Therapeutic monoclonal antibodies
© Oldřich Farsa 2020
ANTIBODIES
●
produced by the B cells (= B lymfocytes, a subgroup of agranulocytes) of the immune
system.
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like weapons of our defense system and can be described as “ homing devices ” that target
antigens and destroy them.
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immunoglobulins of 5 classes:
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immunoglobulin G and D (IgG and IgD,  75%)
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immunoglobulin A (IgA  15%)
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immunoglobulin M (IgM  15%)
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immunoglobulin E (IgE < 1%).
They differ from each other in size, charge, carbohydrate content, and amino acid
composition. Within each class, there are subclasses that show slight differences in structure
and function from other members of the class.
Antibody structure
●
normally depicted as a capital letter Y configuration
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IgG is the most predominant antibody: a tetrameric molecule consisting of two identical heavy
(H) polypeptide chains of about 440 amino acids and two identical light (L) polypeptide chains
of about 220 amino acids
●
four chains are held together by disulfide bonds and noncovalent interactions
●
within the light and heavy chains are domains, which consist of about 110 amino acids of
similar polypeptide sequence: constant domains C H1, C H2, and C H3 of the heavy chain
C L domain of the light chain
●where the sequence is variable, variable domains, one each on the heavy and light chain: V H
and V L
●
the variability is confined to particular regions of the variable domain, called the
complementarity - determining regions. These regions have the appropriate 3D structure to
bind to antigens.
Antibody
structure
Typical quartenary structure of immunoglobulins
Antibody structure
An antibody can be cleaved by enzymes such as papain and pepsin into
different fragments.These different fragments are the following:
• Variable Fragment (Fv): The tips of the two Y arms vary greatly from one
antibody to another. They are the regions that bind to epitopes of antigens
and bring them to the natural killer cells and macrophages for
destruction.
• Antigen - Binding Fragments (Fab), Fab′ , and F (ab′) 2 : Various parts that
contain the variable fragment.
• Constant Fragment ( Fc): This is the stem of the letter Y . It is the part that
is identical for all antibodies of the same class; for example, all IgGs have
the same Fc. The Fc fragment is the part that links the antibody to other
receptors and triggers immune response and antigen destruction.
Different fragments of the antibody
molecule
PapainPapai
n
Monoclonal antibodies for human use as defined by the Pharmacopoeia (EP 7.5)
Anticorpora monoclonalia ad usum humanum
Monoclonal antibodies for human use are preparations of an immunoglobulin or a fragment
of an immunoglobulin, for example, F(ab′)2, with defined specificity, produced by a single
clone of cells. They may be conjugated to other substances, including for radiolabelling.
They can be obtained from immortalised B lymphocytes that are cloned and expanded as
continuous cell lines or from rDNA-engineered cell lines.
Currently available rDNA-engineered antibodies include the following antibodies.
Chimeric monoclonal antibodies: the variable heavy- and light-chain domains of a human
antibody are replaced by those of a non-human species that possess the desired antigen
specificity.
Humanised monoclonal antibodies: the 3 short hypervariable sequences (the
complementarity-determining regions) of non-human variable domains for each chain are
engineered into the variable domain framework of a human antibody; other sequence
changes may be made to improve antigen binding.
Recombinant human monoclonal antibodies: the variable heavy- and light-chain domains
of a human antibody are combined with the constant region of a human antibody.

The EP does not suppose fully animal mabs in current use in humans.
Examples of mabs in therapeutical use
woldwide
Examples of particular therapeutical
monoclonal antibodies
Antineoplastics
Growth factors inhibitors
bevacizumab
Avastin®
●
humanized: Immunoglobulin G 1 (human-mouse monoclonal rhuMAb-VEGF gamma-chain
anti-human vascular endothelial growth factor), disulfide with human-mouse monoclonal
rhuMAb-VEGF light chain, dimer
●
angiogenesis inhibitor
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antibody against vascular endothelial growth factor (VEGF)
●
vascular endothelial growth factor (VEGF), a pro-angiogenic factor, binds to 2 receptors
VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), activates receptor tyrosin kinase (RTK) and
induces angiogenesis
●
VEGF and its receptors are often over-expressed in cancers, that is why angiogenesis was
proposed as a target site of anti-cancer therapy by Folkman and col. in 1970th
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bevacizumab approved in USA in 2004 for treatment of metastatic colorectal cancer
combined with fluorouracil; later against non small cells lung cancer (2006) and breast
cancer (2008); for the same purposes approved also in EU
●
its efficacy, either alone or in combnation, was demonstrated also in many other cancers
including neuroendocrinous ones, which are often resistant to classical chemotherapy
●
recently, falsifications of Avastin ® has been catched in U.K.
