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Current
Pharmaceutical Design
ISSN: 1381-6128
Current Pharmaceutical Design
Volume 15, Number 23, 2009
Contents
Recombinant Immunotoxins – The Next
Generation
Executive Editor: Stefan Barth
Editorial: Pp. 2650-2651
Recombinant Immunotoxins for the Treatment of Chemoresistant
Hematologic Malignancies Pp. 2652-2664
R.J. Kreitman
[Abstract] [Purchase
Article]
Ribonucleases and ImmunoRNases as Anticancer
Drugs Pp. 2665-2675
S.M. Rybak, M.A.E. Arndt, T. Schirrmann,
S. Dübel and J. Krauss
[Abstract] [Purchase
Article]
Development of Novel, Highly Cytotoxic
Fusion Constructs Containing Granzyme B: Unique Mechanisms
and Functions Pp. 2676-2692
M.G. Rosenblum and S. Barth
[Abstract] [Purchase
Article]
Immunokinases, a Novel Class of Immunotherapeutics
for Targeted Cancer Therapy Pp. 2693-2699
M.K. Tur, I. Neef, G. Jäger, A. Teubner,
M. Stöcker, G. Melmer and S. Barth
[Abstract] [Purchase
Article]
Improved Immunotoxins with Novel Functional
Elements Pp. 2700-2711
C. Hetzel, C. Bachran, M.K. Tur, H. Fuchs
and M. Stöcker
[Abstract] [Purchase
Article]
Fcγ
Receptor 1 (CD64), a Target Beyond Cancer Pp.
2712-2718
T. Thepen, M. Huhn, G. Melmer, M.K. Tur
and S. Barth
[Abstract] [Purchase
Article]
Targeting Phosphatidylserine in Anti-Cancer
Therapy Pp. 2719-2723
H. Kenis and C. Reutelingsperger
[Abstract] [Purchase
Article]
General Articles
Nanocarriers in Ocular Drug Delivery: An
Update Review Pp. 2724-2750
S. Wadhwa, R. Paliwal, S.R. Paliwal and
S.P. Vyas
[Abstract] [Purchase
Article]
Vitamin D Deficiency: The Invisible Accomplice
of Metabolic Endotoxemia? Pp. 2751-2758
P. Lee and L.V. Campbell
[Abstract] [Purchase
Article]
Abstracts
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Editorial: Recombinant Immunotoxins – The Next
Generation
Immunotoxins are anticancer agents with at least
two functional components, one of which binds to a tumor-specific,
internalizing cell-surface antigen and one that confers cellular
toxicity. The two components may be joined covalently in
vitro by chemical conjugation or they may be expressed
as a fusion protein generated by recombinant DNA technology.
The binding moiety is usually a monoclonal antibody derivative
or a cytokine, ultimately of mammalian origin, whereas the
cytotoxic moiety is an enzyme that may originate from any
source, including plants and bacteria as well as mammals.
After selective binding to a tumor-specific receptor, immunotoxins
are internalized and translocated into the cytosol. The cytotoxic
component then induces cell death through its catalytic activity.
Beyond the first recombinant immunotoxins to undergo clinical
studies were based on Pseudomonas exotoxin A, a format
pioneered by Pastan and colleagues. One of the major challenges
with this format was its immunogenicity – treated patients
produced neutralizing antibodies against the toxin, reducing
the effectiveness of multiple treatment cycles. This special
issue of Current Pharmaceutical Design considers recent developments
in immunotoxin technology that aim to overcome this difficulty
by incorporating modified or novel cytotoxic components into
immunotoxins. The new generation of recombinant immunotoxins
includes molecules that incorporate cytotoxic human enzymes,
or non-human enzymes with reduced immunogenicity.
