Cancer Research: C5a Inhibition

 

STEP ONE

Be informed.  Read information below.

STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

STEP THREE

Submit "New Patient Form", current labwork, & imaging results prior to consultation.

STEP FOUR

Establish if you are a candidate for this therapy

in accordance with the Georgia

"Access to Medical Treatment Act".

 

----------------------------------------------------------------------------------------------------------------------------------------------------------

*** SPECIAL NOTE FROM DR. WICHMAN ***

The following excellent article was reproduced from Expert Review of Clinical Immunology at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637164/:

 

Expert Rev Clin Immunol. Author manuscript; available in PMC 2016 Feb 1.
 
Published in final edited form as:
PMCID: PMC4637164
NIHMSID: NIHMS732917

Immunological Effects and Therapeutic Role of C5a in Cancer

Victoria R. Darling, Ralph J. Hauke, Stefano Tarantolo, and Devendra K. Agrawal
Author information ► Copyright and License information ►
 
 
Go to:

Introduction

C5a is an anaphylatoxin formed via the cleavage of the complement protein, C5 [1]. The C5, a 1,676 amino acid protein, is primarily produced in the liver and resides in the circulation system as a zymogen [2,3]. When cleaved by a C5 convertase, C5 produces two active derivatives, C5a and C5b. The C5b complexes with C6-9 of the complement system to form the membrane attack complex (MAC), while C5a functions as an anaphylatoxin [4].

C5a is composed of 74 amino acids but is rapidly metabolized into a 73 amino acid form, C5a des-Arg. C5a is converted to C5a des-Arg via carboxypeptidases, which are located on cell surfaces and in plasma and function by removing the C terminal arginine group from C5a [5]. Both C5a and C5a des-Arg can act on two different types of 7-transmembrane domain receptors, CD88 and C5a receptor-like 2 (C5L2). CD88 is a G protein-coupled receptor, while C5L2 is a non-G protein-coupled receptor [6]. CD88 and C5LR receptors are generally expressed on similar cell types; however, fewer C5LR receptors tend to be expressed than CD88. The cell types on which these two receptors are expressed include both myeloid and non-myeloid-derived cells, including lymphocytes, phagocytes, and hepatocytes [7]. C5a binds with a similar affinity to CD88 and C5L2 receptors. But, while C5a and C5a des-Arg have approximately the same binding affinity for C5L2 receptors, C5a des-Arg has a considerably lower affinity for CD88 receptors than C5a [6]. Therefore, C5a des-Arg is a less potent form of C5a [8]. As a part of the complement system, C5a is produced in serum during activation of the complement cascade. However, C5a can also be produced locally via cleavage by phagocytes and thrombin [9, 10].

Go to:

C5a in the Complement System

C5a is one of over 30 proteins of the complement system. The proteins of the complement system are produced in liver and macrophages and are activated sequentially as a part of the innate immune response. Functioning as opsonins, pro-inflammatory molecules, and in the formation of the membrane attack complex, most proteins of the complement system circulate within the blood stream as zymogens. Traditionally, C5a can be produced via complement system activation, which occurs via three pathways: the classical, alternative, and lectin pathway (Table 1). All three pathways result in the cleavage of C3 into C3a and C3b and therefore, also result in the activation of C5, C6, C7, C8, and C9 [4, 11]. More recent research indicates that C5 can also be activated by two additional mechanisms: thrombin and activated neutrophils functioning as a C5 convertase (Table 1) [9, 10].

