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ANTIBODY-BASED THERAPIES
Antibodies: properties and functions
Polyclonal and monoclonal antibodies
The production of the monoclonal antibodies
Passive immunisation
The therapeutic utility of monoclonal and polyclonal
antibodies
Modified antibodies, chimeric antigen receptors
The properties and the functions of
IMMUNOGLOBULINS
(review with some extras)
The effector funkctions
of antibodies
mast cell
activation
•
•
•
•
•
IgG - gamma (γ) heavy chains
IgM - mu (μ) heavy chains
IgA - alpha (α) heavy chains
IgD - delta (δ) heavy chains
IgE - epsilon (ε) heavy chains
Light chain types
• kappa (κ)
• lambda (λ)
Isotypes
Human Immunoglobulin Classes
The properties of the antibody isotypes
Antigen – antibody interaction
Valency and avidity
Valency: number of
connections between the
antigen and the antibody
Affinity: the „strength” of the
connection between one
epitope and one antigene
binding site
Avidity: the overall strength of
the interactions between
epitopes and binding sites
(like the teeth of the zipper or
velcro)
Antibody isotypes and functions
The significance of the Fc part
The different isotypes
show different
distributions in the
body
Fc receptors
FcγR : γ  IgG binding and transfer Fc receptors
FcεR : ε  IgE binding
FcαR : α  IgA binding
FcRn
„neonatális” Fc receptor
Placental cells, endothelial
and epithelial cells, etc.
IgG transfer, IgG salvage
pIgR
poly Ig receptor
Epithelial cells
IgA, IgM transfer
pIgR: IgA transport, secretory IgA
SS
The pIgR is cleaved by proteases, so the
IgA can detach from the cell surface. The
remaining receptor fragment on the IgA is
called: secretory fragment
SS
ss
J
SS
S
S
ss
S
S
SS
SS
SS
ss
SS
J
S
S
S
S
S
S
Epithelial
cell
pIgR internalizes
with the bound IgA
ss
SS
B
S
S
S
S
S
S
J
J
S
S
ss
J
SS
SS
SS
ss
ss
S
S
J
S
S
S
S
J
S
S
S
S
SS
SS
The pIgR bound IgA
transported across
the cell: transcytosis
M
U
C
I
N
SS
Submucosal B cells (BALT, GALT)
produce dimeric (trimeric) IgA
“poly Ig receptors” (pIgR)
located at the
basolatheral surface of
the epithelial cells.
They can transport IgA
(and IgM) to the apical
surface
What is transported by pIgR (poli immunoglobulin receptor)?
Immunoglobulins with J-chain: (polimer) isotypes: IgA, IgM
(DO NOT MIX this J chain with the J region of Ig gene rearrangement (VJ/VDJ rearrangement)l!!!)
Where to transport?
• epithelial (e.g. gut- and lung) surfaces
• secretums (saliva, breastmilk)
The IgM can form pentameric
or hexameric structures
without the J-chain also, but
this polimer is not transported
by pIgR.
Hexameric IgM can activate
the complement system
better than the pentameric
(an order of a magnitude).
IgG can form hexamers on antigenic surfaces with
dense repetitive epitopes
14 MARCH 2014 VOL 343 SCIENCE
• Superior complement activation
• No J-chain  no pIgR transport
IgG can be transported also
FcRn, FcγRn
Transport of monomeric IgG and albumin
expressing cell types:
- endothelial cells
- epitelial cells (digestive-, respiratory- and urogenital system)
- lots of cells of the immune system: e.g. professional APCs
neonatal Fcg receptor (FcRn)
Human FcgRn
Human MHC
Class I
(structural similarity, but different function)
The function of
FcRn
(rescue)
pinocitosis
FcRn binds the IgG with high
affinity in acidic environment
and rescue it from the
degradation
Transcytosis
IgG can be transported to various compartments in the body this way:
• blood (circulation)  tissues
• tissues  epithelial surfaces
(There are more IgG in the urogenital- or in the upper respiratory tract than IgA)
• mothernal circulation  foetal circulation
The half life of IgG is rised by FcRn
MONOCLONAL
and
POLYCLONAL ANTIBODIES
Both type are used therapeutically
Antigen: any kind of material that
can be recognised specifically by
the immune system
Antigen
Antigen determinants or epitops:
Different parts of the antigen where
the antigen specific antibodies are
binding
Antibody response
Ag
Immunserum
B cell set
Activated B cells
Policlonal
antibodies
Antibody producing
plasma cells
Ag
Antigen-specific
polyclonal antibodies
(the products of different B cell clones!)
