Transcript Bispecific antibodies - Lectures For UG-5
Slide 1
Bispecific antibodies
Touqeer Ahmed Ph.D.
Atta-ur-Rahman School of Applied Bioscience,
National University of Sciences and Technology
24rd September, 2014
Slide 2
Bispecific antibodies
•
•
•
•
Bispecific antibodies (bsAbs) contain two different
binding specificities within a single molecule and
these can specifically bind two different molecules
together
They have been used extensively for research and
therapeutic approaches
In principal, bsAbs could be used in any application
where two molecules need to be cross-linked
noncovalently within a distance of about 10 to 20
nM.
Types (based on how are these produced)
1. Chemically cross-linked antibodies
2. Hybrid hybridoma antibodies
Slide 3
Type1-Chemically Cross-Linked Antibodies
•
In this protocol two antibodies or their Fab
fragments are cross-linked using the hetero
bifunctional compound N-succinimidyl-3-(2pyridyldithiol) propionate (SPDP)
•
This reagent binds randomly to ε-amino
groups on lysine residues and forms disulfide
bonds between antibodies
•
The resulting bispecific molecules consist of
aggregates of antibodies of varying size
linked together at random sites.
Slide 4
Epsilon Amino Group on Lysine
Slide 5
Type-2 Antibodies Obtained from Hybrid Hybridoma
• Differentially labeled hybridomas are
fused and cells staining for both
fluorochromes are isolated using a
fluorescence-activated cell sorter
(FACS)
• One hybridome is labeled with
fluorochrome FITC (green) and the
other one is labeled with TRITC
(red). These are fused with poly
ethylene glycol (PEG)
• Double labeled hybridoma are
selected by FACS
Slide 6
Purification of Bispecific Antibodies
• Using affinity column chromatography and
using both antigens one after the other or
using two separate columns for the
purification of bispecific antibodies
• Specificity checking in ELISA
Slide 7
Uses of Antibodies
Slide 8
Application of Monoclonal Antibodies
Therapeutic Field
Anti-tumor therapy
Antibodies against tumor antigen are produced and they are
converted into Immunotoxins
Immunosuppression
Using monoclonal antibodies against TCR, BCR, Co-receptor
complex and cytokines etc
Muromonab-CD3 (OKT3)
Antithymocyte globulins
Alemtuzumab
Drug toxicity reversal
Toxicity produced by drugs is treated using monoclonal
antibodies against drugs, so that the functions of drugs are
blocked and effect reversed.
Slide 9
Application of Monoclonal Antibodies
Therapeutic Field
Anti-tumor therapy
•
Monoclonal antibodies have become an active area of drug development for
anticancer therapy and other non-neoplastic diseases, because they are directed at
specific targets and often have fewer adverse effects. They are created from B
lymphocytes (from immunized mice or hamsters) fused with “immortal” Blymphocyte tumor cells.
•
Currently, several monoclonal antibodies are available in the United States for the
treatment of cancer. Trastuzumab, rituximab, bevacizumab, and cetuximab are
important examples.
• Alemtuzumab: is effective in treatment of B-cell chronic lymphocytic leukemia
that no longer responds to other agents
• I131-tositumomab: is used in relapsed non-Hodgkin lymphoma.
• Gemtuzumab ozogamicin: which is a monoclonal antibody conjugated with a
plant toxin that binds to CD33 (a cell-surface receptor that is present on the
leukemia cells of 80 percent of patients with acute myelocytic leukemia)
Slide 10
Trastuzumab
• In patients with metastatic breast cancer, overexpression of
transmembrane human epidermal growth factor–receptor protein
2 (HER2) is seen in 25 to 30 percent of patients.
• Trastuzumab [tra-STEW-zoo-mab], a recombinant DNA–produced,
humanized monoclonal antibody, specifically targets the
extracellular domain of the HER2 growth receptor that has intrinsic
tyrosine kinase activity.
• The drug, usually administered with paclitaxel, can cause regression
of breast cancer and metastases in a small percentage of these
individuals.
• Trastuzumab binds to HER2 sites in breast cancer tissue and inhibits
the proliferation of cells that overexpress the HER2 protein, thereby
decreasing the number of cells in the S phase.