Table 1
Pathways activating C5a and activating stimuli
Go to:

Activation of C5

The Classical, Lectin, and Alternative Pathways

The classical pathway via antibody-antigen interactions activates the complement system. Antibody-antigen complexes are recognized by C1 fragment of the complement system which when bound to the Fc region of an antibody is capable of cleaving C4 protein and activating the rest of the complement cascade. The lectin pathway via two possible interactions activates the complement system: mannose-mannose binding lectin (MBL) interactions and oligosaccharides-ficolin interactions. Mannose and specific oligosaccharides displayed on the cell surface of foreign bodies (i.e. fungi or bacteria) are recognized by the host proteins, MBL and ficolin, respectively. Both MBL and ficolin are complexed with mannan-binding lectin serine protease 1 (MASP1) and mannan-binding lectin serine protease 2 (MASP2). Upon the recognition and binding of MBL to mannose and ficolin to an oligosaccharide, MASP1 and MASP2 are activated. MASP2 functions to cleave both C4 and C2, thus producing C3 convertase (C4bC2a). The complement system is activated by the alternative pathway via the spontaneous hydrolysis of C3 into C3a and C3b [4, 12, 13].

Thrombin and Phagocytic Cells Functioning as C5 Convertase

Huber-Lang and colleagues [10] examined the concentration of C5a in mice with a genetic deficiency of C3. Without C3, C5 convertase of the classical and lectin pathways (C4bC2aC3b) and C5 convertase of the alternative pathway (C3bBbC3b) could not be formed. Interestingly, functionally active C5a may still be produced in the genetically deficient mice in both the in vitro and in vivo studies [10]. Furthermore, the mice genetically deficient in C3 had a 3 fold higher activity of plasma thrombin than those without C3 deficiency. These findings suggested that the C5 convertase activity necessary for the production of C5a can be elicited in thrombin [10]. Thrombin, the final product of the extrinsic coagulation pathway, is itself activated by tissue damage. Thus, the coagulation pathway and complement pathway are perhaps interwoven at this C5 junction.

Huber-Lang and colleagues [9] have also implicated activated rat alveolar macrophages and human neutrophils as cells with C5 convertase capabilities. By incubating rat alveolar macrophages and human neutrophils with C5, C5a was generated. It is proposed that C5a is produced by local cleavage of C5 via phagocytes, which utilize an inducible serine protease [9]. Serine proteases are enzymes that cleave peptide bonds via the use of the amino acid, serine, which serves as the necessary electron donor [14].

Go to:

Function of C5a

C5a is a powerful anaphylatoxin that functions in multiple ways to induce inflammation; C5a acts as a chemotactic agent for inflammatory cells, stimulates respiratory burst, cytokine and chemokine release, and functions to increase vascular permeability [15]. Additionally, C5a has been found to stimulate angiogenesis (Figure 1) [16].

Figure 1
Activation of the complement cascade and its role in tumorigenesis. The complement cascade can be activated via the classical, lectin, or alternative pathway; each of which results in the production of tumorigenic complement proteins. C3, C3a, C5, C5a, ...

A cornerstone of the immunological effect of C5a is its ability to stimulate the release of histamine from mast cells. Histamine, a vasoactive amine, stimulates vasodilation and contraction of venular endothelial cells, thus increasing vascular permeability. This effect takes place in coordination with the function of C5a as a chemotactic factor by facilitating the extravasation of the leukocytes, such as basophils, neutrophils, monocytes, and eosinophils, attracted by C5a [3]. Furthermore, histamine also stimulates the production of VEGF-A, which induces angiogenesis. Ryuji and co-investigators [16] have found C5a to directly stimulate angiogenesis via promoting the migration of human microvascular endothelial cells (HMEC-1) both in vitro and in vivo [16]. Thus, C5a could indirectly induce angiogenesis either via production of VEGF-A or directly via its effect on the migration of endothelial cells.

C5a also stimulates the lipoxygenase pathway of arachidonic acid metabolism. This pathway involves the conversion of phospholipids into arachidonic acid, which is metabolized into two types of eicosanoids, leukotrienes and lipoxins. It is unclear whether C5a stimulates the lipoxygenase pathway directly by simply binding to its CD88 receptors or involves other mechanisms. This obviously requires further attention. None-the-less, in this process neutrophils and macrophages serve as local sources of the eicosanoids. Leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) are eicosanoids that both function in chemotaxis and leukocyte adhesion. Leukotrienes C4, D4, and E4 stimulate vasoconstriction, an initial and ephemeral stage of inflammation, and increased vascular permeability. Lipoxins serve as inflammatory antagonists and gradually begin to be produced as cells switch from the production of inflammatory mediators to that of anti-inflammatory mediators [3].

