ToC | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9
In vivo tumor imaging using near infrared labeled EndostatinIntroduction: Endostatin is a 20 kDa
C-terminal fragment of collagen XVIII and is a potent inhibitor
of angiogenesis. The exact mechanism of action for endostatin
is unknown. Imaging technologies that use near-infrared (NIR)
fluorescent probes are well suited to the laboratory setting.
The goal of this experiment was to determine if endostatin
labeled with a NIR probe could be detected in an animal after
a sub-cutaneous injection.
Methods: Endostatin was conjugated to Cy5.5 monofunctional
dye and purified from free dye by gel filtration. LLC a murine
tumor, and BxPC-3 a human pancreatic tumor, were implanted
in C57BL/6 and SCID mice respectively. Tumors were allowed
to grow to 350mm2 when mice were injected with endostatin-Cy5.5
and imaged at various time points. Imaging was performed using
a lightproof box affixed to a fluorescent microscope mounted
with a filter in the NIR bandwidth (absorbance max 675nm and
emission max 694nm). Images were captured by a CCD and desktop
computer and stored as 16 bit Tiff files.
Results: The endostatin-Cy5.5 was quickly absorbed
and rendered a NIR fluorescent image of the entire animal
within 2 hours. Ex vivo imaging of multiple organs at 24 hours
demonstrated no NIR image. In contrast, both the LLC and the
BxPC-3 tumors emitted persisting NIR signal at 24 hours. Unlike
previous analogous studies with GSAO-Cy5.5, whose tumor image
faded with time, the endostatin-Cy5.5 emitted NIR signal from
the tumor up to 7 days after injection, at the last time point
examined. Cy5.5 dye alone had no tumor signal enhancement
over background at any time point.
Conclusion: This study demonstrates that endostatin
covalently bound to Cy5.5 will migrate from a distant sub
cutaneous injection site to a tumor. This may indicate that
the mechanism of action of endostatin is within the tumor
or tumor vasculature and is not a systemic effect. This imaging
technique is ideal for in-laboratory animal imaging and will
be useful as the Radiation Oncology Branch begins the molecular
credentialing project.
Background: Interleukin-12 (IL-12) is a cytokine
with promising antiangiogenic activity mediated by the induction
of interferon-g (IFN-g) and antiangiogenic chemokines. Although
IL-12 does not have a direct effect on endothelial cell (EC)
proliferation or migration, it triggers ECs to release IFN-g
that can activate immune cells. Stimulated immune cells release
factors that can inhibit EC proliferation suppressing angiogenesis.
We developed a bifunctional fusion protein, murine recombinant
IL-12 vascular homing peptide (mrIL-12vp) that simultaneously
targets vascular and immune cell compartments. The fusion
protein contains a small peptide sequence, arginine-glycine-aspartic
acid (RGD), a ligand for the integrin avb3 that specifically
directs mrIL-12vp to avb3 expressed on angiogenic endothelial
cells.
Objective: We tested the hypothesis that directly targeting
IL-12 to avb3 integrin on proliferating endothelial cells
would be more effective at inhibiting angiogenesis and less
toxic than systemic administration of non-targeted IL-12.
Methods: mrIL-12vp was over expressed from CHO cells and an
IL-12 ELISA verified production. ELISA was also used to validate
activity of mrIL-12vp using production of IFN-? as a readout.
Immunoflourescence labeling was used to verify specific targeting
of mrIL-12vp to avb3 integrin expressed on malignant ECs and
other tumor cells. Toxicity experiments were performed in
DBA mice to evaluate tolerability of several doses of mrIL-12vp
vs. mrIL-12. Corneal pocket neovascularization assays compared
the antiangiogenic effects of mrIL-12vp with those of mrIL-12
in response to angiogenic stimulation. Results: mrIL-12vp
elevated levels of IFN-g in vitro and in vivo indicating that
it has biological activity indistinguishable from that of
mrIL-12. Immunofluorescent assays using fluorescence-labeled
anti-IL-12 antibody showed mrIL-12vp only bound avb3+ cells
but not avb3- cells, indicating that mrIL-12vp specifically
targets avb3. Systemic administration of mrIL-12vp (0.5 µg/day
IL-12 equivalent) or mrIL-12 (0.5 µg/day) by continuous subcutaneous
infusion in DBA mice resulted in hepatic necrosis in the mice
treated with mrIL-12 but not mrIL-12vp. In two mouse models,
corneal angiogenesis induced by bFGF showed at least a four-fold
greater inhibition in angiogenesis in the mice treated at
equivalent dosages of mrIL-12vp compared to those treated
with mrIL-12. mrIL-12vp is a potent antiangiogenic agent with
a favorable toxicity profile in mice.