In the first review, RJ Kreitman [1] summarizes results from
recent clinical studies with immunotoxins based on Pseudomonas
exotoxin A. These studies involved immunotoxins targeting
CD25 (LMB-2) and CD22 (BL22 and HA22) in patients with hematologic
malignancies, and showed promising results against a range
of leukemias and lymphomas. The review considers recent strategies
to reduce the immunogenicity of the bacterial toxin, e.g.
by removing immunodominant epitopes or by suppressing the
patient’s immune system nonspecifically.
Several research groups have investigated the possibility
of replacing immunogenic bacterial toxins with human enzymes,
to generate fully human immunotoxins. Rybak et al.
[2] were the first to consider this approach, using human
ribonucleases (RNases) as the cytotoxic component. Their review
discusses the most recent developments in the field of immunoRNase
fusion proteins, focusing on the results of pre-clinical studies
featuring immunoRNases targeting CD22 and CD30.
Similarly, Rosenblum et al. [3] discuss preclinical
data on immunotoxins incorporating the human protease Granzyme
B. The authors present several examples including the specific
targeting of melanoma by gp240, neovascularized tumors by
KDR/flk-1 receptors on vascular endothelial cells, Hodgkin
lymphomas by CD30 receptors and distinct subtypes of acute
myeloid leukemia by CD64.
Tur et al. [4] discuss a novel approach in which
tumor cells are prompted to undergo apoptosis through the
reactivation of a suppressed pro-apoptotic kinase. The immunotoxin
in this case consists of the human CD30 ligand joined to a
constitutively active deletion mutant of death-associated
protein kinase 2 (DAPK2), whose activity is suppressed in
Hodgkin lymphoma cells. Using this approach, the authors were
able to show that the immunotoxin was active against only
those cells expressing CD30 on the surface and lacking DAPK2
activity, resulting in exquisite sensitivity and selectivity
towards Hodgkin lymphoma cell lines.
The bacterial toxins present in the first-generation immunotoxins
contain specific functional domains that facilitate active
translocation of the internalized immunotoxins into the cytoplasm.
Such domains are not found in any of the human enzymes that
have been used to replace bacterial toxins, thus reducing
their cytotoxic efficacy. Hetzel et al. [5] summarize
recent research focusing on the development of novel functional
elements that can be incorporated into fully human immunotoxins
to achieve higher specific toxicities without increasing their
immunogenicity.
Cell-specific targets play a crucial role in the development
of novel immunotherapeutics. Thepen et al. [6] provide
an overview of the role of macrophages as dysregulated immune
effector cells. Macrophages can be targeted specifically by
molecules that bind to Fc? receptor 1 (CD64). Onlγ
activated macrophages are eliminated by an immunotoxin targeted
in this fashion, whereas non-activated, resting macrophages
are not affected at all. The authors discuss these encouraging
results in the context of therapy for chronic inflammatory
diseases such as atopic dermatitis and rheumatoid arthritis.
Finally, Kenis et al. [7] describe the potential
value of phosphatidylserine as a novel anticancer target.
This lipid is normally found on the inner surface of the plasma
membrane but it is exposed on the surface of apoptotic cells
as well as tumor cells and tumor vasculature, and can be recognized
by the protein Annexin A5 which is currently used as a marker.
The therapeutic potential of immunotoxins based on AnxA5 is
discussed.
Together, these reviews provide a snapshot of the current
state of the art in immunotoxin research and possible future
trends in targeting strategies, enhanced cytotoxicity and
fine-tuning of the immune response. These exciting developments
show how the immunotoxins of the future could become more
specific and efficacious while off-target effects are reduced
or even eliminated, thus making cancer therapy more successful
and less arduous for the patient.
References
[1] Kreitman RJ. Recombinant immunotoxins for the treatment
of chemoresistant hematologic malignancies. Curr Pharm Des
2009; 15(23): 2652-2664.
[2] Rybak SM, Arndt MAE, Schirrmann T, Dübel S, Krauss
J. Ribonucleases and immunoRNases as anticancer drugs. Curr
Pharm Des 2009; 15(23): 2665-2675.
