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Amanda Hidalgo
Writing 116
19 November 2013
Translating Integrin Receptors For Detection of Cancer
Introduction:
Integrins are a group of heterodimer cell surface receptors that are considered as the
central extracellular matrix receptors. They convey cell-matrix and cell-to-cell
interactions that involve adhesion, invasion, proliferation, and migration. In particular,
alpha-v-beta-6 (avb6) and alpha-v-beta-3(avb3) integrins are targets for cancer therapy.
Their expression increases the cellular components by the start of carcinoma types to
invade tumor cells. Clinical studies tested antibodies and peptides that bind to the
integrin receptors such as the fibronectin III domain, AgRP peptide, and cystine knots, as
a method to detect cancer; Specifically, the identification of pancreatic cancer by
translation of the cysteine knots binding to integrin receptors like, avb6 and avb3.
Unfortunately, there are some cases in which avb3 is expressed more in binding with
specific antibodies and peptides rather than avb6 integrin. This is because avb3 integrin
binds to a vast range of integrin ligands and sequences to regulate cell growth and
survival. This review will also include the effectiveness of yeast surface display, fine
epitope mapping, and down regulation of the integrin receptor, so in future purposes it
will be relevant for potential therapeutic approaches.
The Effectiveness of Peptides and Antibodies to Bind with Alpha-v-Beta-3 Integrin
The avb6 and avb3 integrin receptors are targets for cancer and applicable for advantages
in therapies. In one of the studies, researchers conducted phage cloning in order to bind
to the avb3 and avb6 receptors. By using the phage cloning, phage FNfn10 showed
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similarities to the avb3 integrin receptor rather than the avb6 integrin receptor. Kogelberg
et al investigated how the single-chain fv-antibody binds to avb3 integrin from the
specific loop of a foot-and-mouth-disease virus. From the collected data, a series of antiavb3 scFvs was generated by the insertion of a 17-mer peptide of VPI, composing the
prevention of a 20-mer peptide into the CDR-H3. (2) As a result from Kogelberg et al
study, the FNfn10-3JCLI4 is a possible effecting agent because of “its ability to
recognize avb3 on tumor vasculature”.
The figure on the left represents the prevention of glass capillary tube formations of
FNfn10-3JCL14. By the in vitro inhibition, there are still investigations in which to
explore the improvements of phage vector FNfn10-3JCL14 as binding agent with the
avb3 integrin. Not only did Kogelberg et
al study the significance of avb6, but
Ylipalosaari et al studied how the avb6
integrin down-regulates a matric
metalloproteinases expression in oral
squamous cell carcinoma cells (OSCC).
The matrix metalloproteinases (MMPs) are able to divide into various subdivisions and
have enzymatic activity. This is useful in the expression in OSCCs because the upregulation of avb6 promotes OSCC invasion in vitro and in vivo which indicates the
transfected OSCC cells to form a proteolytic cascade resulting in activation of the uPA to
MMP-3 to MMP-9 pathway (6). Ylipalosaari et al studied concluded in the research that
the “inhibition of avb6 induces MMP-13 protein expression by Western blotting, gelatin
zymography, and reverse zymography”. The research allowed the treatment with anti-
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avb6 antibody to be in C1 and VB6 cell lines.
With the TIMP-1 levels being
distinguishable in C1 and VB6 cells the avb6 antibody up-regulated MMP-9 expression
in order to invade OSCCs.
Yeast Surface Display
Most of the studies use a yeast surface display to examine the high specificity and affinity
of the integrin receptors. The yeast surface display is generated for the cystine-knot
library in order for avb3 integrin receptor to be tested for the ability for bind to express
human cancer cells. The figure below explains how Silverman et al conducted binding
titrations. The data represents the arbitrary mean of fluoresce units for 10^4 cells bound
with varying concentrations of the AgRP peptides and stained with fluorescein-labeled
anti-6x-His antibody. (5) Thus, the results showed that the AgRP peptide is able to bind
the integrin receptor with high affinity and can be potential in tumor imaging in animal
models to further out targeting of integrins on cancer cells. This will create cystine-knot
scaffold to be of greater importance clinically and scientifically.
The yeast surface display is effective for “finer, residue-level resolution of antibodyantigen binding interactions”(1). In spite of Silverman et al research using yeast surface
display, Lipovsek et al used a different approach on using the yeast surface display.
Lipovsek et al investigated Fn3 variants with sub-nanomolar affinity can be selected from
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libraries with only one or two randomized loops (3). Fn3 variants will bind with protein
and integrin receptors in order to recognize distinct structures of cancer cell targets.