ramucirumab
syn. IMC-1121B
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humanized
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angiogenesis inhibitor
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targeted against VEGFR-2 receptor
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VEGFR-2 is selectively expressed in cancer endothelial cells, simultaneously it is in a
direct contact with blood  promising therapeutical target
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antibodies against Flk-1 isoform antagonised binding of VEGF to the receptor, signal
transduction by means of VEGFR-2 and VEGF induced endothelial cells growth 
antiangiogenic, antitumor and antimetastatic activity
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clinical trials: phase 2 for breast cancer , phase 3 for non small lungs cells carcinoma
combined with docetaxel, phase 2 for prostate cancer combined with mitoxantron and
prednisone, phase 3 for gastric cancer etc.
trastuzumab
Herceptin ®
●
humanized
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IgG1 κ anti – HER2
●
family of receptors for epidermal growth factor includes 4 structurally very similar receptors:
Erb/HER (EGFR; HER-1, and ERBB1), human EGFR-2 (HER-2 and
●
ERBB2), HER-3, a HER-4, transmembrane glycoproteins containing a domain binding an
intracellular ligand and an intracellular receptor tyrosine kinase (RTK) domain
●
deregulation of Erb/HER pathway by over-expression or by constitutive activation can trigger
a cancer process including angiogenesis and metastasising and brings a bad prognosis in
many types of human cancers
●
early studies with trastuzumab as a single agent in HER-2-positive metastatic breast cancer
achieved overall responses of 11.6 and 15% for patients who had progressed after
chemotherapy. As a first-line treatment for metastatic breast cancer, trastuzumab showed
response rates of 26% in HER-2-positive patients and responses of 35% in patients with 3+
HER-2 overexpression by immunohistochemistry and 34% in patients positive for HER-2 gene
amplification by fluorescence in situ hybridisation (FISH).
●
a pivotal phase III trial of trastuzumab in combination with chemotherapeutic agents
demonstrated an overall response rate of approximately 50% (versus 32%), longer duration of
response (time to progression; 7.4 versus 4.6 months), longer survival (overall survival:
25.1 versus 20.3 months) and a 20% reduction in risk of death compared to chemotherapy
alone in HER-2 overexpressing metastatic breast cancer.
cetuximab
Erbitux®
●chimeric IgG1 antibody
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binds to the EGFR with an affinity that is approximately 5- to 10-fold higher than that of
endogenous ligands
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blocks binding of endogenous EGFR ligands resulting in inhibition of the function of the
receptor
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further induces the internalisation of EGFR, which can lead to down-regulation of EGFR
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also targets cytotoxic immune effector cells towards EGFR-expressing tumour cells
(antibody dependent cell-mediated cytotoxicity, ADCC)
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in both in vitro and in vivo assays, cetuximab inhibits the proliferation and induces apoptosis
of human tumour cells that express EGFR. In vitro cetuximab inhibits the production of
angiogenic factors by tumour cells and blocks endothelial cell migration. In vivo cetuximab
inhibits expression of angiogenic factors by tumour cells and causes a reduction in tumour
neo-vascularisation and metastasis.
