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Antibody

Structure & Function

School of Chemical & Life Sciences (Chemical) CLB335 Molecular Biotechnology

Antibodies

• Main mediator of humoral immunity

• Belong to a family of globular proteins called immunoglobulins

(secreted form of immunoglobulins)

• Secreted soluble proteins produced by plasma cells when B cells

bind to specific antigens

Structure of Antibody

• Y-shaped molecule with two identical heavy polypeptide chains

(termed H chains) and two identical light polypeptide chains

(termed L chains) held together by covalent disulphide bond

• Each light chain is about 25 kilodalton (kD) while each heavy

chain is 55 to 70 kD depending on the class of immunoglobulin

Light chains

(depicted in green)

Heavy chains

(depicted in blue) 3-dimensional structure of IgG

obtained from X-ray crystallography

Structure of Antibody

• Light chains exist in 2 distinct forms called kappa (κ) and

lambda (λ) which are present in different ratios depending on the

type of species (e.g. mouse has 95% κ while human has 60% κ)

• Heavy chains exist in 5 distinct forms called γ, μ, α, δ and ε

which give rise to the 5 classes of immunoglobulins and their

different biological functions

• Both light chains and heavy chains consist of two distinct

regions – one constant region (C) on the C-terminal half of the

chain and a variable region (V) on the N-terminal half

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Structure of Antibody

• Variable regions (V) of light and heavy chains are about 110

amino acids long and denoted by VL and VH respectively

• Constant region (C) on the light chain is also about 110 amino

acids long and while that for the heavy chain is about 330 or 440

amino acids long depending on the class of immunoglobulins; they

are denoted by CL and CH respectively

• The variable region is the part of the molecule that binds to the

epitope of an antigen and give rise to its specificity

Hypervariable Regions

• The variable regions of the light and heavy chains can be further

divided into the hypervariable and framework regions

• Each hypervariable region contains three highly variable amino

acid sections known as the complementary-determining regions

(CDRs)

• Each CDR is about 10 amino acid residues in length and are

denoted by CDR1 (from the N-terminal end), CDR2 and CDR3

• The complementary-determining regions (CDRs) are located

around amino acid residues 30, 50 and 95; and form protruding

loops on the surface of the domains which are complementary to

the configuration of the antigen that binds to the antibody (i.e. the

CDRs determine the specificity of the antibody)

Framework Regions

• The complementary-determining regions (CDRs) are flanked by

relatively constant regions with little variability in the amino acid

sequence known as the framework regions, which accounts for

about 85% of the variable region

Hypervariable

regions of light

chain

Variable regions

of light chain

Variable regions of

heavy chain

Hypervariable

regions

Hypervariable

regions of

heavy chain

Antigen-binding

site

Globular Domain

• In addition to the inter-chain disulphide bonds that hold the L and

H chains together, intra-chain disulfide bonds also exist within

the chain forming loops of peptide chains

• Both the light and heavy chains contain repeating segments of

approximately 110 amino acids that fold independently into a

compact globular structure known as domains.

• Each light chain consists of 2 domains – 1 variable (VL) and 1

constant (CL) domain

• Each heavy chain consists of 4 or 5 domains – 1 variable

domain (VH) and 3 or 4 constant domains (CH1, CH2, and CH3) (Both μ and ε chains have one variable and four constant domains)

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Globular Domain

• Variable domains are responsible

for antigen binding while the

constant domains of the heavy

chains (excluding CH1)

determines the other biological

properties of the antibody

Light Chains

Heavy

Chains

Antigen-binding Site

CH1

CH2

CH3

CH1

CH2

CH3

CL CL VH VH

VL VL

Hinge Region

• The hinge region is a short segment of

amino acids (predominantly cysteine and

proline residues) found between the CH1

and CH2 regions of the heavy chains

• The hinge region confers flexibility to the

antibody for binding with antigens as it

allows variability of the angle between the

arms of the Y-shaped antibody as

well as rotational flexibility of

each individual arm

Functions of Antibody Functions of Antibody

• Opsonization

Microorganisms or other foreign particles coated with antibodies are

especially susceptible to phagocytosis as the antibodies bind to the Fc

receptors of phagocytes such as dendritic cells, macrophages and neutrophils

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Functions of Antibody

• Immune Complex Formation

Cross-linking of antigens and antibodies

can result in large aggregates known as

immune complexes. Precipitation can

occur if the complexes become large enough.