When using the yeast surface display, the Fn3 variants that were chosen bond with a hen
egg white lysozyme. The hen egg white lysozyme is used as an antigen extensively in
structural studies of antibody-antigen binding (3). With the first set of selected antibody
mimics that bonded with the hen egg white lysozyme, yeast populations displayed
libraries BC7, FG7, 2L14, and BF14 to show the “affinity-maturation” from libraries BFs
1,2, and 3. Out of the libraries, FG7 constructed the random residues in loop FG of
human FN3 to be closely related as a selected clone. The construction of the residues in
loop FG of human FN3 makes the option of binding properties to be instantaneous and
accurate for the purification of individual clones. Researchers are expected to confirm
the structural studies of variants, such as FN3, with cystine pairs in complex with
lysozymes to contribute in binding surfaces on antibody-mimic molecules. Lipovsek et al
explained in the study how in vitro of the FN-3 based antibody could have “the tendency
of the system to re-evolve the disulfide after the cystine positions are randomized during
affinity maturation” (3). Thus, integrin receptors will improve on higher affinity
maturation and specificity.
Enhancement AVB3 By Engineering Fibronectin Type III Domain
Richards et al investigated that “high- affinity and high-specificity variants of FNfn10 to
a particular integrin could be engineered by optimizing residues surrounding the integrinbinding RGD sequence in the flexible FG loop”(4). They determined that the fibronectin
is able to bind with the avb3 integrin in vivo by means of the RGD sequence. The
modified FNfn10 with higher specificity and affinity for avb3 can result in residues of
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amino acids surrounding the RGD sequence in a FG loop. By doing this, avb3 is capable
of binding to a great range of RGD ligands, which are useful in staining human umbilical
vein endothelial cells in cytometric assay. Results of the study showed FNfn10-3JCLI4
were able to bind to avb3 in half-log increments from .0001ng/ml to 0.1ng/ml,
corresponding to molar concentrations of 8pM to 8nM(4). “The biotinylated FNfn103JCLI4 or FNfn10-WT wee added to wells coated with 5ng/ml concentrations of
different purified integrins”(4). This is significant by which the plate-bound FNfn103JCLI4 was detected for those that have been coated with avb3, thus creating a high
affinity. The avb3 binding with the FNfn10 clones that were discovered in the RGD
sequence has similarities with the purified monomeric FNfn10-protiein by the stability
and cell adhesion effect in vitro of formation of the capillary tubes. As an outcome of
Richard et al study, using bio-panning screening can purify FNfn10-3JCLI4 protein to
bind to immobilized human avb3 integrin with much higher affinity that FNfn10-WT and
exhibit only background binding to other purified integrins (4).
Conclusion
Conducting more research on avb3 and avb6 integrin receptors will help improve
therapeutic procedures such as cancer. Avb3 integrin a cell surface receptor tht
multifunction’s in normal cell growth that may contribute to oncogenesis. Through the
effectiveness of peptides and antibodies, using yeast surface display methods, and
engineering proteins to associate the binding of either avb3 and avb6 integrins,
researchers can explore various approaches in cancer cells and tumor vasculature. They
are able to pinpoint the target cells and modify antibodies or sequences of RNA/DNA to
inhibit the high affinity and specificity of the integrin maturation. These methods could
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potentially help patients with cancer and possibly prevent individuals in promoting
cancer by the significance of human integrin receptors eliminating the danger cancer
cells.
References
1. Chao G, Cochran JR, Dane Wittrup K. 2004. Fine epitope mapping of antiepidermal growth factor receptor antibodies through random mutagenesis and
yeast surface display. J Mol Biol 342(2): 539-50.
2. Kogelberg H, Tolner B, Thomas GJ, Di Cara D, Minogue S, Ramesh B, Sodha S,
Marsh D, Lowdell MW, Meyer T, et al. 2008. Engineering a single-chain fv
antibody to αvβ6 integrin using the specificity-determining loop of a foot-andmouth disease virus. J Mol Biol 382(2): 385-401.
3. Lipovšek D, Lippow SM, Hackel BJ, Gregson MW, Cheng P, Kapila A, Wittrup
KD. 2007. Evolution of an interloop disulfide bond in high-affinity antibody
mimics based on fibronectin type III domain and selected by yeast surface
display: Molecular convergence with single-domain camelid and shark antibodies.
J Mol Biol 368(4): 1024-41.
4. Richards J, Miller M, Abend J, Koide A, Koide S, Dewhurst S. 2003. Engineered
fibronectin type III domain with a RGDWXE sequence binds with enhanced
affinity and specificity to human αvβ3 integrin. J Mol Biol 326(5): 1475-88.
5. Silverman AP, Levin AM, Lahti JL, Cochran JR. 2009. Engineered cystine-knot
peptides that bind αvβ3 integrin with antibody-like affinities. J Mol Biol 385(4):
1064-75.
6. Ylipalosaari M, Thomas GJ, Nystrom M, Salhimi S, Marshall JF, Huotari V,
Tervahartiala T, Sorsa T, Salo T. 2005. αvβ6 integrin down-regulates the MMP13 expression in oral squamous cell carcinoma cells. Exp Cell Res 309(2): 27383.