●
cetuximab approved by both FDA and EMA for treatment of metastasising colorectal
carcinomas expressing EGFR
panitumumab
Vectibix ®
●fully human monoclonal IgG2 antibody against EGFR
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produced in a mammalian cell line by recombinant DNA technology
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binds to the ligand binding domain of EGFR and inhibits receptor autophosphorylation
induced by all known EGFR ligands
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its binding to EGFR results in internalisation of the receptor, inhibition of cell growth,
induction of apoptosis, and decreased interleukin 8 and vascular endothelial growth factor
(VEGF) production
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in vitro assays and in vivo animal studies have shown that panitumumab inhibits the growth
and survival of tumour cells expressing EGFR
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no anti-tumour effects of panitumumab were observed in human tumour xenografts lacking
EGFR expression
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the addition of panitumumab to radiation, chemotherapy or other targeted therapeutic
agents, in animal studies resulted in an increase in anti-tumour effects compared to
radiation, chemotherapy or targeted therapeutic agents alone
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unwanted skin reactions occurred in 93 % of patients
Antitumour mabs, which target various receptors and signaling
proteins on the cell surface
etaracizumab
Abegrin®
●
syn. vitaxin, MEDI-522
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humanized
●against αvβ3 integrin
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integrins: a family of receptors on the cell surface, which are responsible for exchange of
information between cells and an extracellular matrix, which surrounds them (ECM)
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heterodimers composed from 1 – 10 -subunits and 1 - 8 -subunits
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every subtype has its specificity for a different protein of ECM
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signals, which influence growth, migration ability, differentiation, invasivity and survival of cells,
are generated in a cell in response to binding of ECM components
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integrins play an important role in tumour biology; useful target of anti-cancer therapy
●V3 integrins are more expressed in developing vessels than in “adult” ones; they are
supposed to be an important factor of angiogenesis
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vitronectin is the primary ligand, they also interact with fibronectin and thrombospondin
●relationship of V3 with i.a. vascular endothelial growth factor (VEGF) was demonstrated
●administration of a murine monoclonal antibody against V3 (LM609) interrupted cancercaused
angiogenesis on a chicken chorioallantoic membrane
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the ability of the substance to stop cancer vascularisation and cause its regression without a
damage of normal matured vessels was verified in murine models of various cancers in vitro
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etaracizumab is a fully humanized form
●expression of V3 murine ovarian cancer and simultaneously effect of etaracizumab against
it were demonstrated
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clinical tests of phases 1 – 2 for treatment of various cancers (colorectal, malignant
melanoma, androgen-independent prostate cancer, kidney cancer, lymphoma) and
autoimmune inflammatory diseases (plaque psoriasis, rheumatoid arthritis) were finished
alemtuzumab
MabCampath ®
●humanised IgG1 monoclonal antibody specific for a lymphocyte cell surface glycoprotein
of 21 000 < Mr < 28 000 (CD52) expressed primarily on the surface of several types of
leukocytes
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produced in mammalian cell (Chinese Hamster Ovary) suspension culture in a nutrient
medium
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alemtuzumab was generated by the insertion of six complementarity-determining regions
from an IgG2a rat monoclonal antibody into a human IgG1 immunoglobulin molecule
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causes the lysis of lymphocytes by binding to CD52, a highly expressed, non-modulating
antigen which is present on the surface of essentially all B and T cell lymphocytes as well as
monocytes, thymocytes and macrophages
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the antibody mediates the lysis of lymphocytes via complement fixation and antibodydependent
cell mediated cytotoxicity
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the antigen has been found on a small percentage (< 5%) of granulocytes, but not on
erythrocytes or platelets
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alemtuzumab does not appear to damage haematopoietic stem cells or progenitor cells
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indicated for the treatment of patients with B-cell chronic lymphocytic leukaemia (B-CLL) for
whom fludarabine combination chemotherapy is not appropriate
rituximab
Mabthera ®
●
chimeric mouse/human monoclonal antibody representing a glycosylated immunoglobulin
with human IgG1 constant regions and murine light-chain and heavy-chain variable region
sequences
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produced by mammalian (Chinese hamster ovary) cell suspension culture and purified by
affinity chromatography and ion exchange, including specific viral inactivation and removal
procedures
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binds specifically to the transmembrane antigen, CD20, a non-glycosylated phosphoprotein,
located on pre-B and mature B lymphocytes
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the antigen is expressed on >95 % of all B cell non-Hodgkin’s lymphomas
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CD20 is found on both normal and malignant B cells, but not on haematopoietic stem cells,
pro-B cells, normal plasma cells or other normal tissue
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this antigen does not internalise upon antibody binding and is not shed from the cell surface.
