Download humanized mAb

Antibody (immunoglobulin)
Serum protein electrophoresis (SEP)
 Basic structure and function of
immunoglobulin.
 Monoclonal antibody, its modification and
application

Serum protein electrophoresis
Buffer pH 8.6
Serum electrophoresis
Immunoglobulin (antibody, BCR): universal adaptor
典型抗體分子的構造(1)
 BCR
and Ab
N端
構造
功能
異
C端序
列
作用
機制
同
免疫球 辨識
蛋白 抗原
C端
Proteolytic digestion of IgG
Variable region
Fab: papain digest
F(ab')2: pepsin
digest
Constant region
(Fc)
Different
domains have
different
functions
抗體分子與專一性抗原之交
互作用(1)
 局部區域的高度
變異序列形成抗
原結合位
Hypervariable region
Framework region
CDR (complementaritydetermining region)
General structures of five classes of
antibody
Structure and formation of secretory
IgA
Antigenic determinants of
immunoglobulin
Isotypes: all variants
present in serum of normal
individual
types: heavy and light chain
classes: IgG, IgA, IgM, IgD, IgE
subclasses: IgG1, IgG2, IgG3, IgG4
subgroups: VHI, VHII, VHIII
Allotypes: genetically controlled
alternative forms
Idiotypes: individually specific to
each immunoglobulin molecule
Immunoglobulin superfamily
Generation of diversity (G.O.D.)
VDJ rearrangement of heavy chain
 VJ rearrangement of light chain
 random combination of heavy and light
chain
 Imprecision of rearrangement

V-region genes are constructed from gene segments
Overview of B
cell
development
Model of allelic exclusion
Immunoglobulin concentration
changes along the age
Serum immunoglobulin vary among
different healthy persons
IgG: 800-1600 mg/dl
IgA: 140-400 mg/dl
IgM: 50-200mg/dl
IgD: 0-40 mg/dl
Antibody response
Clonal selection
 Primary and secondary antibody response
Isotype switch: IgM----->IgG
Affinity maturation: Somatic mutation