The blood plasma of the immunised animals is a simple source for antigenspecific antibodies.
You should immunise more animals for prolonged usage of the antibodies
- The standardisation of the antibodies is difficult
- The amount of the antibodies are limited
A proliferating immortal antigen-specific B cell would provide infinite source
of antigen-specific antibodies.
Monoclonal antibody production
Georges J.F. Köhler, Cesar Milstein (Nobel prize 1984)
Antigen
Monoclonal antibodies
Polyclonal antibodies
Monoclonal antibodies
The products of one B cell clone
Homogenous (antigen specificity, affinity, isotype…)
They can be produced in large quantity, and similar quality for
extended period of time (principally infinite)
It needs the production of proliferating, immortal
antigen specific B cell
„immortalisation”?
- can happen „in vivo” in pathologic conditions
e.g. myeloma multiplex: malignant proliferation of a B cell clone  large quantity
of a monolonal antibody in the circulation
Possibilities?
Transformation/immortalisation by viruses
e.g. EBV – can transform human B cells
- The selection of the antigen specific clone is difficult
- The virus can appear in the antibody product as contamination
Possibilities?
Fusion with an immortal cell line
Requirements for the fusion partner:
- it should not produce it’s own antibodies
- the immortal status should be maintained in the hybrid
- it should not influence the antibody production of the
specific B cell
- selectability: some easy way to get rid of the aspecific
unnecessary cells
There are few good fusion partner cell line:
Mouse:
Sp2/0-Ag14 (SP2 cell line)
Human:
no good general fusion partner exists!
heteromyelomes, triomes can be used:
(The fusion partner could be a hybrid of 2-3 cell type by own.
Sometimes mice-human hybrid cells)
CB-F7, K6H6B5, H7NS, NAT-30, HO-323, A4H12
Fusion possibilities?
Micromanipulation
(needs expensive complicated hardware)
„FuCell”
antigen
B cell
fluorescent or paramagnetic
label
- The separation of the antigen specific B cells are mediated by
the antigen themself
- The B cells are placed in the vicinity of the fusion partner
- The fusion is mediated by electrical pulse
- As the B cell is antigen specific, there is no need for multistep
selection - the requirements of the fusion partner are not so strict
The process of the fusion in the instrument
The production of the monoclonal antibodies
Generally: Hybridoma production, cell fusion with mouse SP2 cell line
Hybridoma technology
 Antigen specific immune cells with limited lifespan (B cells) are fused with
immortal proliferating tumour cells. Stable, proliferating, antigene specific antibody
producing hybridoma cells can be separated with a careful multistep selection.
 The end products are specific antibody producing hybridoma clones, and
monoclonal antibodies.
Monoclonal antibody production in brief
serum,
policlonal
antibodies
immunisation
myeloma
antigen specific
activated B cells
spleen
- Immunisation of a laboratory animal
- Isolation of a lymphoid organ (e.g. spleen)
with antigen specific B cells
fusion
selection
- Fusion of the cells with the immortal
tumour cells
Antibody-selection
- Multistep selection of the specific antibody
producing hybridomas
The hybridoma cells continuously secrete
antigen-specific antibodies into the cell
culture medium
Recloning the antibody-producing clones
freezing
In vitro cultures
The main steps of the hybridoma production
spleen cells
myeloma cells (SP2)
PEG, Sendai virus,
Dying within days
Antigen specific
antibody producing
hybrids
Dividing (proliferating) cells
unnecessary hybrids
1st main selection step
spleen cells
HGPRT+ TK+
Discarding the unnecessary
myeloma cells
myeloma cells (SP2)
HGPRT- TK-
drug sensitive
(„HAT” selection)
HGPRT+ TK+
HGPRT- TK-
2nd main selection step:
Selecting the specific antibody producing cells
Antigen specific
antibody producing
hybridoma cells
The antibodies in the cell
culture medium can react
with the antigen
Other antibody
producing or non
antibody producing
hybridoma cells
No specific antibody in the
cell culture medium
Discarded
Further culturing,
Cell cloning
Cell cloning
The antigen specific
hybridome cells are
producing policlonal
antibodies. Some of them
are weak binder of the
antigen (low affinity) – not
useful
The high affinity antibody producing hybridoma cells can be selected
from the mixed hibridoma cells by cell cloning
(antibodies can be screened by ELISA)
Positive clones can be stored froen in liquid nitrogen for yeas or for decades
Difficulties, annoyances:
The transformed cells and the hybridomas with multiple
chromosome sets are genetically unstable  chromosome loss
They should be regularly tested for antibody production.