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CD20 does not circulate in the plasma as a free antigen and, thus, does not compete for
antibody binding
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the Fab domain of rituximab binds to the CD20 antigen on B lymphocytes and the Fc domain
can recruit immune effector functions to mediate B cell lysis. Possible mechanisms of effectormediated
cell lysis include complement-dependent cytotoxicity (CDC) resulting from C1q
binding, and antibody-dependent cellular cytotoxicity (ADCC) mediated by one or more of the
Fcγ receptors on the surface of granulocytes, macrophages and natural killers (NK) cells.
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rituximab binding to CD 20 antigen on B lymphocytes has also been demonstrated to induce
cell death via apoptosis
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indicated for the treatment of follicular lymphoma, CD20 positive diffuse large B cell nonHodgkin’s
lymphoma, chronic lymphocytic leukaemia (CLL) and severe active rheumatoid
arthritis
ofatumumab
Arzerra ®
●human monoclonal antibody (IgG1) that binds specifically to a distinct epitope
encompassing both the small and large extracellular loops of the CD20 molecule
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produced in a recombinant murine cell line (NS0)
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indicated for the treatment of chronic lymphocytic leukaemia (CLL) in patients who are
refractory to fludarabine and alemtuzumab
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authorised under a so-called ‘conditional approval’ scheme: further evidence on this
medicinal product is awaited by the European Medicines Agency (EMA)
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the binding of ofatumumab to the membrane-proximal epitope of the CD20 molecule
induces recruitment and activation of the complement pathway at the cell surface, leading
to complement-dependent cytotoxicity and resultant lysis of tumour cells
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induces appreciable lysis of cells with high expression levels of complement defence
molecules
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induces cell lysis in both high and low CD20 expressing cells and in rituximab-resistant
cells
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in addition, the binding of ofatumumab allows the recruitment of natural killer cells
allowing the induction of cell death through antibody-dependent cell-mediated cytotoxicity
catumaxomab
Removab ®
●trifunctional rat-mouse hybrid IgG2 monoclonal antibody specifically directed against
the epithelial cell adhesion molecule (EpCAM) and the CD3 antigen
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produced in a rat-mouse hybrid-hybridoma cell line
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EpCAM: a superficial glycoprotein of human cells expressed in some „normal“ ones,
but namely in cancer cells („pancarcinomatic“ antigen – discovered 1979; in nearly u
100 % ovary carcinomas resistant to chemotherapy)
Tertiary structure of EpCAM
containing 3 extracellular
domains with cysteine -S-Sbridges
and sites of glycosylation
●
CD3 is one of the signal transducing modules expressed on mature T-cells
●
a component of the T-cell receptor, which is a multimeric cell-surface complex and is
normally responsible for the recognition of antigen associated with the major
histocompatibility complex (MHC)
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if a ligand such as catumaxomab is bound to CD3, the T cell is stimulated to perform its
effector functions (cytokine release and/or cytotoxicity)
Structure and mechanism of action of
catumaxomab
●
the anti-tumour activity of catumaxomab has been demonstrated in vitro and in vivo.
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effective catumaxomab-mediated killing of tumour cells in vitro was observed for target
cells with low and high expression of the EpCAM antigen, independent of the primary
tumour type
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the in vivo anti-tumour activity of catumaxomab was confirmed in an immunologically
compromised mouse model of ovarian carcinoma, where tumour development was
delayed by an intraperitoneal treatment with catumaxomab and human peripheral blood
mononuclear cells
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catumaxomab is indicated for the intraperitoneal treatment of malignant ascites in patients
with EpCAM-positive carcinomas where standard therapy is not available or no longer
feasible
adecatumumab
syn. MT201
●human IgG1
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against epitelial cell adhesion molecule (EpCAM) only
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phase II clinical tests against liver metastases of any cancer was proceeding from March
2009 to November 2011
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single-agent adecatumumab therapy has shown dose- and target-dependent clinical activity
in EpCAM-positive MBC, albeit no objective tumor regression. Further investigation of
adecatumumab in patients with EpCAM-overexpressing tumors and lower tumor burden are
proposed.
