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Synergistic anti-tumor responses against metastatic cancer using antibodies to CD40 and IL2: coordination of dendritic cell and T cell arms of the immune responseBackground: Due to the weak immunogenicity
of human cancers, the coordinate engagement of professional
antigen-presenting cells (i.e. dendritic cells) and cell-mediated
effector cells (i.e. CD8+ T cells) may be vital for inducing
effective anti-tumor responses. CD40 is critical for, but
not limited to, dendritic cell differentiation and function
whereas IL2 is pivotal for, but also not limited to, T cell
expansion.
Objective: To determine if the combination of CD40 stimulation
with IL2 may result in synergistic anti-tumor effects in vivo.
Methods: Mice were given metastatic renal cell carcinomas
and, after significant tumor growth, were subsequently treated
with agonistic antibodies to CD40 (intraperitoneal administration;
10-100 µg/mouse/day for 14 days) plus human recombinant
IL2 (intraperitoneal administration; 300,000 IU/mouse BID,
twice a week for a total of 8 injections).
Results: In this study, protective effects were observed when
either anti-CD40 or IL2 were administered as single agents
to these advanced tumor-bearing mice. However, a marked and
highly synergistic effect on tumor regression was observed
following combined administration of CD40 agonist monoclonal
antibody and IL-2 such that 70% of the advanced tumor-bearing
mice survived the tumor and were immune to subsequent re-challenge
with the original tumor. No overt toxicity was noted after
treatment. Marked increases in both dendritic cell and CD8+
T cell numbers were observed after combination treatment compared
to either treatment alone (p<0.005 and p<0.0001, respectively)
and the recovered CD8+ T cells from this treatment group alone
produced significantly greater amounts of interferon-gamma
specifically in response to the tumor (p<0.05). The anti-tumor
effects were independent of NK cells but were dependent on
both interferon-gamma and IL12 since therapeutic synergy was
not observed in interferon-gamma or IL12 knockout mice.
Discussion: Combination therapy using co-stimulatory molecules
and cytokines such as CD40 stimulation in combination with
IL2 administration can result in synergistic and efficacious
immune responses against advanced metastatic cancer in mice.
We have evaluated tumors that recur following distinct immunotherapies that preferentially induce NK- or T cell-mediated in vivo control of the NXS2 neuroblastoma in tumor bearing mice. Recurrent tumors that had escaped an NK-dependent antitumor response were generated by intentionally using a sub-optimal regimen of hu14.18-IL2, a humanized IL2 immunocytokine targeted to the GD2 ganglioside, which initially induced a clear antitumor response followed, however, by delayed tumor recurrence in some mice. Importantly, we demonstrated that such recurrent NSX2 tumors revealed markedly enhanced (5-7 fold) MHC class I antigen expression. The H-2high phenotype of these tumors is likely dependent on immune selective pressures in vivo since it is not maintained following 1 week in tissue culture. Additionally, this enhanced H-2 antigen expression on NSX2 cells was associated with reduced susceptibility to both NK cell-mediated tumor cell lysis and antibody-dependent cellular cytotoxicity in vitro. In contrast to the increased MHC class I antigen levels of NSX2 cells escaping after sub-optimal hu14.18-IL2 therapy, most NSX2 tumors that have escaped F1t3-Ligand treatment, which was able to induce a T cell-dependent memory response, revealed a decreased expression of MHC class I antigens. While NSX2 tumors are highly susceptible to destruction following either hu14.18-IL2 or Flt3-Ligand mediated therapies, these results indicate that some tumor cells may respond to these in vivo immunotherapies by modulating their level of H-2 expression up or down, allowing them to respectively escape either NK cell-or T cell-mediated antitumor responses.
Therapeutic approaches directed towards blocking the epidermal growth factor receptor (EGFR) based on its anti-proliferative activities are currently being evaluated clinically in cancer. Chemokines have been shown to affect the progression of various tumors, particularly metastatic spread and angiogenesis. The aim of this study was to assess the effects of a combined blockade with antibodies to EGFR and interleukin 8 (IL8). IL8 has been reported to augment the progression of some human tumors, and we therefore evaluated the anti-tumor effects of a human IL8 antibody, ABXIL8 in combination with anti-EGFR, ABXEGFR in a SCID mouse model bearing a metastatic human breast carcinoma. Whereas anti-IL8 alone had no appreciable anti-tumor effect, the combination of anti-IL8 significantly enhanced the anti-tumor effects of anti-EGFR, resulting in greater survival of SCID tumor bearing mice. This effect on survival was correlated with decreased metastatic spread in mice receiving both antibodies. Moreover, the combination of anti-IL8 and anti-EGFR markedly inhibited matrix metalloproteinase activity associated with MDA-231 cells to a greater degree than either antibody singly. Combined administration of these two human antibodies may thus provide a more effective approach for treatment of metastatic human breast carcinoma.