Background: Angiogenesis, inflammation
and immune dysregulation are important features of the pathogenesis
of cancer, and these responses have been associated with activities
of a diverse array of angiogenesis and immunoregulatory factors.
The limited effectiveness of therapy targeted against individual
angiogenesis and immunomodulatory factors or receptors has
been found to arise from expression of this diversity of factors.
The underlying basis for expression of such a diverse repertoire
of factors in cancer is not well understood, but the identification
of common mechanisms could provide a more limited number of
targets for biologic therapy. Alterations in signal pathway
and transcription factor activation have been implicated in
oncogenesis, and such alterations within common pathways could
regulate multiple genes, including angiogenesis and inflammatory
factors.
Methods: mRNA differential display, microarray and
ELISA were used to identify a repertoire of cytokines and
angiogenesis factors expressed by murine and human SCC. Comparison
of sequences of the promoter regions revealed common transcription
factor binding sites, and the role of these transcription
factors and upstream signal pathways in expression of cytokines
was determined using genetic mutational analysis and inhibitors.
Results: IL-6, IL-8, GRO, GM-CSF and VEGF were detected
in tumor, cell lines or serum of patients with head and neck
squamous cell carcinomas, and their homologues were expressed
with metastatic progression of murine SCC. These factors were
found to be co-expressed as a result of signal activation
of transcription factors Nuclear Factor kappa B and Activator
Protein-1 by IL-1, Epidermal Growth Factor and MET receptors.
Inhibition of these receptors and common signal pathways inhibited
cell survival, proliferation, inflammation, angiogenesis and
tumorigenesis.
Discussion: The molecular mechanisms responsible for
oncogenesis of SCC promote co-expression of a diverse array
of proinflammatory and proangiogenic cytokines. Inhibition
of common signal receptors, kinases and transcription factors
which regulate cytokine factors and other genes inhibit tumorigenesis
and may provide a target for biological therapy.
Tumor lysate-pulsed dendritic cells (TP-DC) serving as stimulators can educate naive primary lymphocytes in vitro, which can result in the generation of tumor-specific proliferative, cytokine producing, and cytolytic T cells. Moreover, immunization of syngeneic mice with TP-DC has resulted in potent specific priming and antitumor effects on micrometastatic pulmonary nodules in several histologically-distinct tumors, which are mediated by CD8+ and, to a lesser extent, CD4+ host-derived T cells. We have also found tumor lysates to be equivalent to apoptotic tumor cells as a source of TAA for pulsing of DC. The systemic administration of relatively low doses of recombinant IL-2 in combination with tumor TP-DC has resulted in markedly enhanced therapeutic effects against well-established tumors at either subcutaneous or pulmonary sites. We have also evaluated whether KLH can augment the antitumor efficacy of TP-DC immunization in vivo. In addition to being used as a “surrogate antigen” in vaccine approaches to measure immunologic response in cancer patients, KLH has also been shown to be a strongly immunogenic carrier protein to elicit T cell help. Indeed, the addition of KLH to TP-DC immunization can both profoundly augment IFN-gamma production by tumor-specific T cells and result in enhanced antitumor therapeutic efficacy in vivo. We have also shown for the first time that secondary lymphoid tissue chemokine (SLC) can induce a strong antitumor response that results in significant infiltration of immune effector cells into treated tumors and that genetic modification of DC to express SLC can enhance their capacity to elicit tumor rejection in vivo. Of importance, we have shown for the first time that SLC-secreting DC can effectively prime tumor-reactive T cells at the tumor site in the complete absence of functional lymph nodes. The potential of combining DC-based vaccines with bone marrow transplantation (BMT) for the treatment of metastatic disease is currently being considered for clinical evaluation, based on our recent successful results from preclinical studies. Indeed, in a lymphopenic environment, naive T cells can undergo homeostasis-driven proliferation and can acquire increased sensitivity to antigen stimulation. Our new findings demonstrate that it is possible to promote effective antitumor immunity in a defined lymphopenic environment following BMT through DC-based immunization. We have also focused effort on designing alternative strategies to overcome the potential limitation of sufficient tumor from an individual to produce the DC-based vaccine. In preclinical studies, we have found that approaches or agents that selectively elicit apoptosis of tumors in vivo may profoundly augment both the therapeutic efficacy and immune stimulatory capacity of injected DC alone.