Slide 11
Heterodimer formation of members of the HER family and
downstream signaling
Signaling downstream of HER family activation is dependent on heterodimerization of the HER family member triggered by
ligand binding to the extracellular ligand-binding domain (with the exception of HER2, which has no identified ligand and is
always in an open conformation that allows dimerization). Phosphorylation of the HER kinase domains (with the exception
of HER3, which does not have a kinase domain) initiates a downstream cascade resulting in VEGF transcription and other
physiological responses required for carcinogenesis.
Abbreviations: AR, amphiregulin; BTC, betacellulin; EPG, epigen; EPR, epiregulin; HB-EFG, heparin-binding EGF-like ligand;
NRG, neuregulin.
Nature Reviews Clinical Oncology 9, 16-32 (January 2012)
Slide 12
Cell Division in Eukaryotes
•
•
In eukaryotic cells, cell division
starts with the most important
process of DNA replication
The cell cycle consists of
four distinct phases:
–
–
–
–
G1 phase
S phase (synthesis)
G2 phase
M phase (mitosis)
G1, S and G2 phases are collectively known as interphase
M phase is composed of two tightly coupled processes:
•Mitosis, in which the cell's chromosomes are divided between the two daughter cells
•Cytokinesis, in which the cell's cytoplasm divides in half forming two distinct cells
Slide 13
Trastuzumab:
Mechanism of action:
• How the antibody causes its anticancer effect remains
to be elucidated.
Several mechanisms have been proposed:
– for example, down-regulation of HER2-receptor
expression
– an induction of antibody-dependent cytotoxicity,
– or a decrease in angiogenesis due to an effect on
vascular endothelial growth factor.
• Efforts are being directed toward identifying those
patients with tumors that are sensitive to the drug.
Slide 14
Trastuzumab
• Pharmacokinetics: Trastuzumab is administered IV. Trastuzumab
does not penetrate the blood-brain barrier.
• Adverse effects:
– The most serious toxicity associated with the use of trastuzumab is
congestive heart failure. The toxicity is worsened if given in
combination with anthracycline.
– Extreme caution should be exercised when giving the drugs to patients
with preexisting cardiac dysfunction.
– Other adverse effects include infusion-related fever and chills,
headache, dizziness, nausea, vomiting, abdominal pain, and back pain,
but these effects are well tolerated.
– Cautious use of the drug is recommended in patients who are
hypersensitive to the Chinese hamster ovary cell components of the
proteins or to benzyl alcohol (in which case sterile water can be used in
place of the bacteriostatic solution provided for preparation of the
injection).
Slide 15
Rituximab
• Rituximab (ri-TUCKS-ih-mab) was the first monoclonal antibody to
be approved for the treatment of cancer.
• It is a genetically engineered, chimeric monoclonal antibody
directed against the CD20 antigen that is found on the surfaces of
normal and malignant B lymphocytes.
• CD20 plays a role in the activation process for cell-cycle initiation
and differentiation.
• The CD20 antigen is expressed on nearly all B-cell non-Hodgkin
lymphomas but not in other bone marrow cells.
• Rituximab has proven to be effective in the treatment of
posttransplant lymphoma and in chronic lymphocytic leukemia.
Slide 16
Rituximab
Mechanism of action:
The Fab domain of rituximab binds to the CD20 antigen
on the B lymphocytes, and its Fc domain recruits
immune effector functions, inducing complement and
antibody dependent, cell-mediated cytotoxicity of the B
cells.
The antibody is commonly used with other
combinations of anticancer agents, such as
cyclophosphamide, doxorubicin, vincristine (Oncovin),
and prednisone (CHOP).
Slide 17
Rituximab
Pharmacokinetics:
Rituximab is infused IV and causes a rapid
depletion of B cells (both normal and
malignant). The fate of the antibody has not
been described.
Slide 18
Rituximab
Adverse effects:
Severe adverse reactions have been fatal. It is important to
infuse rituximab slowly. Hypotension and bronchospasm may
occur.
Chills and fever commonly accompany the first infusion,
especially in patients with high circulating levels of neoplastic
cells, because of rapid activation of complement, which
results in the release of tumor necrosis factor α and
interleukins.
Pretreatment with diphenhydramine, acetaminophen, and
bronchodilators can ameliorate these problems.