- recloning
The biochemistry of the „HAT” selection
Hypoxanthine, Aminopterin, Thymidine containing medium
nucleotide precursors
enzyme inhibitor
The „HAT” medium
and the two main pathway of the DNA synthesis
”salvage” pathway
reusage of nucleic acid degradation
products: purins, hypoxanthine, thymidine
HGPRT, TK
(hypoxanthine guanine phosphoribosyl transferase,
thymidine kinase)
DNS
nucleoside triphosphates
aminopterin
other enzymes
”basic” building blocks, simple sugars, amino
acids
”de novo” pathway
Only this pathway is
functioning in the SP2
myelome cells!
A slightly detailed picture:
”salvage” pathway
purins, e.g.
hypoxanthyne
thymidine
HGPRT
TK
dGTP
IMP
dTMP
DNS
dATP
phosphorybosyl
pyrophosphate,
glutamine
dTTP
dCDP
OMP
dCTP
aminopterin
”de novo” pathway
HAT  Hipoxantin, Aminopterin, Timidin
aspartic acid,
carbamoil phosphate
IMP – inozin monofoszfát
OMP – orotidin monofoszfát
HGPRT – hipoxantin-guanin foszforibozil transzferáz
TK – timidin kináz
The applications of the monoclonal antibodies in the clinical
practice
DIAGNOSTICS
•Blood typing, tissue typing (anti-A, anti-B, anti-D ellenanyagokkal)
•Analysis of complex antigenic mixtures (sera, liquor)
•Analysis of cell types and functions with cellular markers
•Immunohistochemistry
•Limphome typing by CD markers
THERAPEUTIC POSSIBILITIES (several hundred products exist)
•Cell separation
•CD34+ bone marrow stem cell separation
•Tumourtherapy (e.g, targeted chemotherapy)
•anti CD20 antibodies against B cell Non-Hodgkin
• Therapeutic inhibition of the cellular functions by blocking specific receptors or
the neutralisation of the ligands
•Tumourtherapy
•Immunosuppression
•T cell suppression after tissue/organ transplantation
Therapeutic antibodies
Monoclonal antibodies, as
medicaments?
The commonly available animal (mouse) antibodies can act
as antigens inside the human body (foreign proteins!!!)
How could it be coped with?
The „evolution” of the therapeutic monoclonal antibodies
mouse /rat
chimera
Human
Humanized
The glycosylation of the humanized antibodies are still similar to the producing cell type.
Real human monoclonals can be established by human hybridomes.
The most frequently used therapeutic
monoclonal antibody medicaments
- Tumourtherapy
Cell type specific targeting of the tumours by
selective chemotherapy
- Immunosuppression
Cell type specific immunosuppression, receptor
antagonists, ligand neutralisation
Monoclonal antibodies in tumour therapy
1. „Naked MAb”, non-conjugated antibodies
Anti-CD20 (rituximab – Mabthera/Rituxan, humanized): B-cell Non-Hodgkin lymphoma
Anti-CD52 (alemtuzumab – Mabcampath, humanizált): chronicus lymphoid leukaemia (3rd line,
chlorambucil and fludara resistent cases)
Anti-ErbB2 (trastuzumab – Herceptin, humanized): mammary tumour
Anti-VEGF (bevacizumab – Avastin, humanized): colorectalis tum. (+ Lucentis!)