DCs can be utilized either as vectors or as targets for therapy. Patients with metastatic melanoma received CD34-DC vaccine, that contains Langerhans cells and Interstitial DCs. DCs were pulsed with MART-1, tyrosinase, MAGE-3, gp100 and Flu-MP peptides, and KLH. DCs induced an immune response to control antigens in 16/18 patients. An enhanced immune response to 1 or more melanoma antigens (MelAg) was seen in these 16 patients. The 2 patients failing to respond experienced rapid tumor progression. 6/7 patients with immunity to 2 or less MelAg had progressive disease 10 weeks after study entry, in contrast to tumor progression in only 1/10 patients with immunity to > 2 MelAg. The tumor immunity score correlated with clinical outcome. Since tumor immunotherapy targets autologous antigens we can learn from systemic autoimmunity such as SLE. As opposed to normal monocytes, SLE monocytes induce proliferation of allogeneic CD4 T cells. SLE sera induce monocyte differentiation towards DCs in IFN-a dependent mechanism. Spiking autologous serum with IFN-a reproduces DC differentiation. 50% of SLE patients have high serum levels of IFN- a, which could explain T/B lymphopenia.Yet, plasmacytoid DCs, a major IFN-a source, are 80% decreased. pDCs and IFN- a may play a role in SLE pathogenesis and therapy.
Dermal administration of a melanoma vaccine
should result in activation and trafficking of epidermal Langerhans
cells to draining lymph nodes. There, lymphocytes may be activated
and induced to proliferate. Despite this central role of the
draining node in the response to vaccination, little is known
about the T-cell response in these nodes, after tumor vaccines.
Thus, we have performed two clinical trials in which a lymph
node draining a vaccine site was harvested using sentinel
node technology (sentinel immunized node, SIN), and was evaluated
by ELIspot and by tetramers for reactivity to defined melanoma
antigens, in conjunction with evaluation of the peripheral
blood.
All vaccines used a mixture of 4 melanoma peptides, from tyrosinase
and gp100, restricted by HLA-A1, A2, or A3, plus a modified
tetanus helper peptide that induces Th1-type responses.
In the first of these trials (Mel31), two vaccine delivery
systems were evaluated: (1) dendritic cells pulsed with peptides,
and (2) injection of an emulsion of peptides plus GMCSF in
incomplete Freund’s adjuvant. Patients had measurable
stage IV disease or unresectable stage III disease. The emulsion
of peptides and GMCSF was substantially more immunogenic that
the dendritic cell vaccine, with CTL detected in 80-90% of
patients receiving the former vaccine. CTL were detectable
twice as often in the SIN as in the PBL.
In the second trial (Mel36), we evaluated the same peptides
in GMCSF-plus-adjuvant, in high-risk patients (Stage IIB,
III, or IV) after resection, but without clinical evidence
of disease. Interestingly, CTL responses detected in the peripheral
blood were much more consistent with CTL responses detected
in the SIN. This finding suggests that, in the absence of
measurable tumor, CTL are not depleted from the peripheral
circulation in the same way that appears to occur in patients
with advanced melanoma. In this study, we also evaluated the
impact of low-dose interleukin-2 on the CTL response detected
in the SIN and in the peripheral blood. These results will
be presented.
Our newest trial will include a panel of 12 melanoma peptides,
as a prelude to more complex multi-epitope vaccines incorporating
epitopes for both CD4+ and CD8+ T-cells.