An agglutination reaction can also occur if the immune complex involves

the cross-linking of cells or particles.

• Virus & Toxin Neutralization

Antibodies can neutralize viruses and toxins by direct binding to these

antigens (which will be cleared by the complement pathway) and prevent

their adherence to surface receptors on target cells

Functions of Antibody

• Complement Fixation

Complement pathway

(classical) is triggered

by the binding of C1q

(one of the three proteins

forming the complex C1)

to antibodies attached

on the surface of

pathogen

C1q is only activated

when it binds at least

two Fc pieces

Immunoglobulins

• Group of glycoproteins present in the serum and tissue fluid of

all mammals

• Can be expressed as secreted or membrane-bound forms

• Membrane-bound form are present on the surface of B cells

where they act as receptors for specific antigens

• Activation (binding) of B cells by antigens result in the

development of plasma cells which secrete antibodies (secreted

form of immunoglobulins)

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History of Immunoglobulins Tiselius and Kabat discovered γ-globulin (IgG)

from the electrophoresis of rabbit serum

1940

1990

1950

1960

1970

1980 Cesar Milstein and George Köhler created hybridoma

cells to produce monoclonal antibodies (Mab)

Rodney Porter and Gerald Edelman received the

Nobel Prize for elucidating the chemical structure

of antibody

IgE was isolated from pollen proteins by the Japanese

scientist couple Teruka and Kimishige Ishizaka.

Immunoglobulins

• Immunoglobulins can be classified into 5 different classes with

distinct characteristics. They are IgG, IgA, IgM, IgD and IgE.

• The heavy (H) chains are structurally and functionally distinct

for each class and are designated by Greek letters that

correspond to the immunoglobulin class.

No. Immunoglobulin Class

(Isotype) Heavy Chain

1 IgG γ (gamma)

2 IgA α (alpha)

3 IgM μ (mu)

4 IgD δ (delta)

5 IgE ε (epsilon)

Immunoglobulins IgG

• Biological & Structural Properties

Molecular weight ≈ 150,000 Da with two γ heavy chains (≈ 50,000 Da each)

and two lights chains (≈ 25,000 Da each) held together by disulfide bonds

Contains 4 subclasses IgG1, IgG2, IgG3 and IgG4 in the approximate

proportions of 65, 25, 5 and 5% respectively

Constitute 15% of the total serum proteins in humans and 75% of the total

serum antibodies

Has the longest half life of all Ig isotypes – approximately 23 days

Only isotype that can pass through the human placenta and provide

immunity to the fetus in utero

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IgG

• Biological Functions

Major antibody synthesized during secondary immune response

Activation of complement (classical pathway)

Neutralization of toxins and venom by blocking the active sites (passive

immunization)

Agglutination (clumping) and immobilization of microorganisms

Opsonization of microorganisms to enhance phagocytosis

Involve in antibody-dependent cell-mediated cytotoxicity (ADCC) where the

antibodies coated on the target cells bind with receptors on natural killer cells

ADCC

• Triggered by the interaction of antibody (IgG) with the CD16

receptor on the surface of NK cells

• Mechanism of attack is similar to that of cytotoxic T cells which

involve the release of cytoplasmic granules containing perforin

and granzymes, inducing apoptosis

IgM

• Biological & Structural Properties

First immunoglobulin to be produced upon initial exposure to an antigen

(elevated levels of IgM usually indicate recent infection or immunization)

Macroglobulin of high molecular weight (900,000 Da)

Pentameric molecule with 5 units of four chain structure (two L and two H

chains joined together by disulfide bonds between their Fc portions and by a

polypeptide chain called the J chain)