ipilimumab
Yervoy ®
●fully human anti-CTLA-4 monoclonal antibody (IgG1κ)
●
produced in Chinese hamster ovary cells by recombinant DNA technology
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cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a negative regulator of T-cell activation
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ipilimumab is a T-cell potentiator that specifically blocks the inhibitory signal of CTLA-4,
resulting in T-cell activation, proliferation, and lymphocyte infiltration into tumours, leading
to tumour cell death
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the mechanism of action of ipilimumab is indirect, through enhancing T-cell mediated
immune response
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indicated for the treatment of advanced (unresectable or metastatic) melanoma in adults
who have received prior therapy
lexatumumab
● IgG1 human monoclonal antibody – TRAIL (=Tumor necrosis factor-related apoptosisinducing
ligand) agonist
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TRAIL induces apoptosis in tumorigenic and transformed cell lines (cultures), but rarely in
normal cells
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TRAIL interacts with the specific domain death receptors, DR4 and DR5, and triggers
trimerization and formation of a primary complex (DISC = Death Inducing Signaling Complex)
in the intracellular lipid rafts in the cytoplasm. DISC synthesis involves the recruitment of
caspase 8, mediated by the adaptor proteins, into the death domain of the activated receptor.
Activated caspase 8 triggers the extrinsic apoptic pathway by the direct activation of efectors,
e.g. caspases 3 and 7. Except of it, caspase 8 can initiate the intrinsic apoptic pathway be the
activation of pro-apotic Bid protein. Both pathways lead to caspase 3 activation and to
(tumour) cell death.
●
●
lexatumumab was intended for therapy of urinary bladder and prostate cancer and in this
indication successfully tested in rabbits
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clinical tests of the phase I n childern and young adoults with solid carcinoma were being
performed between December 2006 and December 2011
●
the primary aim was to determine the tolerance of the adult maximum tolerated dose and
dose limiting toxicities of lexatumumab in patients with refractory pediatric solid tumors.
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the secondary aim was to quantify tumour response to lexatumumab
Antiinfective mabs
tefibazumab
Aurexis®
●humanized IgG1
●Mr  147 590
●
treatment of Staphylococcus aureus infections
●
targets fibrin-binding surface epitope of the clumping factor A (CfA) expressed on the
surface of most Staphylococcus aureus strains
●
CfA belongs among surface adhesins of the cell of S. aureus of the MSCRAMMs type
(microbial surface components recognizing adhesive matrix molecules), which are a family of
cell surface staphylococcal adhesins whose primary function is to facilitate bacterial
attachment to the extra-cellular matrix of the host during the colonization phase of infection,
and which also can facilitate colonization of implants and damaged endothelial surfaces
●
CfA binds fibrinogen
●
Proposed mechanism of action for MSCRAMM® protein antibodies; inhibition of
microbial adhesion and subsequent immune clearance, or phagocytosis
Endothelium surface with fibrinogene molecules
CfA
tefibazumab
●
phase II clinical tests against staphyloccoccal bacteriaemia were succesfully finished
●
in June 2006, Inhibitex was seeking to outlicense certain development rights to the
drug. Pending the outcome of partnering discussions, Inhibitex suspended the initiation
of any additional clinical trials of tefibazumab.
●
Inhibitex was later acquired by Bristol-Myers Squibb
pagibaximab
syn. BSYX-A110
●
chimeric
●
against staphylococcal lipoteichoic acid (LTA) –
an important constituent of the cell wall of
staphylococci; LTA is anchored in the cell
membrane with its lipophilic part; it inhibits
bacteria phagocytosis in vitro, induces cytokines
cascade and is supposed to be necessary for
staphylococci survival, also helps staphylococci
to permeate blood-brain barrier (BBB)
●
prevention of staphylococcal sepsis in
premature neonates with very low birth weight
●
security and efficacy has been verified by phase
2 – 3 blinded clinical study: Three once-a-week
90 or 60 mg/kg pagibaximab infusions, in highrisk
neonates, seemed safe and well tolerated.
No staphylococcal sepsis occurred in infants who
received 90 mg/kg.