Cardiac arrhythmias can also occur.
Bispecific antibodies
Touqeer Ahmed Ph.D.
Atta-ur-Rahman School of Applied Bioscience,
National University of Sciences and Technology
24rd September, 2014
Slide 2
Bispecific antibodies
•
•
•
•
Bispecific antibodies (bsAbs) contain two different
binding specificities within a single molecule and
these can specifically bind two different molecules
together
They have been used extensively for research and
therapeutic approaches
In principal, bsAbs could be used in any application
where two molecules need to be cross-linked
noncovalently within a distance of about 10 to 20
nM.
Types (based on how are these produced)
1. Chemically cross-linked antibodies
2. Hybrid hybridoma antibodies
Slide 3
Type1-Chemically Cross-Linked Antibodies
•
In this protocol two antibodies or their Fab
fragments are cross-linked using the hetero
bifunctional compound N-succinimidyl-3-(2pyridyldithiol) propionate (SPDP)
•
This reagent binds randomly to ε-amino
groups on lysine residues and forms disulfide
bonds between antibodies
•
The resulting bispecific molecules consist of
aggregates of antibodies of varying size
linked together at random sites.
Slide 4
Epsilon Amino Group on Lysine
Slide 5
Type-2 Antibodies Obtained from Hybrid Hybridoma
• Differentially labeled hybridomas are
fused and cells staining for both
fluorochromes are isolated using a
fluorescence-activated cell sorter
(FACS)
• One hybridome is labeled with
fluorochrome FITC (green) and the
other one is labeled with TRITC
(red). These are fused with poly
ethylene glycol (PEG)
• Double labeled hybridoma are
selected by FACS
Slide 6
Purification of Bispecific Antibodies
• Using affinity column chromatography and
using both antigens one after the other or
using two separate columns for the
purification of bispecific antibodies
• Specificity checking in ELISA
Slide 7
Uses of Antibodies
Slide 8
Application of Monoclonal Antibodies
Therapeutic Field
Anti-tumor therapy
Antibodies against tumor antigen are produced and they are
converted into Immunotoxins
Immunosuppression
Using monoclonal antibodies against TCR, BCR, Co-receptor
complex and cytokines etc
Muromonab-CD3 (OKT3)
Antithymocyte globulins
Alemtuzumab
Drug toxicity reversal
Toxicity produced by drugs is treated using monoclonal
antibodies against drugs, so that the functions of drugs are
blocked and effect reversed.
Slide 9
Application of Monoclonal Antibodies
Therapeutic Field
Anti-tumor therapy
•
Monoclonal antibodies have become an active area of drug development for
anticancer therapy and other non-neoplastic diseases, because they are directed at
specific targets and often have fewer adverse effects. They are created from B
lymphocytes (from immunized mice or hamsters) fused with “immortal” Blymphocyte tumor cells.
•
Currently, several monoclonal antibodies are available in the United States for the
treatment of cancer. Trastuzumab, rituximab, bevacizumab, and cetuximab are
important examples.
• Alemtuzumab: is effective in treatment of B-cell chronic lymphocytic leukemia
that no longer responds to other agents
• I131-tositumomab: is used in relapsed non-Hodgkin lymphoma.
• Gemtuzumab ozogamicin: which is a monoclonal antibody conjugated with a
plant toxin that binds to CD33 (a cell-surface receptor that is present on the
leukemia cells of 80 percent of patients with acute myelocytic leukemia)
Slide 10
Trastuzumab
• In patients with metastatic breast cancer, overexpression of
transmembrane human epidermal growth factor–receptor protein
2 (HER2) is seen in 25 to 30 percent of patients.
• Trastuzumab [tra-STEW-zoo-mab], a recombinant DNA–produced,
humanized monoclonal antibody, specifically targets the
extracellular domain of the HER2 growth receptor that has intrinsic
tyrosine kinase activity.
• The drug, usually administered with paclitaxel, can cause regression
of breast cancer and metastases in a small percentage of these
individuals.
• Trastuzumab binds to HER2 sites in breast cancer tissue and inhibits
the proliferation of cells that overexpress the HER2 protein, thereby
decreasing the number of cells in the S phase.