Mainly found in serum due to its large size

Relatively short-lived with a half life of approximately 5 days

Does NOT pass through the human placenta and presence in cord blood

indicates acute fetal infection

High avidity but low affinity to antigens due to presence of multiple binding

sites

Structure of IgM

Antigen binding sites

μ heavy chain

Light

chain

= disulfide bond

J chain

All the Fc portions

are joined together

by disulfide bonds

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IgM

• Biological Functions

Efficient agglutinating agent due to its pentameric form – macromolecular

bridges can be formed between epitopes on distant molecules

Most efficient initiator of complement (classical pathway)

Primary antibody (isohemagglutinin) against the red blood cell antigens of the

ABO blood groups – responsible for the clumping (agglutination) of red blood

cells if the recipient of a blood transfusion receives blood that is not compatible

with their blood type

IgA

• Biological & Structural Properties

Major immunoglobulin found in external secretions

such as saliva, mucus, sweat, gastric fluid and tears

Molecular weight ≈ 165,000 Da with two heavy α

chains and two lights chains

Relatively short-lived with a half life of

approximately 5.5 days

Contains 2 subclasses IgA1 and IgA2

Present in small amount in serum as a monomer

(one four-chain unit) but found predominantly as a

dimer in mucous secretions known as secretory IgA

(sIgA)

Secretory IgA (sIgA) contains two IgA monomers

connected by a J chain and contains a secretary

component which protects the dimeric IgA from

proteolytic cleavage

IgA

Dimeric IgA are produced by plasma cells in the lamina propria (connective

tissue adjacent to mucosal surfaces)

Dimeric IgA can bind to the polymeric immunoglobulin receptor on the

(basolateral) surface of epithelial cells before being taken up into the cell via

endocytosis

The receptor-IgA complex passes through the cellular compartments before being

secreted into the lumen containing a portion of the receptor known as the

secretory component

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IgA

• Biological Functions

Primary immunologic defense against local infections in areas such as the

respiratory or gastrointestinal tract

Protects mucosal surface

Activates complement via the alternative pathway

Efficient antiviral agent and protects mucosal surfaces via immune exclusion

and intracellular neutralization mechanisms

Immune exclusion involves the inhibition of contact between pathogen and the

mucosal epithelial cell surface

Intracellular neutralization involves the inhibition of key viral replication

steps (such as removal of capsid) from the interaction between sIgA and the

endocytosed virus

IgA Protection Mechanisms

Immune

Exclusion

Intracellular

Neutralization

B Cell Maturation IgD

• Biological Properties & Functions

Present in trace amounts in serum

Monomer with a molecular weight ≈ 180,000 Da with two heavy δ chains and

two lights chains

Co-expressed with IgM on the surface of mature B cells

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IgE

• Biological Properties & Functions

Mainly found in the linings of the respiratory and gastro-intestinal tract

Molecular weight ≈ 200,000 Da with two heavy ε chains and two lights

chains

Present in the least abundance and has the shortest half life (2 days) among

the 5 classes of immunoglobulins

High affinity to receptors on mast cells and basophils – trigger Type I

hypersensitivity reactions (allergy) when IgE bind to these cells

Plays an important role in parasite immunity through the activation of

eosinophils

IgE-Mediated Hypersensitivity

Hypersensitivity

Immunity Against Parasitic Worm Summary

Properties IgG IgM IgA IgD IgE

Structure Monomer Pentamer Dimer Monomer Monomer

Molecular Weight

(Da) 150,000 900,000 400,000 180,000 200,000

Half Life (days) 23 5 6 3 2

Serum

Concentration

(mg/ml)

12 1.2 2 0.03 0.00004

Placental Transport Yes No No No No

Activate

Complement

Yes

(Classical)

Yes

(Classical)

Yes

(Alternate) No No

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Antigenic Determinants

• Immunoglobulins are glycoproteins which can function as

antigens themselves

If a rabbit is immunized with human antibodies, the antibodies will be

recognized as a foreign antigen by the rabbit’s immune system resulting in

the production of anti-human antibodies (anti-serum)