Structure of lipoteichoic acid from Streptococcus
pneumoniae
raxibacumab
ABthrax ®
●
human
●IgG1 against the „protective antigen“, which is a part of anthrax toxin
●
succesfully tested in rabbits and monkeys infected with inhaled spores of Bacillus anthracis
●
succesfull phase 3 clinical tests in healthy volunteers: a „security study“
●
raxibacumab has demonstrated efficacy in a post-exposure setting even after toxins were
released into the bloodstream
●
in the U.S. Strategic National Stockpile (SNS) as a tool for safeguarding citizens against
anthrax bioterorism attack
edobacomab
syn. E5
●
mouse
●
IgE against bacterial endotoxin
●
endotoxin = lipopolysaccharide part of the outer membrane of bacteria; primarirly
iniiates „gramnegative sepsis“: triggers the cascade of pro-inflammatory cytokines (eg.
TNF), which in its result stimulates several pro- and anti-inflammatory cascades 
systemic signs typical for sepsis
●
a compound binding (= neutralizing) an endotoxin could be capable to stop this cascade
●
endotoxins of various G- bacteria markedly differ one from each other, E5 is however
bound to the „nucleus“ - a sequence that is common for all
●
phase 3 clinical study: E5 did not improve short-term survival compared with placebo
bavituximab
Tarvacin ®
●
chimeric
●against phosphatidylserine (PS) bound to 2-glycoprotein I
●
phosphatidylserine – the most widely spread anionic phospholipide of the plasmatic
mebrane
R
1
O
O
H
O
OR
2
O P
OH
O
O H
NH2
OH
O
Phosphatidylserin
●
PS is normally tightly segregated to the internal surface of the plasma membrane in most cell
types, including the vascular endothelium
●
PS asymmetry is maintained by ATP-dependent aminophospholipid translocases (Mg2+ATPases)
that catalyze the transport of aminophospholipids from the external to the internal
leaflet of the plasma membrane
●
loss of PS asymmetry occurs during apoptosis, necrosis, cell activation and transformation,
resulting in the exposure of PS on the external surface of the cells
●
similar situation occurs in viruses infected cells, in which replication proceeds: activation of the
cell by a virus causes intracellular Ca2+ loss  triggering of PS „exporteurs“ & inhibition of its
„import“ inside the cell by means of translocases  „externalisation“ of PS
●
potential anti-tumour and antiviral drug (HCV, HIV)
●
approx. 15 finished and ongoing phase 1 and 2 clinical studies against hepatitis C and/or HIV
infection and various tumours (breast, non-small cell lung, liver, prostate, pancreatic) in USA,
India, Georgia, Russia and Ukraine
Drugs for chronic inflammatory diseases
Tumour Necrosis Factor
There are two types of tumour necrosis factor: TNF -α and TNF - β . Of the two, TNF - α has
been studied in more detail. TNF - α is a 157 amino acid polypeptide. It is a mediator of
immune regulation, including the activation of macrophages and induction of the proliferation
of T cells. Another TNF - α function is its cytotoxic effects on a number of tumour cells. Recent
research, however, concentrates on its property in the stimulation of inflammation, particularly
in the case of rheumatoid arthritis, Crohn disease, ulcerative collitis etc. Clinical trials are
being conducted with drugs to block TNF - α with anti - TNF - α monoclonal antibodies. These
antibodies target the excessive levels of TNF - α in the synovial fluid of joints or in the
intestinal mucosa and provide relief to sufferers of rheumatoid arthritis or of an inflammatory
disease of GIT.
TNF-
inhibitors
„Anti-TNF molecules“ - are bound to TNF and neutralize its activity
Infliximab (Remicade ® ) : mouse/human chimaera, where variable regions of murine
antibody are linked to constant regions of human IgG1
Adalimumab (Humira ® ): recombinant human antibody of IgG1 type expressed in Chinese
Hamster Ovary cells
Golimumab (Simponi ® ): recombinant human antibody of IgG1 type produced by a murine
hybridoma cell line with recombinant DNA technology
Certolizumab pegol (Cimzia ® ): recombinant, humanised antibody Fab' fragment against
TNF expressed in Escherichia coli and conjugated to polyethylene glycol (PEG).
Etanercept (Enbrel ® ): soluble dimeric fusion protein in which human p75 TNF receptor is
linked to Fc domain of human IgG1
Usage: treatment of rheumatoid arthritis, inflammatory intestinal disease (ulcerative colitis,
Crohn disease...) and many other inflammatory diseases.