[3] Rosenblum MG, Barth S. Development of novel, highly cytotoxic
fusion constructs containing granzyme B: unique mechanisms
and functions. Curr Pharm Des 2009; 15(23): 2676-2692.
[4] Tur MK, Neef I, Jäger G, Teubner A, Stöcker
M, Melmer G, Barth S. Immunokinases, a novel class of immunotherapeutics
for targeted cancer therapy. Curr Pharm Des 2009; 15(23):
2693-2699.
[5] Hetzel C, Bachran C, Tur MK, Fuchs H, Stöcker M.
Improved immunotoxins with novel functional elements. Curr
Pharm Des 2009; 15(23): 2700-2711.
[6] Thepen T, Huhn M, Melmer G, Tur MK, Barth S. Fc? receptor
1 (CD64), a target beyond cancer. Curr Pharm Des 2009; 15(23):
2712-2718.
[7] Kenis H, Reutelingsperger C. Targeting phosphatidylserine
in anti-cancer therapy. Curr Pharm Des 2009; 15(23): 2719-2723.
Stefan Barth
Department of Pharmaceutical Product Development
Fraunhofer Institute for Molecular Biology and Applied Ecology
Aachen, Germany
Stefan.Barth@ime.fraunhofer.de
and
Department of Experimental Medicine and Immunotherapy
Chair of Applied Medical Engineering
Helmholtz Institute for Biomedical Engineering
University Hospital RWTH Aachen, Germany
Barth@hia.rwth-aachen.de
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Recombinant Immunotoxins for the Treatment of Chemoresistant
Hematologic Malignancies
R.J. Kreitman
Recombinant immunotoxins are proteins composed of fragments
of monoclonal antibodies fused to truncated protein toxins.
No agents of this class are approved yet for medical use,
although a related molecule, denileukin diftitox, composed
of interleukin-2 fused to truncated diphtheria toxin, is approved
for relapsed/refractory cutaneous T-cell lymphoma. Recombinant
immunotoxins which have been tested in patients with chemotherapy-pretreated
hematologic malig-nancies include LMB-2 (anti-CD25), BL22
(CAT-3888, anti-CD22) and HA22 (CAT-8015, anti-CD22), each
containing an Fv fragment fused to truncated Pseudomonas exotoxin.
Major responses were observed with LMB-2 in adult T-cell leukemia,
chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma,
Hodgkin’s disease, and hairy cell leukemia (HCL). BL22
resulted in a high complete remission rate in patients with
HCL, particularly those without excessive tumor burden. HA22,
an improved version of BL22 with higher affinity to CD22,
is now undergoing phase I testing in HCL, CLL, non-Hodgkin’s
lymphoma, and pediatric acute lymphoblastic leukemia.
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Ribonucleases and ImmunoRNases as Anticancer Drugs
S.M. Rybak, M.A.E. Arndt, T. Schirrmann,
S. Dübel and J. Krauss
Ribonucleases degrade RNA, now considered an important
drug target. The parent member of this protein superfamily
is bovine pancreatic RNase A that functions as a digestive
enzyme. Other physiological roles and activities have been
ascribed to more recently discovered members of this superfamily.
Angiogenin was isolated by following angiogenic activity from
cell culture media conditioned by colon cancer cells. ONCONASE
kills tumor cells in vitro and in vivo and
has advanced to a phase IIIb confirmatory clinical trial for
the treatment of unresectable malignant mesothelioma. All
three of these RNA degrading enzymes have been used to generate
immunoRNases; chemical conjugates and ligand-RNase fusion
proteins, for cancer therapy. The properties of each of these
RNases are described along with the increasingly sophisticated
construction of recombinant immunoRNases. The advantages of
using RNase as an antibody payload is compared to using plant
or bacterial toxins in the construction of immunotoxins, a
related strategy for specifically killing malignant cells.