• Antigenic determinants of immunoglobulins can be classified into

three categories – isotypic, allotypic and idiotypic

Isotypic Determinants

• Isotypic determinants are the antigenic specificities that

distinguish the different classes and subclasses of immunoglobulins

present in all normal individuals of a given species

• Isotypic determinants are located on the constant region of the

heavy chain which determine the five classes of immunoglobulins

(IgG, IgA, IgM, IgD and IgE) and their effecter functions

• Antibodies to isotypes are used for the quantitation of

immunoglobulin classes and subclasses in the diagnosis of various

immunodeficiency diseases (e.g. B cell leukemia)

Allotypic Determinants

• Allotypic determinants are the antigenic specificities that

distinguish immunoglobulins of the same class between different

groups of individuals in the same species

• Allotypic differences arise from genetic variations of the alleles

encoding the antigenic determinants and occur mostly in the

constant regions of the heavy chain

• Antibodies to allotypic determinants may be produced by a

pregnant mother in response to paternal allotypic determinants in

the fetal immunoglobulins (may be used for paternity testing)

IgG1

Person 1

IgG1

Person 2

Idiotypic Determinants

• Idiotypic determinants are the antigenic specificities exclusive to

each individual immunoglobulin molecule

• Idiotypic determinants are located at the hypervariable regions of

the light and heavy chains, i.e. the antigen binding sites

• Anti-idiotypic antibodies stimulate B cells to make antibody and

can be used as a vaccine against highly dangerous pathogens

Mouse IgG1

against antigen A Mouse IgG1

against antigen B

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Polyclonal vs. Monoclonal Antibodies

• Polyclonal antibodies are

heterogeneous mixtures of antibodies

produced from multiple clones of

plasma cells, with different specificity

for the various epitopes on an antigen

• Monoclonal antibodies are identical

antibodies produced from a single

clone of plasma cells with specificity

for one particular epitope on an

antigen

Polyclonal Antibodies

Monoclonal Antibodies

History of Monoclonal Antibodies

Köhler and Milstein developed the hybridoma technology which allows

monoclonal antibody to be produced

First antibody Orthoclone OKT3® approved for therapeutic use

First chimeric antibody ReoPro® produced by Centocor was approved

by FDA

First humanized antibody Zenapax® was approved by FDA for the

prevention of acute kidney transplant rejection

First human antibody Humira® produced by Abbott Laboratories was

approved by FDA for the alleviation of symptoms associated with

rheumatoid arthritis

Vectibix® produced by Amgen became the first antibody produced from

transgenic humanized mouse to be approved for therapeutic use

1975

1986

1994

1997

2002

2006

Hybridoma Technology

• Monoclonal antibodies are typically produce from hybridoma

cells made by fusing myeloma (cancerous) cells with spleen cells

from a mouse that has been immunized with the desired antigen:

Antigen is injected into mouse to stimulate the production of antigen-specific

(antibody-producing) plasma cells

Plasma cells are isolated from the mouse’s spleen and fused with myeloma

cells using polyethylene glycol which change the membrane’s permeability

Cells are grown in HAT (hypoxanthine, aminopterin and thymidine) medium

which is selective for fused cells.

Surviving cells are cultured and supernatants from each clone are placed into

antigen-coated wells to identify the cell producing the specific antibody

Identified hybridomas producing the desired antibody are grown in large

quantities to expand the cell population

Antigen is injected into mouse

Culture of myeloma cells in vitro

Plasma cells are derived

from spleen of mouse

Fusion of myeloma and plasma cells

using polyethylene glycol

Culture in HAT medium

selective for hybridoma cells

Identification of hybridomas

producing antibodies of

desired specificity

Clonal expansion of

identified clones

Purified

Monoclonal

Antibodies

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Therapeutic Monoclonal Antibodies Monoclonal Antibodies

Antibody

Engineering

Antibody Engineering

• Antibody engineering involve the use of recombinant DNA

technology and other techniques to modify properties of antibody

for therapeutic use. This include:

Humanization of murine antibodies to minimize undesired

anti-murine immune responses

Affinity maturation strategies to improve potency and efficacy

Production of antibody fragments or bispecific antibodies

with different recognition sites

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Murine Antibody

• First generation of Mab produced from mouse hybridomas

Murine Antibody

• First generation of Mab produced from mouse hybridomas

• Recognized as non-self by the host immune system resulting in

the production of human anti-mouse antibodies (HAMA)

causing severe allergic reactions

• Fail to trigger a number of effector functions associated with the

constant region of native antibody

• Short half life of 30 – 40 hours

• Example: Orthoclone OKT3®

Chimeric Antibody

• 2nd generation of Mab consisting of murine variable regions and

human constant regions

• Approximately 70% human with half life and effector functions

similar to human antibody

• Less immunogenic than murine antibodies but can still trigger

antibody responses

• Example: Rituxan (Rituximab), Remicade (Infliximab)

Humanized Antibody

• >90% human where the murine complementary determining

regions (CDRs) are engrafted onto a human framework region

• High antigen affinity with limited HAMA response

• Example: Herceptin (Trastuzumab)

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Human Antibody

• Antibody with complete human polypeptide

• Can be derived from phage display technology or genetically

engineered mice

• Phage display technology involve the use of bacteriophage to

infect bacteria cells and produce bacteriophages expressing the

antibody on their coat

• Example: Humira® (Adalimumab), Prolia® (Denosumab)

Phage Display Technology

• Use of recombinant DNA technology to create bacteriophages

with the desired antibody fragments displayed on the surface of

their protein shells

• Genes encoding the heavy and light chain variable regions are

amplified from human B cells and randomly joined together

before fusing to the bacteriophage genes encoding the protein coat

• Bacteriophages are then allowed to replicate in a bacterial cell

culture to produce a collection of recombinant phage, each

displaying a different antigen-binding domain on its surface

Phage Display Technology

• Phage display technology involves three main steps:

Construction of antibody library

Phage selection using immobilized antigens whereby phages

that do not bind are washed away while the binding phages are

eluted and further amplified for subsequent screening rounds.

This process is also known as biopanning.

Screening of eluted phages using enzyme-linked

immunosorbent assay (ELISA). Once the desired specificity is

obtained, the genes of antibody variable regions can be cloned

into whole human IgG expression vectors and transfected into

cell lines to produce fully human monoclonal antibodies

Phage Display Technology

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Transgenic Mouse

• Mouse’s genes encoding the heavy

and light chains are knocked out

(inactivated) and replaced with

human gene fragments

• Human antibodies are produced

by the genetically engineered

mouse when immunized with

foreign antigens

• The B cells harvested from the

immunized mice are immortalized

by fusion with a myeloma cell

line, as in traditional hybridoma

technology.

Antibody Fragments

Antibody Fragments

• Antibody fragments are increasingly used in place of intact

antibody for therapeutic and diagnostic purpose for the following

reasons:

Can be fused with a range of molecules to enhance therapeutic effects and

improve drug delivery

Low molecular weight allow for more efficient penetration into tissues

Elimination of Fc-associated effector functions (e.g. complement fixation)

in antigen-antibody binding studies

Lower immunogenicity than intact antibody for in vivo studies

• Can be generated by digestion with reducing agents and

proteases, or expressed in genetically modified microorganisms

Antibody Fragments

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Antibody Fragments

Antigen

Binding Sites

Antigen

Binding Sites

Mercaptoethanol

Reduction

Papain

Digestion

Pepsin

Digestion

Two Fab

Fragments

One Fc

Fragment

One F(ab)2 Fragment

Subfragments

of Fc

Four H & L chains

F(ab’)2 Fragment

• F(ab')2 Fragment

Approximately 110 kDa

Retain antigen binding capability (two antigen-binding regions)

Can form immunocomplexes and cause precipitation

Cannot trigger effector functions associated with Fc fragment (e.g. ADCC)

Reduced immunogenicity due to the lack of the Fc complement-activating

region

Can be generated by digestion with pepsin (non-specific endopeptidase)

Complement Fixation Fab Fragment

• Fab Fragment

Approximately 50 kDa

Univalent fragment linked by an intramolecular disulfide bond

Composed of one constant and one variable domain of each of the heavy

and the light chain (variable regions of the heavy and light chains can be

fused together to form a single-chain variable fragment)