Slide 11
Heterodimer formation of members of the HER family and
downstream signaling
Signaling downstream of HER family activation is dependent on heterodimerization of the HER family member triggered by
ligand binding to the extracellular ligand-binding domain (with the exception of HER2, which has no identified ligand and is
always in an open conformation that allows dimerization). Phosphorylation of the HER kinase domains (with the exception
of HER3, which does not have a kinase domain) initiates a downstream cascade resulting in VEGF transcription and other
physiological responses required for carcinogenesis.
Abbreviations: AR, amphiregulin; BTC, betacellulin; EPG, epigen; EPR, epiregulin; HB-EFG, heparin-binding EGF-like ligand;
NRG, neuregulin.
Nature Reviews Clinical Oncology 9, 16-32 (January 2012)
Slide 12
Cell Division in Eukaryotes
•
•
In eukaryotic cells, cell division
starts with the most important
process of DNA replication
The cell cycle consists of
four distinct phases:
–
–
–
–
G1 phase
S phase (synthesis)
G2 phase
M phase (mitosis)
G1, S and G2 phases are collectively known as interphase
M phase is composed of two tightly coupled processes:
•Mitosis, in which the cell's chromosomes are divided between the two daughter cells
•Cytokinesis, in which the cell's cytoplasm divides in half forming two distinct cells
Slide 13
Trastuzumab:
Mechanism of action:
• How the antibody causes its anticancer effect remains
to be elucidated.
Several mechanisms have been proposed:
– for example, down-regulation of HER2-receptor
expression
– an induction of antibody-dependent cytotoxicity,
– or a decrease in angiogenesis due to an effect on
vascular endothelial growth factor.
• Efforts are being directed toward identifying those
patients with tumors that are sensitive to the drug.
Slide 14
Trastuzumab
• Pharmacokinetics: Trastuzumab is administered IV. Trastuzumab
does not penetrate the blood-brain barrier.
• Adverse effects:
– The most serious toxicity associated with the use of trastuzumab is
congestive heart failure. The toxicity is worsened if given in
combination with anthracycline.
– Extreme caution should be exercised when giving the drugs to patients
with preexisting cardiac dysfunction.
– Other adverse effects include infusion-related fever and chills,
headache, dizziness, nausea, vomiting, abdominal pain, and back pain,
but these effects are well tolerated.
– Cautious use of the drug is recommended in patients who are
hypersensitive to the Chinese hamster ovary cell components of the
proteins or to benzyl alcohol (in which case sterile water can be used in
place of the bacteriostatic solution provided for preparation of the
injection).
Slide 15
Rituximab
• Rituximab (ri-TUCKS-ih-mab) was the first monoclonal antibody to
be approved for the treatment of cancer.
• It is a genetically engineered, chimeric monoclonal antibody
directed against the CD20 antigen that is found on the surfaces of
normal and malignant B lymphocytes.
• CD20 plays a role in the activation process for cell-cycle initiation
and differentiation.
• The CD20 antigen is expressed on nearly all B-cell non-Hodgkin
lymphomas but not in other bone marrow cells.
• Rituximab has proven to be effective in the treatment of
posttransplant lymphoma and in chronic lymphocytic leukemia.
Slide 16
Rituximab
Mechanism of action:
The Fab domain of rituximab binds to the CD20 antigen
on the B lymphocytes, and its Fc domain recruits
immune effector functions, inducing complement and
antibody dependent, cell-mediated cytotoxicity of the B
cells.
The antibody is commonly used with other
combinations of anticancer agents, such as
cyclophosphamide, doxorubicin, vincristine (Oncovin),
and prednisone (CHOP).
Slide 17
Rituximab
Pharmacokinetics:
Rituximab is infused IV and causes a rapid
depletion of B cells (both normal and
malignant). The fate of the antibody has not
been described.
Slide 18
Rituximab
Adverse effects:
Severe adverse reactions have been fatal. It is important to
infuse rituximab slowly. Hypotension and bronchospasm may
occur.
Chills and fever commonly accompany the first infusion,
especially in patients with high circulating levels of neoplastic
cells, because of rapid activation of complement, which
results in the release of tumor necrosis factor α and
interleukins.
Pretreatment with diphenhydramine, acetaminophen, and
bronchodilators can ameliorate these problems.
Cardiac arrhythmias can also occur.