Anti-EGFR (cetuximab – Erbitux, chimera): colorectalis tumours (+ Vectibix, recomb. human!)
2. Conjugated antibodies
Anti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin)
Anti-CD20 + Iodine-131 (tositumomab – Bexxar, chimera!)
Trifunctional antibody
ADDITIONAL APPROACHES WHICH UTILIZE
MONOCLONAL ANTIBODIES.
Radio-immunotherapy
Pl. Zevalin, Bexxar – monoclonal + isotope
Antibody-directed enzyme prodrug therapy (ADEPT)
The antibody bound enzyme process the applied prodrug to
form active drug metabolite locally
Immuno-liposomes
Liposome packed genes (tumour suppressor genes) or abortive
nucleotides, or drugs targeted with monoclonal antibodies
Other targeted manipulations
pl. abciximab (ReoPro): inhibits thrombocyte aggregation
Immunosuppressive antibodies and
receptor-Fc fusion products
1. Anti-TNF-α antibodies
infliximab (Remicade): 1998, chimera
adalimumab (Humira): 2002, recombinant human
2. Etanercept (Enbrel) – dimer fusion protein,
TNF-α receptor + IgG Fc-part
IgG Fc-part:
increased half life and spreadability in the body: FcRn
Anti-TNF-α therapy:
• Rheumatoid arthritis
• Spondylitis ankylopoetica (SPA - M.
Bechterew)
• Psoriasis, arthritis psoriatica
• Crohn-disease, colitis ulcerosa
IMMUNSUPPRESSIVE ANTIBODIES 2.
-
Muromonab-CD3 (OKT-3) mouse! IgG2a
anti-CD3 pan-T cell antigen, before or after transplantation as immunosuppression; as
mouse protein rarely applied novadays; the humanized version is being tried in type I
diabetes mellitus
-
Omalizumab (Xolair):
anti IgE humanized IgG1
Ind.: allergic asthma, Churg-Strauss syndrome
-
Daclizumab (Zenapax):
anti IL-2 receptor humanized
Indication: transplantation – against rejection
(inhibition of T-limphocyte activation)
-
Basiliximab (Simulect): as daclizumab, but chimera!
-
Efalizumab (Raptiva): anti-CD11a, humanized, ind.: psoriasis
The side effects of the immunsuppressive antibodies could be similar to the
symptomes of the immundefficiecies (e.g. opportunistic pathogenic diseases)
NOMENCLATURE OF THE MONOCLONAL ANTIBODIES
An International Nonproprietary Name (INN) is an official generic and nonproprietary name
given to a pharmaceutical drug or active ingredient.
Prefix
Target substem
old
new
Előtag— Cél
-anibi-
angiogenesis (inhibitor)
Stem
meaning
-aForrás
-b(a)-
bacterium
-ci(r)-
-c(i)-
circulatory system
-i-
primate
-fung-
-f(u)-
fungus
-o-
mouse
-gr(o)-
-gr(o)-
growth factor
-u-
human
-ki(n)-
-k(i)-
interleukin
-xi-
chimeric (human/foreign)
-ki(n)- interleukin
-les-
—
-li(m)-
-l(i)-
-zu-
humanized
immune system
-vet-
veterinary
musculoskeletal system
-xizu-*
chimeric/humanized hybrid
mab
systemtumor
-co(l)-nervous
colon
-n(e)-*
-os-
-s(o)-
bone
-toxa-
-tox(a)-
toxin
colonic tumor
-neu(r)idegrendszer
testicular tumor
Stb.. ovarian tumor
-go(v)-ma(r)-
-t(u)-
melanoma
-pr(o)-
prostate tumor
-tu(m)-
miscellaneous tumor
-v(i)-
-xi--axo-kiméra
-mab
-pab
rat/mouse hybrid
(see trifunctional antibody)
-zu-humanizált
stb.