Retain antigen binding capacity but does NOT form immunocomplexes

Can be generated by digestion with papain (non-specific thiol-endopeptidase)

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Fc Fragment

• Fc Fragment (≈ 50 kDa)

Approximately 50 kDa

Also known as the fragment crystallizable (Fc) region

Binds to various receptors of the immune cells (e.g. mast cells, natural killer

cells) to trigger the immune response

Part of the antibody that activate the complement system

LUCENTIS® (ranibizumab injection)

• Approved for the treatment of wet type age-

related macular degeneration (common form of

age-related vision loss)

• Binds to and inhibits vascular endothelial growth

factor A (VEGF-A) which trigger the growth of

new blood vessels in the retina

• Fab fragment derived from the same parent

mouse antibody as Avastin® (bevacizumab) –

removal of Fc portion minimize the risk of

systemic exposure

• Costs about $2,000 per dosage (40 times more

than Avastin®)

Bispecific Antibodies

• Comprise two different binding specificities fused into a single

molecule

• Can be designed to bind either two adjacent epitopes on a single

antigen to increase avidity (strength of antigen-antibody binding)

or to bind two different antigens for different functions (e.g.

recruitment of natural killer or cytotoxic T-cells while targeting

toxins and tumor cells)

• Majority of bispecific antibodies are designed to bind to the CD3

T-cell co-receptor and thereby recruit cytotoxic T-cells (CTLs) to

the tumor site.

Removab® (Catumaxomab)

• Approved within the European Union in 2009 for the treatment of

malignant ascites (collection of liquid in the abdominal cavity)

• Bispecific with two different antigen-binding sites – one binds

EpCAM while the other binds CD3

• Comprises of a mouse κ-light chain, a rat λ-light chain, a mouse

IgG2a-heavy chain and a rat IgG2b-heavy chain

• Trifunctional antibodies which can simultaneously bind to a tumor

cell, a T cell and an accessory cell of the innate immune system

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Removab®

Nomenclature

• Antibodies are named in accordance to the guidelines developed

by the US Adopted Name (USAN) Council

• The name of the antibody is made up of 4 key elements:

A starting prefix of at least two syllables to be determined by manufacturers

An infix representing the target disease

An infix representing the source of the antibody

A stem (suffix) of –pab for polyclonal antibodies and –mab for

monoclonal antibodies

• A second word is added to the name if the antibody is

conjugated to another chemical or radiolabeled

Example

Bevacizumab (Avastin®)

Proprietary Trade Name

Suffix

Infix

(Source of antibody)

Infix

(Target Class)

Prefix

Avastin® is a humanized monoclonal antibody for the treatment of

metastatic cancer which inhibits the formation of new blood vessels

Target Class Infix

Source Infix Old New

Bacterial -ba(c)- -b(a)- Chimera -xi-

Circulatory

Cardiovascular -ci(r)- -c(i)- Hamster -e-

Fungal -fung- -f(u)- Fully Human -u-

Interleukin -ki(n)- -k(i)- Humanized -zu-

Inflammatory lesions -les- — Mouse -o-

Immune System -li(m)- -l(i)- Primate -i-

Neural -ne(u)(r)- -n(e)- Rat -a-

Bone -os- -s(o)-

Toxin -toxa- -tox(a)-

Tumour (colonic) -co(l)-

-t(u)- Tumour (ovarian) -go(v)-

Tumour (prostate) -pr(o)-

Tumour (misc.) -tu(m)-

Viral -vi(r)- -v(i)-

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Question

Generic Name Trade Name Source Indication / Use

Abciximab ReoPro® Platelet aggregation inhibitor

Capromab pendetide Prostascint®

Denosumab Prolia®

Efungumab Mycograb®

Palivizumab Synagis®

Tefibazumab Aurexis®

Daclizumab Zenapax®

Chimeric

Mouse Prostate cancer detection

Human Osteoporosis, bone metastases etc.

Human Invasive Candida infection

Humanized Prevention of respiratory

syncytial virus infection

Humanized Staphylococcus aureus infection

Humanized Prevention of organ transplant

rejections

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