Stem cell drug resistance gene therapy has
yet to produce definitive results in patients. Drug resistance
gene transfer into stem cells is designed to protect stem
cells from cytotoxic damage and to allow enrichment of stem
cells during myelosuppression. In contrast, most cancer gene
therapy is designed to directly attack tumjor cells. While
the first goal is to improve tolerance to chemotherapy, successful
stem cell selection may lead to the use of these genes to
enrich for cells carrying a second therapeutic correction
gene be it to correct a genetic disorder, or target a malignant
cell by introduction of a cytokine, suicide enzyme, antisense
to an oncogene, or a wild type tumor suppressor gene. The
most common approaches to gene therapy include murine oncoretroviruses
although a number of investigators have introduced adenovirus,
adeno-associated virus or plasmids by electroporation. For
stem cells, retroviruses are the most promising, but these
other methods have yielded recent success. More recently,
lentivirus have been used because of the potential to introduce
these vectors without the requirement of cell division and
the use of cytokines.
In our own studies, we have used a series of mutant forms
of MGMT identified as resistant to the alkyltransferase [AGT]
inhibitor, O6-benzylguanine [BG]. These mutants target the
region of the AGT protein that is responsible for the transfer
of the alkyl group to cysteine, the key residue in the active
site. The most potent mutants consist of one or more [up to
5] amino acid substitutions in the active site region of the
protein that collapses the pocket making it inaccessible to
BG, either by becoming more polar or by reducing the size
of the pocket.. The most potent of these contains 5 amino
acid substitutions and was identified using Darwinian molecular
evolution and selection from a pool of over ten million mutants
[3]. Others, containing G156A and P140K substitutions are
also potent BG-resistant mutants [4]. Gene transfer of one
modified murine retroviral backbone, MFG, into which has been
placed the of one mutant MGMT, either G156A or P140 K results
in a 120 to >2000 fold resistance to BG inhibition and an
increase in the IC50 of about 6 fold for BCNU and up to 20-fold
for methylating agents such as temozolomide [5]. We have made
a series of observations in this system that proof its value
in vitro and in animal models. First, we showed that transduced
murine and human hematopoietic progenitor cells are resistant
to BG & BCNU. We next showed that mice transplanted with transduced
cells are protected from the lethal effects of BCNU and BG
and that these cells are selected for during drug treatment.
In fact we found that the mice were protected from myelosuppression
and gained in cell counts with each dose of treatment. We
then moved to a setting of cell infusion without prior myeloablation,
and asked whether it was possible to enrich for transduced
cells in mice that received only a small inoculum of cells.
In a study performed in mice that had not received myelosuppression
prior to cell infusion, animals infused with as few as 50,000
transduced cells could be completely repopulated with MGMT+
cells after 2-3 doses of BG& BCNU [6]. When we asked how much
enrichment at the level of the long term repopulating cell
had taken place, we calculated that the enrichment was 1000
fold in favor of the transduced cells. These mice can serve
as the donors for secondary transplant recipients, and these
secondary recipients continue to express the MGMT gene and
to contain a high proportion of the transduced cells, indicating
that repopulating stem cells have been transduced and that
these cells express the MGMT gene. Next , we turned to a pre-clinical
model of stem cell drug resistance gene therapy. In nude mice
carrying the SW480 human colon cancer xenograft, improved
tolerance to drugs and better tumor control over more cycles
of chemotherapy was observed [7]. We have now initiated a
clinical trial in which patients with advanced malignancy
will have collection of CD34 cells that are then transduced
with the MFG G156A mutant MGMT and reinfused into the patient.
Patients will then receive standard phase II doses of BG [120
mg/m2] and BCNU [33 to 40 mg/m2]. The BG dose was selected
based on our phase I trial that indicated that tumor alkyltransferase
activity was completely inhibited without toxicity [8]. During
sequential treatments with BG and BCNU, monitoring of marrow
CD34 cells will determine whether transduced cells can be
identified and whether there is enrichment of these cells
over time. Our results suggest that MGMT gene transfer remains
a promising approach to stem cell drug resistance gene mediated
selection that may make possible significant degrees of stem
cell protection and selection in vivo with application in
both chemotherapy treatment for cancer patients as well as
therapeutic introduction of other genes.
Purpose: To determine the maximal
tolerated dose (MTD) of CV706, a replication-competent cytolytic
adenovirus, when delivered into the prostate by a transrectal
ultrasound-guided transperineal technique in patients with
prostate cancer (PCa) that is locally-recurrent following
radiation therapy. Secondary objectives included evaluation
of antitumor activity as determined by serum PSA and evaluation
of post-treatment prostate biopsies, time to progression,
PSA velocity, and evaluation of biodistribution and immune
response to therapy.