mammary tumor
-me(l)-
-vi(r)* under discussion as of December 2009
hamster
inflammatory lesions
-ne(u)(r)-
-go(t)-
-u- humán
-ci(r)- kardiovaszkuláris -o- egér
variábilis
—
-co(l)-
-e-
rat
Utótag
-ba(c)-
-mulvariable
Source substem
meaning
virus
e.g. Abciximabab- + -ci(r)- + -xi- + -mab, monoclonal chimera antibody
used in cardiovascular disease
Prefix
variable
Target substem
Source substem
old
new
meaning
-anibi-
—
angiogenesis (inhibitor)
-a-
rat
-ba(c)-
-b(a)-
bacterium
-e-
hamster
-ci(r)-
-c(i)-
circulatory system
-i-
primate
-fu(ng)-
-f(u)-
fungus
-o-
mouse
-ki(n)-
-k(i)-
interleukin
-u-
human
-le(s)-
—
inflammatory lesions
-xi-
chimeric (human/foreign)
-li(m)-
-l(i)-
immune system
-zu-
humanized
-mu(l)-
—
musculoskeletal system
-xizu-*
chimeric/humanized hybrid
-ne(u)(r)-
-n(e)-*
nervous system
-o(s)-
-s(o)-
bone
-axo-
rat/mouse hybrid
-toxa-
-tox(a)-
toxin
-co(l)-
colonic tumor
-go(t)-
testicular tumor
-go(v)-
ovarian tumor
-ma(r)-
-t(u)-
mammary tumor
-me(l)-
melanoma
-pr(o)-
prostate tumor
-tu(m)-
miscellaneous tumor
-vi(r)-
-v(i)-
virus
meaning
'Suffix' (stem)
-mab
Passive immunisation
mouse monoclonal
antibodies
immunisation
humanized, mouse monoclonal
antibodies (recombinant methods)
immunisation
PROTECTED
INDIVIDUAL
serum antibodies
ENDANGERED
INDIVIDUAL
Human immunoglobulin
transgenic mouse
human monoclonal
antibodies
• Only the effector functions are activated
• Immediate effect
• Temporary protection
Immunoglobulin degradation!
Therapeutic usage of policlonal antibodies
Plasma products from blood donors are used regularely for human therapy
fresh freezed plasma or IVIG (intravenal immunoglobulin product)
treatment in:
• Immundefficient or weakened
persons (childrens) (passive
immunisation)
• antibody mediated (e.g. type II
hypersensitivity) autoimmune diseases
(overloading the FcRn system – the IgG
autoantiodies can also degrade without
efficient FcRn function)
Passive immunisation II.
Type
Usage
Intramuscular
(small amount can enter, low
effectivity)
HBV-Ig; Varicella-Zoster-Ig;
Anti-D profilaxy
Intravenal (IVIG)
Humoral immundefficiencies
(Bruton-agammaglobulinaemia;
hypogammaglobulinaemic
immunodefficiencies)
Anti venom therapy
(e.g. snake bite)
Natural passive immunisation:
Placental IgG transfer – protection of the newborn in the first few months
Breastmilk: IgA – protection of the upper gastrointestinal/respiratory tract
Passive immunisation III.
Policlonal antibodies
Antivenom – „antisera” against
snakes, scorpions, spiders, poisonous
sea creatures
Slow gradual hyperimmunisation of large animals
(usually horses)
Purified plasma immunoglobulin fraction –
decreased possibility of immunisation against the
horse serum proteins:
Repeated usage can be possible:
(slow, precautious application of antivenom, Epinephrine (adrenaline) should
always be drawn up in readiness)
Passive immunisation in EBOLA infection
• monoclonal antibody mix:
was shown effective in „In vitro” tests and laboratory animals. Experimental vaccines were autorized for the
emergency situations in the past years.
MB-003: humanized and human–mouse chimera mAb: c13C6, h13F6 and c6D8
ZMAb: 3 mouse mAb: m1H3, m2G4 and m4G7
ZMapp: 3 chimera: c2G4, c4G7 (ZMAb), c13C6 (MB-003)
This mAbs were proven the most effective during the previous epidemics
mAb genes can be transfected into agrobacteria with viral vector. The target place is the modified Ti (tumour
inducing) plasmide of the agrobacterium. Tobacco plant cells can be infected with the modified agrobacterium. The
genes are inserted in the plant genome. Antibodies can be easily purified from the plant.
