Download 5 practice - monoclonal antibody

Monoclonal antibodies
- products of a single B-lymphocyte clone
- homogeneous (antigene-specificity, affinity, isotype)
- in human body: only under pathological circumstances
e.g. in gammopathy
(malign growth of a certain plasma-cell clone)
-their advantage versus polyclonal antibodies:
antibodies of the given specificity and isotype can be
produced in large amount and of the same quality
only one
B-lymphocyte clone
more
B-lymphocyte clone
monoclonal antibody
Polyclonal antibody
Features of polyclonal and monoclonal antibodies
Polyclonal antibody
Monoclonal antibody
(low affinity)
Monoclonal antibody
(high affinity)
Number of recognized
antigen determinants
several (frequent
cross-reactions)
one (but frequent
cross-reactions)
mostly one
Specificity
polyspecific
often polyspecific
monospecific
Affinity
Varying (diverse
antibodies)
low
high
Concentration of nonspecific
immunoglobulines
high
low
low
Yield
high
low
low
Cost of preparation
low
high
high
Standardisability
Impossible (or
uneasy)
easy
easy
Amount
limited
limitless
limitless
Applicability
method-dependent
low
excellent
Procedure of monoclonal antibody
production
Hybridoma technology
- immunisation of a mouse/rat with a specific antigen
-removal of the spleen or lymph nodes of the mouse,
homogenisation
- fusion of mouse plasma cells (with spleen origin) + mouse tumor
cells (plasmocytoma/myeloma cells with B-cell origin)
- identification of antibody producing clones. The newly formed
hybridomas are proliferating continuously and producing antibodies
which concentrate in the medium.
Procedure of monoclonal antibody production II.
Immunisation
Selection of hybridoma cells
Spleen
B cells,
aminopterine
*HGPRT+
Myeloma cell
HGPRT-
PEG fusion
HAT selection
Testing supernatants for specific antibody production
HAT= hypoxanthine, aminopterine, thymidine
*Hypoxantine-guanine phosphoribosyltransferase
(1)Immunisation of a mouse
(2) Isolation of B cells from the spleen
(3) Cultivation of myeloma cells
(4) Fusion of myeloma and B cells
(5) Separation of cell lines
(6) Screening of suitable cell lines
(7) in vitro (a) or in vivo (b)
multiplication
(8) Harvesting
Factors influencing the efficacy of monoclonal antibody production
1. Activation of antigen-specific B-lymphocytes
- mouse: inbred lines with characterized genetic information,
small amounts of antigen is required for immunisation
The followings influence the efficacy of hybridoma cell separation:
a) the way of immunisation (using adjuvants, place of injection: intraperitoneal, foot,
tail-vein (caudal vein), spleen)
b) number of repeated shots to reinforce (boost) the immune response
c) number of days elapsed between the last vaccination and the fusion (2-4 days)
2. Fusion partners
- Sp2/0-Ag14 tetraploid,
non-antibody producing plasmocytoma cells from BALB/c mice
(these plasmocytoma cells have HGPRT /hypoxantine-guaninephosphoribosyl-transferase/ and thymidine kinase deficiency)
Possible use of monoclonal antibodies
- Identifying cell types
Immunohistochemistry
Characterization of lymphomas with CD (cluster of differentiation) markers
- Isolation of cells
Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral
blood!)
- Blood group determination (with anti-A, anti-B and anti-D monoclonals)
-Analysis of a mixture of antigens
- Identification of cell surface and intracellular antigens
Investigation of T-cell activation
- Targeted chemotherapy
CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma
Prevention of organ rejection after transplantation targeting T cells (anti-T cell
monoclonals)
- Drug elimination with antibodies
Anti-digoxin antibodies for the treatment of digoxin-intoxication
Monoclonal antibodies as drugs?
In immunized (with human antigens) mice the
produced antibodies will contain mousespecific proteins, and therefore, they will elicit
an immune response upon administering in
human subjects.
(see immunogenicity-determining factors!)
How we can solve this problem?
Evolution of monoclonal antibodies
Mouse
Chimeric
Human
Humanized
Humanizing monoclonal antibodies
1. CAMPATH-1H anti-CD52 monoclonal rat antibody
Repeated treatments with non-human antibodies induced strong immune
response in patients. (Problem: HAMA = human anti-mouse antibodies)
The antigen binding region of the rat antibody is exchanged with the antigen binding
region of a human antibody – in the resulting antibody, only the CDR will be rat
protein (chimeric antibodies).
(Problem: HACA = human anti-chimera antibodies, although they are less
immunogenic)
2. In vitro phage display: recombinated VDJ regions are expressed on the surface of
the filamentous phage capsid. Then, these phages with high-affinity for a given
antigen are separated by affinity chromatography. DNA (encoding the human
immunoglobulin genes) from these phages are expressed in transgenic mice.