Monoclonal antibody
Myeloma (HGPRT-)
Spleen from immunized
mice
HAT medium
Selection
Limiting dilution and
cloning
Method
Immunization with antigen
Spleen and myeloma cell fusion
Polyethylene glycol (PEG): polymer of ethylene oxide
HAT medium: selection of hybridoma
Screening
Cloning by limiting dilution: Poisson distribution
Large scale production and storage: can be purified by protein A
affinity column
cell line (10-50 mg/ml)
ascites (1-10 mg/ml)
Main pathway (de novo)
~~~~~~~~~~~~~~~~~~~~~~
DNA
RNA
Aminopterin Block
N U C L E O T I D E S
HGPRT
TK
Hypoxanthine
Thymidine
Secondary Pathway
Normal
splenocyle
Hybrid cell
spontaneous death
ACTIVE GROWTH
Enzyme-deficient
(HGPRT-) myeloma cell
HAT-dependent death
Application of monoclonal antibodies:
universal adaptor
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Antigen purification: affinity column
Diagnosis: detect Ag or Ab
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pathogen
cell surface marker
soluble antigens e.g. AFP
cancer diagnosis: immunoimaging
Immunotherapy
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Cancer therapy
Autoimmune disease treatment
Allergy prevention
Tissue transplantation
Application of monoclonal antibody
Application of mAb as
immunotherapeutic drugs
Monoclonal antibodies that recognize tumorspecific antigens might be used in a variety of
ways to help eliminate tumors
MAT: the magic bullet
Therapeutic results were disappointing
until recently.
 Two major hurdles were encountered.

 Biologic
activity in humans severely limited
due to the host’s immune response to foreign
mAbs (production of human anti-mouse Abs).
Causes neutralization of activity and toxicity.
 Large-scale production
Mouse mAb may cause serum
sickness in vivo
Approaches to reduce or eliminate
human anti-mouse antibodies.

Chimeric Antibodies
 Cloned
antibody genes are generated in which
the variable regions from the original mouse
mAb are combined with human antibodyconstant regions.
 Highly effective in reducing human antimouse antibodies (HAMA). However, the
variable (Ag-recognition) domains are still
foreign; HAMA often still occurs neutralizing
efficacy.
Generation of chimeric mouse-human
mAb
Antibodies can be engineered to
reduce their immunogenicity in
humans
humanized
chimaetic
Red: mouse
White: human
Chimeric Antibodies
Humanized mAbs

All portions of the mAb not required for
antigen binding, including framework
residues in the variable region, are
replaced with human sequences.
 Gene
engineering, expression, evaluation
 < 10% of optimized mAb retains mouse
sequence

Highly-effective (but time-consuming)
approach
抗體分子與專一性抗原之交
互作用
 局部區域的高度
變異序列形成抗
原結合位
Hypervariable region
Framework region
CDR (complementaritydetermining region)
Humanized
mAb
CDR:
complementary
determination
region
Antibodies can be engineered to
reduce their immunogenicity in
humans
humanized
chimaetic
Red: mouse
White: human
Modification of mouse mAb
Immunotoxin:
magic bullet
Antibodies in action
MAT in Organ Transplantation
Rejection of allografts is an immunological
response exerted primarily by T-cells. mAb
approaches against T-cell antigens has proven
effective.
 CD25 (IL2-Receptor a chain). Highly effective
approach for the prevention of graft rejection
episodes and graft loss (e.g., renal).

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basiliximab (chimeric)
dacliximab (humanized)
MAT in Coronary Artery Disease

Atherosclerotic lesions (plaques) involve platelet
aggregation followed by coagulation and
thrombus formation. Platelet receptors involved
in early steps of plaque formation.

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basis of “one-aspirin-a-day” concept
limited efficacy in preventing platelet aggregation
Abciximab (chimeric mAb) has been
developed/approved as an effective treatment
for the prevention of thrombosis and plaque
formation in high risk patients.
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Targets the platelet receptors (integrins)
Prevents platelet aggregation
MAT in Autoimmune Diseases
TNFa is a proinflammatory cytokine that has
been implicated as a causal factor in a variety of
inflammatory diseases including Rheumatoid
Arthritis.
 Infliximab is a chimeric mAb targeted to TNFa.
has displayed remarkable activity against
Rheumatoid Arthritis and other inflammatory
diseases (Crohn’s Disease).

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Approved in 1998. First for Crohn’s Disease; now R.A.
Antibodies can be used to alleviate
and suppress autoimmune disease
Anti-inflammatory effects of anti-TNF-a therapy in rheumatoid arthritis
MAT in Cancer (I)
Most intensively studied area (25 years of
research).
 Cancer poses the highest hurdles.
 Also being evaluated in modified versions
involving the conjugation to radio-ionizing
particles, immunotoxins,
immunoliposomes.

MAT in Cancer (II)
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Rituximab (chimeric mAb)
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Targets CD20; a cell surface protein expressed exclusively on B
cells.
Approved for the treatment of B cell malignancies (NHL, CLL,
ALL).
Trastuzumab (humanized mAb)

Her2 is a form of the EGF-Receptor that undergoes amplification
in approximately 25% of breast cancers.
1st identified as a rat oncogene called neu (HER2/neu)
 Patients with amplified HER2 expression suffer a very poor
prognosis.

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Trastuzumab (Herceptin) has displayed impressive activity
against HER2+ breast cancer both alone and in combination.
Summary
Antibodies are universal adaptors which can bind
to any specific target both in vitro and in vivo.
 Humanized monoclonal antibody reduce its
immunogenecity without change its
specificity.
 MAT provides another way to develop new
therapy against cancer, autoimmune
disease…etc. (serum therapy)

New approaches under development
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Phage-display libraries expressing human VH
and VL chains are assembled into libraries and
expressed as combinatorial matrices.
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Assayed for immunoreactivity against antigen of
interest.
Transgenic Mouse Approach
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A procedure in which the endogenous mouse Ig gene
loci is replaced by homologous recombination with
their human homologs.
Immunization of transgenic mouse followed by
standard hybridoma procedures produces a fully
humanized mAb.
Advantages of phage display
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More efficiently than through conventional hybridoma system.
Cheaper to produce recombinant antibodies using bacteria, rather
than mammalian cell line.
Easier to maintain and grow bacterial cultures for recombinant
antibody production.
Bypass immunization in antibody selection.
Bypass the use of animal cells for production of antibodies.
Producing the combinatorial library (ideally with 108 to 109
members) of functional antibodies to generate a larger repertoire
of antibodies than those available through conventional
hybridoma technology.
Easy isolation and expression of the cloned gene in a bacterial
host.
Excellent potential to further improve binding properties of the
selected antibody by protein engineering techniques.
PNAS 101:2536, 2004 Feb.
Selection of phage library and
screening of phage antibodies

Two human nonimmune single-chain variable
region fragment (scFv) libraries (a total of
2.7x1010 members) constructed from B cells of
57 unimmunized donors were used for
selection of scFvs against the purified S1-C9
Generation human mAb by
transgenic mice