Small stock was available during the 2014 epidemic. The stock was run out, too few data to evaluate.
• polyclonal immunsera from recovered individuals
Animal experiments with hyperimmunised horses (from 1991)  encouraging results
Human sera from recovered individuals (from the ’90ies)
There are promising results with both type of antibodies, but there are too few
investigations (case numbers) to significantly evaluate the effectivity.
Antibody-dependent enhancement (ADE)
„Sub-neutralising antibodies” can increase the efficiency of virus
infections
- Antibodies react with an inapropriate epitope
- Low affinity antibodies
- Low concentration antibodies
The antibodies can’t achieve neutralisation, but help the virus to enter the cell
Especially problematic in the case of viruses which enter the cell through the endosomal
system (e.g. Dengue vírus).
It was described in connection with other viruses also (HIV, Ebola)
It was shown:
• in vitro cell cultures
• Animal models
‐
‐
The virus titer was increased in monkeys, but the disease score was unchanged
Mouse models – sub lethal Dengue infection turned to lethal in AG129 mouse
strain when applying antibodies
Fc receptor mediated ADE (FcR-ADE),
The Dengue virus use FcR to reach
the compartment of the infection
Model of antibody-dependent enhancement of dengue infection
Antibody (Ab)-dependent enhancement of infection occurs when preexisting antibodies present in the
body from a primary (first) dengue virus (DENV) infection bind to an infecting DENV particle during a
subsequent infection with a different dengue serotype. The antibodies from the primary infection cannot
neutralize the virus. Instead, the Ab–virus complex attaches to receptors called Fcγ receptors (FcγR) on
circulating monocytes. The antibodies help the virus infect monocytes more efficiently. The outcome is an
increase in the overall replication of the virus and a higher risk of severe dengue.
Whitehead, S. S. et al. Prospects for a dengue virus vaccine. Nature Reviews Microbiology 5,518–528 (2007).
Complement receptor mediated enhancment (C-ADE)
can occure also
Base types of the modified monoclonal antibodies
The functionality can be increased and the unvanted
immunogenity can be further decreased with modifications
The unwanted/unnecessary functions can be avoided
(papain)
(pepsin)
(This form can occure naturally in some IgG subclass)
scFV
- single chain variable fragment
Two structural form
Avidity can be further
increased with the
combination of multiple
constructs with peptide linkers
• Neutralisation
• Blocking receptor functions
Bi-specific T-cell engager (BiTE) (di-scFv)
Dual specificity scFv
Chimeic Antigen Receptor (CAR)
Trasfected in effector cytotoxic or toxic cytokine producing helper T cells (TNF, LTA)
(scFV construct, Ig hinge region, intracellular CD3 zeta chain)
first-
secondCARs
linker
„tumor antigen”
recognizing scFV
VH
hinge region
TCR CD3 zeta chain and
the domains of the
costimulatory molecules
VL
third generation
CAR T cells can be co-transfected with inflammatory, cyctotoxic, or other immune
function boosting cytokine genes:
TRUCK (T cells redirected for universal cytokine killing)
TNF
IL-12
IL-23
IL-27
• boosting innate cytotoxic
immune response
• reprogramming or
relesing the
immunosupression
IL-12 or TNF is „toxic” when applied systemic. TRUCK T
cells will produce it locally after CAR engagement (NFAT
minimal promoter or IL-2 promoter driven cytokine production)
In the case of Erb receptors or the cytokine receptors, the cell surface bound
modified ligands can be applied instead of the scFV as a CAR
An artificial soluble antigen recognising CAR can be also co-transfected.
The intracellular part of this CAR can posses „death domains”. The unnecessary
CAR cells can be killed with the application of this artificial soluble antigen at the
end of the therapy.
BLOOD, 24 APRIL 2014 x VOLUME 123, NUMBER 17 2625
Immunological Reviews 2014 Vol. 257: 83–90
Immunological Reviews 2014 Vol. 257: 107–126
Transfus Med Hemother 2013;40:388–402