3. Transgenic mice producing human antibodies: Human germ line (not
recombinated) Ig locus can be expressed as a transgene in knock-out mice where the
Ig genes of the mice were inactivated. Therefore, human Ig gene recombination
occurs in the mouse. Hybridomas can be made from mouse B cells producing human
antibodies, thus large amount of monoclonal antibodies can be prepared.
In vitro phage display
Expressed human antibody
Recombination of
VDJ gene
Phage display vector
Gene3
Human genes encoding
antibodies
Characterization (Separation of
DNA segments encoding human
Igs and multiplicate them in
transgene mice)
Expressed
antibodies on the
surface of the
phage
Selective binding
to antigen
Amplification
Washing
Recovery
Discard
mouse Ig „knock-out”
transgene mouse
(transferring genes encoding human Ig)
VDJ átrendeződés
a transzgén
egérben
Monoclonal antibodies as drugs
- Tumor therapy
Monoclonals made possible the targeted
chemotherapy of tumors. It is cell-type
specific, but not specific to malignant cells!)
- Immunsuppressive monoclonals
Cell-type specific immunsuppression
Monoclonal antibody nomenclature
The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or
nonproprietary names to a group of medicines called monoclonal antibodies.
This scheme is used for the World Health Organization’s International Nonproprietary Names.
Components of nomenclature:
Prefix Target
Source
Suffix
-ki(n)- interleukin as target -u-human
-ci(r)- cardiovascular
-o-mouse
variable
mab
-co(l)- colonic tumor
-xi-chimeric
-neu(r)- nervous system
Etc.
-zu-humanized
Etc.
Example:Abciximabab- + -ci(r)- + -xi- + -mab, it is a chimeric monoclonal antibody
used on the cardiovascular system
Monoclonals in tumor therapy
1. „Naked MAb”, unconjugated antibody
Anti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphoma
Anti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemia
Anti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancer
Anti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!)
Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. humán!)
2. Conjugated antibody
Anti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin)
Anti-CD20 + iodine-131 (tositumomab – Bexxar)
Immunsuppressive monoclonals 1.
1. Anti-TNF-α antibodies
infliximab (Remicade): since 1998, chimeric
adalimumab (Humira): since 2002, recombinant human
2. Etanercept (Enbrel) – dimer fusion protein,
TNF-α receptor + Ig Fc-part
(Not monoclonal antibody, containing only the Fc part of Ig)
Indications of anti-TNF-α therapy:
• Rheumatoid arthritis
• Spondylitis ankylopoetica (SPA - M.
Bechterew)
• Psoriasis vulgaris, arthritis psoriatica
• Crohn-disease, colitis ulcerosa
• (usually - still – not in the first line!)
Immunsuppressive monoclonals 2.
-
Muromonab-CD3 (OKT-3) mouse IgG2a
Against CD3 pan-T-cell antigen, after transplantation; It is rarely (or not) used nowadays (mouse
protein!); ongoing trials in diabetes mellitus, with the humanized version
-
Omalizumab (Xolair):
Anti-IgE humanized IgG1k monoclonal
Ind.: allergic asthma, Churg-Strauss sy.
-
Daclizumab (Zenapax):
anti-IL-2 receptor humanized antibody
Ind.: transplantation
-
basiliximab (Simulect): as daclizumab, but chimeric!
-
efalizumab (Raptiva): anti-CD11a, humanized, used in psoriasis
Molecular targeted drugs
Name
Type
Target
Indications
Alemtuzumab
Monoclonal antibody, humanized
CD52
CLL, CML
Monoclonal IgG1, chimeric
IL-2 R
transplantation
Monoclonal IgG1, chimeric
IL-2 R
transplantation
Monoclonal IgG1, chimeric
CD20
Lymphoma
Monoclonal IgG1, humanized
HER2/neu
Monoclonal IgG4, humanized
CD33
Breast cancer, NSC
lung cancer
leukemia
Monoclonal IgG1, murine
CD20
lymphoma
Monoclonal IgG2, murine
EGFR-TKI
KIT-TKI
EpCAM
EGFR TK
TK
CRC
NSCLC
GIST, CML
(Mabcampath)
Daclizumab
(Zenapax)
Basiliximab
(Simulect)
Rituximab
(Rituxan/Mabthera)
Trastuzumab
(Herceptin)
Gemtuzumab
Calicheamicinnel konjugált
Ibritumomab
(Y90)
Edrecolomab
Gefitinib
Imatinib
Further possibilities with monoclonals
Radioimmunotherapy
As Zevalin, Bexxar – monoclonal + isotope
Antibody-directed enzyme prodrug therapy (ADEPT)
An enzyme is linked to the antibody, and the enzyme will make
citotoxic drug from the later administered prodrug
Immunoliposomes
Targeting nucleotides or drugs in liposomes, linked to an antibody (eg.
tumor suppressor gene or tissue-specific gene transfer)
Non-immunological targets
as abciximab (ReoPro): inhibition of thrombocyte-aggregation