f. To detect HIV / salmonella infection.g. To determine blood glucose levels.

2.Therapeutic applications:

a. Immunosuppressant: inhibition of alloimmune

reactivity.

Acute graft rejection is a T-cell mediated immune response anddepends on presence of IL-2. IL-2 binds to IL-2 receptor. In the

search for more specific immunosupression with MAb L- 2

Receptor (IL-2R), that is expressed on T-cells, which were

chosen as target. Chimeric and humanized MAbs, basiliximab,

and daclizumab were developed to bind to IL-2R. They com-

petitively antagonized IL-2 or caused elimination of activated

T-cells.

b. Anticancer therapy:

Tumor cells are not specifically targeted by one's immune sys-

tem since tumor cells are the patient's own cells. Many tumour

cells display unusual antigens or cell surface receptors that are

rare or absent on the surfaces of healthy cells.E.g. ErbB2-

breast cancer tumor cells.


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Mechanism of antitumor effect:

1. Antibody dependent cellular cytotoxicity (ADCC)2. Complement dependent cytotoxicity (CDC)3. Direct induction of apoptosis mab may be conjugated

with a toxin

c.Antiplatelet therapy

Acute coronary syndromes and percutneous coronary interven-

tion share a common physiological mechanism of intimal dis-ruption and platelet aggregation. Glycoprotein IIb/IIIa receptor

antagonist which interrupt the final common pathway of plate-

let activation and aggregation are used for acute therapy.

Abciximab was the first antagonist to be evaluated . It inhibits

the clumping of platelets by binding to surface receptors that

normally are linked by fibrinogen. It is helpful in preventing

the reclogging of the coronary arteries.

d. Infectious disease

Palivizumab, a humanized MAb directed against Respiratory

Syncytial virus is used for the treatment of premature infants

and infants with bronchopulmonary dysplasia.

e. Opthalmological disorders

Daclizumab has shown to be efficacious for noninfectious

uveitis.

Ranibizumab (Lucentis) is a recombinant humanized IgG1

kappa isotype monoclonal antibody fragment designed for in-

traocular use, which competitively binds and inhibits VEGF.

Therefore, indicated for the treatment of neovascular (wet) age

related macular degeneration.

f. Multiple sclerosis

Natalizumab, a humanized MAb was approved by FDA for re-

lapsing form of multiple sclerosis. The committee consulted by

FDA recommended a risk-minimization program with manda-

tory patient registration and periodic follow-up.

g. Asthma

Omalizumab MAb has shown promise in allergic asthma . It

acts by binding to IgE thus preventing IgE from binding to

mast cells. Omalizumab has shown to reduce serum IgE levels,

reduce inhaled steroid consumption and was also well tolerated

by children and adults.


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h. Psoriasis

MAbs directed against key components of inflammatory pro-

cess have been studied for safer, selective, and effective immu-

nosuppressant agent as psoriasis is a T-cell mediated autoim-

mune disease in which proinflammatory Th-1 cytokine play an

essential role. Efalizumab is a humanized MAb that interrupts

the interaction between the T-cell surface molecule lymphocyte

function associated antigen LFA1 (composed of 2 subunits

CD11a and CD18) and intercellular adhesion molecule1, which

is found on the surface of antigen-presenting cells. It is admin-

istered every other week and carries a risk of tuberculosis reac-

tivation, serious infections, and demyelinating disease.

i. Juvenile diabetes

Anti-CD3 MAb is in phase II trial for type I juvenile DM. This

MAb targets an antigen expressed on T lymphocytes that is re-

sponsible for destruction of islet cells of pancreas and thus

could slow the disease progression.

j. Refractory Wegener's granulomatosis

Humanized antilymphocyte MAbs may provide an effective

treatment in patients with systemic vasculitis which is refracto-

ry or intolerant to steroids or cytotoxic agents.

k. Systemic lupus erythematosus (SLE)

IL-6 levels are elevated in human and murine SLE. Blocking

the action of IL-6 ameliorates disease activity in murine model

of SLE. A humanized MAb is in Phase I of clinical trial.

Rituximab has also been shown to be effective in patients of

SLE with glomerulonephritis, by causing B-cell depletion. B-

cells in SLE display abnormal signaling, express aberrant cell

surface markers and finally produce autoantibody and present

auto antigen to T cells at increased rates.

l. Active immunotherapy

Direct evidence of the importance of gangliosides as potential

targets for active immunotherapy has been suggested by the

observation that human MAbs against these glycolipids induce

shrinkage of human cutaneous melanoma metastasis. Thus, the

cellular over-expression and shedding of gangliosides into the

interstitial space may play a central role in cell growth regula-

tion, immune tolerance and tumor-angiogenesis, thereby repre-senting a new target for anticancer therapy.

Anti-idiotype vaccine has been developed from proteins de-

rived from the outer membrane proteins of Neisseria meningit-

ides B. 1E10 vaccine, is an anti-idiotype vaccine designed to

mimic the N-Glycolyl-GM3 gangliosides. This monoclonal


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antibody is an Ab2-type-antibody which recognizes the Ab1

antibody called P3, the latter is a MAb that specifically recog-

nize gangliosides as antigens. Results of the phase I clinical

trials proved that the three vaccines were safe and able to elicit

specific antibody responses. Phase II trials are being undertak-

en in several neoplastic diseases, with these vaccines.

Vaccination of immunologically responding metastatic colo-

rectal carcinoma patients with SCV 106 leads to slowing of

disease progression, tumor dissemination and significantly pro-

longs survival time.Increased CMI responses to HIV-1 enve-

lope glycoprotein measured by lymphocyte proliferation were

associated with HIV-1 recombinant envelope glycoprotein vac-

cines. HIV-1-specific T helper cell responses can be success-

fully increased by therapeutic immunization in individuals with

chronic infection on suppressive antiretroviral therapy.

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