Combination therapy for early melanoma patients against immune suppression pathway

Abstract

Cancer is currently the leading cause of death in worldwide, Surgery, platinum-based chemotherapy, molecular targeted agents and radiotherapy are the traditional treatment used. However, the prognosis of early melanoma patients, especially those in clinical stage II and III is not as effective as patients in stage I. Therefore, new sorts of treatments are needed to be exploited for these patients.

Immunotherapy is a promising strategy for the treatment of various types of cancer. Programmed death 1 (PD-1) and its ligand PD-L1 play a key role in tumor immune escape and the formation of tumor microenvironment, closely related with tumor generation and development. Blockading the PD-1/PD-L1 pathway could reverse the tumor microenvironment and enhance the endogenous antitumor immune responses. Utilizing the PD-1 and/or PD-L1 inhibitors has shown benefits in clinical trials. An antibody that targets programmed death-1 (PD-1) pathway has been shown to be active towards various types of cancer, including melanoma, resulting in a breakthrough therapy designation granted by FDA. These events pronounce the importance of targeting the PD-1 pathway in the treatment of melanoma cancer

Nevertheless, monoclonal antibody targeting PD-1 therapy inherently carries a number of disadvantages such as their immunogenicity (following repeated administration) and high production costs which may limit their use and broad availability to patients. For example, recently FDA approved anti pd-1 antibody keytruda is only available for patients with metastatic cancer, for it would largely increase patient survival rate and increase tumor regression while it will also trigger severe autoimmune responses. And since PD-L1 expression correlates greatly with PD-1 expression in patient profiles, administration of treatment only targeting PD-1 leaves some patients unresponsive to immunotherapy as well.

Therefore, in this study, we propose developing a combination therapy targeting PD-1/PD-L1 pathway for early stage melanoma patients with limited side effects. The development of this project can be organized into two parts. First, we propose a new design for immune checkpoint PD-1/PDL-1 inhibitor by formulating a nanoparticle that could inhibit the immune suppression. After designing this particle, pharmacodynamics study and in vitro, in vivo functional study will be performed. Secondly, a combinational therapy with anti-PD-1 should be tested in animal model for both its synergistic effect and treatment efficacy.

Collectively, developing a combination therapy by Introducing nanomaterial to traditional cancer immunity treatment would help to solve unwanted side effect raised by antibody blockade action, while broadening and benefiting more patients with sufficient treatment efficacy.

Experimental Plans

Relevant background

Cancer as a chronic, polygene and often inflammation-provoking disease, the mechanism of its emergence and progression is very complicated. There are many factors which impacted the development of the disease, such as: environmental factors, living habits, genetic mutations, dysfunction of the immune system and so on. At present, increasing evidence has revealed that the development and progression of tumor are accompanied by the formation of special tumor immune microenvironment. Tumor cells can escape the immune surveillance and disrupt immune checkpoint of host in several methods, therefore, to avoid the elimination from the host immune system

Drugs targeting PD-1/PD-L1 pathway has been formulated and FDA approved for marketing. For example, pembrolizumab (Keytruda, Merck & Co., Inc., Kenilworth, NJ, USA) is a potent, humanized IgG4 monoclonal antibody against programmed death 1 (PD-1) receptor that directly blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Pembrolizumab has demonstrated robust, durable antitumor activity. However, because of the unmanageable and severe autoimmune side effect of using this medication in patients, it is only approved for advanced or even metastatic melanoma patients. However, it has been reported in literature that, PD-L1 expression level is independent of disease stage, but also correlates greatly with the prognosis and survival of early to mid-stage patients. Therefore, developing a combination therapy for these melanoma patients targeting immune suppression pathway i.e. PD-1/PD-L1 axis is necessary

Spherical nucleic acids (SNAs) are structures with densely packed oligonucleotides with highly oriented functionality. Compared to their free composite oligonucleotides and nanoparticles they derived of, SNA structure confers efficient and rapid cellular uptake with great ability to modulate gene expression. Moreover, previous studies have demonstrated it also have advantages in stimulating both innate (toll-like receptor-9(TLR-9)) and adaptive immunity for cancer treatment. However, given the heterogeneity of in situ cancer population and various evading mechanisms, SNAs could be used as a platform to remove the hurdle of inhibitory signal and modulating immune response against tumor.