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Development of Novel, Highly Cytotoxic Fusion Constructs Containing
Granzyme B: Unique Mechanisms and Functions
M.G. Rosenblum and S. Barth
Recombinant fusion proteins are an expanding, important
class of novel therapeutic agents. The designs of these constructs
typically involve a cell-targeting motif genetically fused
to a highly toxic class of enzymes capable of ruthlessly attacking
critical cellular machinery once delivered successfully to
the cytoplasm of the target cell. Initial development of this
class of constructs typically contained recombinant growth
factors or single-chain antibodies as the cell-targeting motif
fused to highly cytotoxic plant or bacterial toxins. This
review describes second-generation molecules composed of cell-targeting
molecules fused to highly cytotoxic human enzymes capable
of generating intense apoptotic response once delivered to
the cytoplasm. The human serine protease granzyme B has been
shown to be extremely effective as a cytotoxic molecule when
incorporated into numerous cell-targeting constructs. The
biological activity of GrB-containing constructs rivals that
of plant or bacterial toxins and appears to represent a new
generation and class of completely human proteins with unique
biological activities.
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Immunokinases, a Novel Class of Immunotherapeutics for Targeted
Cancer Therapy
M.K. Tur, I. Neef, G. Jäger, A. Teubner,
M. Stöcker, G. Melmer and S. Barth
Certain characteristics of tumor cells make it possible
to develop rational strategies for targeting tumors without
harming normal cells. These include the presence of cell surface
molecules that characterize the current state of the tumor
(e.g. CD30 on Hodgkin lymphoma cells) and the genetic and
epigenetic changes that activate oncogenes and inactivate
tumor suppressor genes (e.g. the inactivation of tumor suppressor
gene DAPK2 in Hodgkin lymphoma cells, which blocks
apoptosis). We have developed a novel tumor-targeting fusion
protein by combining a selective ligand (CD30L) with a constitutively
active version of DAPK2 (DAPK2'-CD30L), thus increasing tumor
specificity and reducing systemic toxicity. We showed that
this immunokinase fusion protein induces apoptosis specifically
in CD30+/DAPK2–
tumor cells in vitro and significantly prolonged
overall survival in a disseminated Hodgkin lymphoma xenograft
SCID mouse model. Therapeutic strategies based on the cell-specific
restoration of a defective, tumor-suppressing kinase demonstrate
the feasibility of targeted therapy using recombinant immunokinases.
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Improved Immunotoxins with Novel Functional Elements
C. Hetzel, C. Bachran, M.K. Tur, H. Fuchs
and M. Stöcker
Immunotoxins (ITs) are protein-based drugs combining a target-specific
binding domain (usually derived from an antibody) and a cytotoxic
domain to kill target cells. They are among the most promising
new therapeutic tools to fight cancer, and several clinical
trials have been completed with encouraging results. Although
the targeted elimination of malignant cells is an elegant
concept, there are numerous practical challenges that limit
the clinical use of ITs, including inefficient cellular uptake,
low cytotoxicity and off-target effects. Here we present some
of the strategies that have been developed to improve the
efficacy of ITs, particularly those involving the incorporation
of functional peptide sequences into recombinant ITs to improve
target binding, modify plasma half life and distribution,
boost tumor penetration, enhance cellular uptake and increase
cytotoxic efficiency.
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Fcγ
Receptor 1 (CD64), a Target Beyond Cancer
T. Thepen, M. Huhn, G. Melmer, M.K. Tur
and S. Barth
Immunotoxins are powerful tools to specifically eliminate
deviated cells. Due to the side effects of the original immunotoxins,
they were only considered for the treatment of cancer as in
these cases, the potential favourable effect outweighed the
unwanted toxic side effects. Over time, many improvements
in the construction of immunotoxins have been implemented
that circumvent, or at least strongly diminish, the side effects.