Materials and Methods: CV706 was constructed by deleting
E3 from an Ad5 adenovirus and adding a minimal promoter-enhancer
construct of the human PSA gene 5’ of E1A resulting in
regulation of E1A expression and, thus, regulated replication.
All patients had biopsy-proven locally-recurrent PCa with
biochemical failure as defined by the ASTRO criteria or a
PSA > 10 ng/ml on 2 successive occasions. Other entry criteria
included: normal serum testosterone, no evidence of metastatic
disease on bone and CT scans. Dose escalation method: single
administration of 1X1011 to 1X1013 viral particles divided
in 20-80, 0.1 cc deposits injected with 10-40 needles. Pre-treatment
planning was performed using a modified 3-D prostate brachytherapy
treatment planning system. Toxicity was graded using the NCI
CTC. Serial post-treatment biopsies were obtained to assess
for treatment effect, PSA staining and evidence of viral replication.
Serial urine and serum samples were also obtained to assist
in PK/PD measurements.
Results: Twenty patients were treated over 5 dose levels.
Treatment was well tolerated. Adverse events consistently
noted included: Day 1, Grade 2 fever (treated with antipyretics),
perineal bruising/edema/pain, urinary irritation (largely
due to the indwelling Foley catheter). Five patients had mild,
transient Grade 1 transaminase elevation. No clinically-significant
alteration in hematology or coagulation parameters noted.
There were 5 reversible Grade 3 toxicities (1 patient with
hypertension, 1 patient with short episode psychosis, 3 patients
with blood clots in urine). There was no dose-limiting toxicity.
Four patients exhibited a PR (> 50% reduction in PSA for at
least 4 weeks). Note that all responders occurred in final
2 dose levels, i.e. 4/11 patients. Viral replication confirmed
by EM of post-treatment biopsy samples. Quantitative PCR documented
CN706 in blood at two different time points, also suggestive
of viral replication.
Conclusions: CV706, administered by a modified prostate
brachytherapy technique, exhibits an acceptable safety profile
with both biochemical and histologic evidence of antitumor
activity in patients with prostate cancer that is locally-recurrent
following radiation. In vivo replication was established in
these patients. As we have recently demonstrated substantial
in vitro and in vivo synergy when CV706 treatment is followed
by radiation, these results support continued clinical development
in a Phase I/II study combining treatment with CV706 plus
radiation thearpy for initial management of patients with
clinically-localized prostate cancer.
Supported by NIH S.P.O.R.E. in Prostate Cancer, NIH GCRC Johns
Hopkins and a clinical research agreement only with Calydon,
Inc.
The goal of cancer treatment is to develop
modalities that specifically target tumor cells, thereby avoiding
unnecessary side effects to normal tissue. Vaccine strategies
that result in the activation of the immune system specifically
against antigens expressed by a cancer have the potential
to be effective treatment for this purpose. An early vaccine
approach that was developed by our group involves the use
of autologous tumor cells that are genetically modified to
express the cytokine granulocyte-macrophage colony stimulating
factor (GM-CSF), as the immunogen to induce systemic antitumor
immunity. Studies in our animal model demonstrated that the
paracrine secretion of GM-CSF at the site of tumor cells induced
a CD4 and CD8 T cell dependent systemic immune response potent
enough to cure mice of most histologic tumor types. We first
tested this vaccine in patients with renal cell carcinoma
(RCC) and found that this vaccine approach can activate RCC
specific immunity in some patients. This induction of antitumor
immunity was associated with clinical responses. Unfortunately,
we also found that it is not technically feasible to develop
an autologous vaccine approach for most types of human cancer
including pancreatic adenocarcinoma. More recent data from
our group and others has demonstrated that the immunizing
tumor cell does not need to be HLA compatible with the host
to induce effective antitumor immunity. These data therefore
provide the immunologic rationale for using allogeneic cells
as the antigen delivery system.
We have completed testing of an allogeneic pancreatic tumor
vaccine approach in patients with stage 2 and 3 pancreatic
cancer. Our findings demonstrated that GM-CSF secreting allogeneic
vaccine cells can induce specific antitumor immunity comparable
to an autologous vaccine. The findings that will be presented
are similar to what we observed in our animal models and in
patients with RCC thereby providing evidence for a similar
mechanism of immune activation. These findings lay the ground
work for further testing of this vaccine approach to determine
if it will result in clinical responses. Furthermore, lymphocytes
isolated from vaccinated patients may assist in the identification
of the immune relevant antigens that are expressed by the
vaccine and patient tumors. Finally, we are utilizing a murine
model of tumor tolerance to develop methods of improving on
the potency of our vaccine approach. Plans for translating
new findings from this model are underway in pancreas and
breast cancer.