Significance

Developing a combination therapy by Introducing nanomaterial to traditional cancer immunity treatment would help to solve unwanted side effect raised by antibody blockade action, while broadening and benefiting more patients with sufficient treatment efficacy.

Innovation

Our immune system have a number of immune mechanism which in theory can attack/destroy tumor cells with those neo antigen, which induce a immune response. One of the major player is the cytotoxic t cells. with the help of several different kinds of immune cells, they could be trained to attack and destroy tumor cells. so if everything is working appropriately, this immune response could be quite potent.

During cancer immunity cycle, t cell has to be activated and trained to be able to target tumor cells, however, tumor cells develop evading mechanism to escape from cytotoxic t cell attacking. PD-1/PD-L1 is one of the inhibitory signal tumor microenvironment use to tur down immune response. Therefore, to boost immune therapy efficiency, it is necessary to suppress inhibitory pathway.

With the recently FDA approved anti pd-1 antibody keytruda for metastatic melanoma, it would largely increase patient survival rate and increase tumor regression, suggesting targeting this pathway is promising even in clinical application. However, since it’s targeting pd1 on the t cell, it has its own limitation that antoimmune side effect is inevitably. And the reason for there is no available anti pdl1 antibody, it’s because limited delivery efficency to target tumor environment. Therefore, in this project we want to benefit from SNA superior structure advantage of highly uptake in tumor environment, and knockdown/interfere pdl1 expression/function to remove the escaping mechanism, and boost t cell killing to remove tumor burden

The concept of T-cell co-stimulation has evolved over time. The two-signal model for T-cell activation was proposed by Kevin Lafferty and co-workers as a model for the activation of naive T-cells. According to this model, T-cells require two signals to become fully activated. (Lafferty and Cunningham, 1975) The first signal, which gives specificity to the immune response, is provided by the interaction of the antigenic peptide−MHC complex with the T-cell receptor (TCR). The second, antigen-independent co-stimulatory signal, is delivered to T-cells by antigen-presenting cells (APCs) to promote T-cell clonal expansion, cytokine secretion, and effector function. (Keir et al., 2008)

Current immunotherapies have been developed based on this model to boost or train immune CD8 T-cells to kill tumors.(Radovic-Moreno et al., 2015)[Ed1] However, tumor cells have progressively developed ways to evade the immune system. One way is through inhibitory second signal pathways like CTLA4 and PD1 interactions. Both clinical and research studies have proved that blocking these pathways could lead to enhanced tumor killing effects. (Blank et al., 2004) However, given the high cellular toxicity of present transfection reagents and the low stability of antibody-based treatment, a more stable and less toxic therapeutic approach is needed.

Previous work from our lab have demonstrated the advantages of using the spherical nucleic acid (SNA) platform as a new strategy in cancer treatment, including for diseases such as glioblastoma multiforme (GBM), retinoblastoma, and prostate cancer.(Jensen et al., 2013; Narayan et al., 2015) Promising data have also confirmed the potential of SNAs in stimulating the immune system as potent cancer vaccines. (paper in progress[Ed2] ). Therefore, developing a new SNA targeting PDL1 as a combinational agent with the current antibody treatment could be an obvious promising step[Ed3] .

Evaluate combination treatment of nanoparticle and anti-PD-1 antibody in melanoma mouse model

i) First and most importantly, given the mechanism of action of anti-PD-1, several studies have tried to determine whether the efficacy of these Abs correlated with PD-L1 ligand expression in the tumor. The first studies provided evidence that there was indeed a strong link between PD-L1 expression by tumor cells and the response to anti-PD-1 Ab. Topalian et al. showed that all the responses to nivolumab were observed in patients whose tumors expressed PD-L1. Likewise, in the KEYNOTE-001 trial, responses to pembrolizumab correlated with PD-L1 expression by tumor cells. A trial assessing the effect of an anti-PD-L1 (MPDL3280A) on different types of cancer found a correlation between the level of PD-L1 present in the intratumoral immune infiltrate (but not by the tumor cells themselves) and clinical response. However, other studies did not confirm this correlation. In a meta-analysis including 1475 patients treated with nivolumab, pembrolizumab or MPDL3280A, response rates were significantly higher in PD-L1-positive tumors (34% versus 19.9%).