In consequence this opens the way to employ these immunotoxins
for the treatment of non-life threatening diseases. One such
category of disease could be the many chronic inflammatory
disorders in which an uncontrolled interaction between inflammatory
cells leads to chronicity. In several of these chronic conditions,
activated macrophages, which are characterised by an increased
expression of CD64, are known to play a key role. In this
review we discus the data presently available on elimination
of activated macrophages through CD64 immunotoxins in several
animal models for chronic disease. A chemically linked complete
antibody with the plant toxin Ricin-A, proved very effective
and provided proof of concept. Subsequently, the development
towards genetically engineered, fully human, multivalent single
chain based immunotoxins that have diminished immunogenicity,
is discussed. The data show that the specific elimination
of activated macrophages through CD64 is indeed beneficial
for the course of disease. As opposed to other methods used
to inactivate or eliminate macrophages, with the CD64 based
immunotoxins only the activated population is killed. This
may open the way to apply these immunotoxins as therapeutics
in chronic inflammatory disease.
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Targeting Phosphatidylserine in Anti-Cancer Therapy
H. Kenis and C. Reutelingsperger
Targeted delivery of cytotoxic agents limits the severe
toxic side-effects of anti-cancer drugs on healthy tissues.
Annexin A5 is a well explored probe to target phosphatidylserine
(PS)-expressing cells in vivo. Our novel understanding
of the cellular and molecular mechanism of annexin A5 as a
cell-entry agent and the finding that PS is expressed on living
tumour as well as endothelial cells in the tumour vasculature,
will allow the development of lead compounds for anti-cancer
therapy.
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Nanocarriers in Ocular Drug Delivery: An Update Review
S. Wadhwa, R. Paliwal, S.R. Paliwal and
S.P. Vyas
Controlled drug delivery to eye is one of the most challenging
fields of pharmaceutical research. Low drug-contact time and
poor ocular bioavailability due to drainage of solution, tear
turnover and its dilution or lacrimation are the problems
associated with conventional systems. In addition, anatomical
barriers and physiological conditions of eye are also important
parameters which control designing of drug delivery systems.
Nanosized carriers like micro/nano-suspensions, liposome,
niosome, dendrimer, nanoparticles, ocular inserts, implants,
hydrogels and prodrug approaches have been developed for this
purpose. These novel systems offer manifold advantages over
conventional systems as they increase the efficiency of drug
delivery by improving the release profile and also reduce
drug toxicity. Conventional delivery systems get diluted with
tear, washed away through the lacrimal gland and usually require
administering at regular time intervals whereas nanocarriers
release drug at constant rate for a prolonged period of time
and thus enhance its absorption and site specific delivery.
This review presents an overview of the various aspects of
the ocular drug delivery, with special emphasis on nanocarrier
based strategies, including structure of eye, its barriers,
delivery routes and the challenges/limitations associated
with development of novel nanocarriers. The recent progresses
in therapy of ocular disease like gene therapy have also been
included so that future options should also be considered
from the delivery point of view. Recent progress in the delivery
of proteins and peptides via ocular route has also
been incorporated for reader benefit.
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Vitamin D Deficiency: The Invisible Accomplice
of Metabolic Endotoxemia?
P. Lee and L.V. Campbell
The aetiology of obesity is multi-factorial. Recent research
has identified a novel association between endotoxaemia (circulating
lipopolysaccharide in the systemic circulation) and low-grade
inflammation in the adipose organ, which may contribute to
obesity. The mechanisms for the low-grade elevation of circulating
lipopolysaccharide in obesity are poorly understood.
Vitamin D has been increasingly recognised for its pleiotropic
actions beyond maintenance of musculoskeletal health. The
parathyroid-vitamin D axis is altered in obesity. Circulating
vitamin D levels are lower in obese individuals. The regulatory
role of vitamin D in the immune system and colonic mucosa
may explain the under-appreciated contribution of vitamin
D deficiency in the obese to the pathogenesis of endotoxaemia
and adipose inflammation.
We propose a hypothetical model linking metabolic endotoxaemia
with vitamin D deficiency in obesity. A therapeutic approach
involving the use of probiotics and vitamin D metabolites
in the obese is described.
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