Pembrolizumab (Keytruda, Merck & Co., Inc., Kenilworth, NJ, USA) is a potent, humanized IgG4 monoclonal antibody against programmed death 1 (PD-1) receptor that directly blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Pembrolizumab has demonstrated robust, durable antitumor activity and a manageable safety profile against several advanced malignancies. Early clinical studies of pembrolizumab employed a body-weight–based dosing strategy of 2 mg/kg every 3 weeks (Q3W) to 10 mg/kg every 2 weeks (Q2W), but in more recent trials a fixed-dose regimen (fixed with respect to body weight) has been introduced.

Given the mechanism of action of pembrolizumab, binding to PD-1 receptors on T cells, independent on direct engagement of the molecule with tumor cells, substantial differences in exposure–response and dose–response are not expected across different tumor types. Indeed, it has been found that the pharmacokinetics (PK) of pembrolizumab are similar across oncology indications. On this basis, selection of a fixed-dose regimen focused on establishing a dose that would provide comparable (central tendency and distribution) exposures as the 2 mg/kg Q3W regimen approved in the United States for melanoma and NSCLC. The fixed dose selected also aimed to maintain exposures within the existing clinical experience range that has been established for melanoma and NSCLC and which has been associated with a lack of clinically important differences in efficacy or safety

the central tendency (mean, median) at 200 mg Q3W is modestly increased (~35%) relative to 2 mg/kg Q3W for all PK measures (AUC_ss, _0-6weeks, C_{max, ss} and C_{trough, ss}), while these values are ~25% of those obtained at 10 mg/kg Q3W. Intersubject variation (% CV) is similar for all regimens and the 10–90% percentiles are largely overlapping for 2 mg/kg and 200 mg Q3W. A minimum effective administration of anti-PD-1 antibody should be tested in mouse model we developed. The administration route, dose amount and dose schedule should be tested in vivo.

Regarding to the in vivo biomarkers they tested for anti-PD1 mAb, A multitude of biomarkers has been studied, predominantly involving indices from the patient’s tumor (tumor cells or cells from the microenvironment) or blood (circulating cells or serum).Treatment with anti-PD-1 Ab was associated with increased circulating IFN-γ, IL-18 and ITAC (an IFN-γ inducible chemokine which is chemotactic for activated T cells) and decreased IL-6. However, no correlation was found with clinical efficacy. The presence of lymphocytes within the tumor is another favorable prognostic factor in numerous cancers treating with checkpoint inhibitor antibody. In patients with metastatic melanoma, an increase in lymphocyte infiltrate in the tumor between baseline and at 3 weeks after treatment initiation correlated with clinical response. Moreover, in melanoma patients, response rate was better in patients with high numbers of peri- and intratumoral CD8 T cells in their pretreatment samples. Analysis of biopsies after treatment showed a correlation between a high ratio of intratumoral CD8/regulatory T cells and tumor necrosis. Moreover, in patients with metastatic melanoma, Tumeh et al. found that clinical response to anti-PD1 therapy (pembrolizumab) correlated with (i) a more clonal (i.e. more restricted, less diverse) TCR repertoire in pretreatment tumor samples and (ii) an increased clonal expansion of T cells in the tumor after anti-PD1 therapy.

ii) Synergistic effect of administrating both nanoparticle and antibody should be tested in vivo. A overall PD-L1 expression level, circulating cytokine, and tumor microenvironment remodeling should be 3 factors to be valued following combination treatment. A tumor regression curve and mouse survival rate should also be performed

[Ed1]Not sure if I would reference this here. As written, it makes it sound like there are disadvantages to our work. I don’t think you should describe it in that way.

[Ed2]`

[Ed3]Add a sentence or two about fundamental scientific issues you will explore.