C5a also has the ability to trigger degranulation of leukocytes, such as neutrophils, and stimulate respiratory burst [17]. Degranulation of neutrophils causes the release of inflammatory substances such as toxic mediators and matrix metalloprotease-9 [18]. Respiratory burst causes the release of reactive oxygen species. Both the mediators released from granules and reactive oxygen species kill harmful entities in the body.

Go to:

Role of C5a in Tumor Suppression

C5a has been experimentally found to inhibit tumor growth via either stimulating innate immune cells or halting cell cycle progression (Figure 2) [19, 20]. However, the inhibitory functions of C5a were observed only when amounts of C5a were low. In fact, not only did C5a in low dose act as a tumor suppressor,, C5a present in high concentrations was actually observed to have significantly accelerated tumor progression [19]. Thus, the concentration of local C5a within a tumor may dictate the effect of C5a on tumor suppression or tumor progression.

Figure 2
Opposing roles of C5a in tumorigenesis. The role of C5a has been found to vary according to its concentration at the tumor site. At low concentrations, C5a inhibits tumor growth via facilitating the infiltration of natural killer (NK) cells and macrophages ...

Gunn and colleagues [19] demonstrated with the ovarian SKOV-3 xenograft model, which uses immune suppressed mice, that low levels of C5a in the tumor microenvironment attract both M1 macrophages and natural killer (NK) cells to tumor sites [19]. The M1 macrophages have cytotoxic effects and function as anti-tumorigenic cells [21]. NK cells are also cytotoxic to tumor cells [22]. When low levels of C5a from lymphoma cells were released into the tumor microenvironment, both increased tumor infiltration and cytotoxic function was observed of macrophages and NK cells, which presumably led to reduced tumor growth. It should be noted that SVOV-3 tumor cells do not express CD88 receptors. Thus, C5a could only have been functioning indirectly to inhibit tumor growth.

Kim and co-investigators [20] also observed a correlation between low levels of C5a and reduced tumor growth using the murine mammary cancer model. Complete tumor regression was seen in 1/3rd of the mice that were administered with cells expressing low levels of C5a. Their results supported the hypothesis that the tumor regression was due to inhibition of the cell cycle and increased rates of apoptosis.

Go to:

Role of C5a in Tumor Promotion

Gunn and colleagues [19] also demonstrated that high levels of C5a (~500ng/ml) stimulate tumor progression [20]. Using immune-competent mice, it was found that higher levels of C5a correlated with more Gr-1+CD11b+ myeloid cells in the spleen and decreased number of CD4+ and CD8+ T-cells in the tumor, tumor-draining lymph nodes and spleen, and ultimately increased tumor growth. However, other research has been done to understand the specific mechanisms by which C5a stimulates tumor growth. The mechanisms, including increased recruitment of myeloid-derived suppressor cell (MDSC), creation of an immunosuppressive microenvironment, increased intra-tumor angiogenesis, and enhanced tumor invasiveness, could be attributed to the effect of C5a in tumor progression [23-25].

Go to:

Antitumor Immunity Suppression via the Inhibition of T-lymphocytes

C5a activates myeloid-derived suppressor cells (MDSCs) both directly and indirectly. The presence of C5a receptors on the cell surface of MDSCs allows C5a to directly bind and stimulate MDSCs. The degree of C5a receptor expression on MDSCs was directly proportional to the extent of tumor infiltration by MDSCs. Therefore, a greater number of C5a receptors lead to greater stimulation of MDSCs by C5a and resultantly, increased immunosuppression [23]. MDSCs are also induced by IL-6, IL-1β and VEGF and C5a stimulates the production of these mediators [24, 26, 27]. Thus, C5a may have the capacity to doubly stimulate MDSCs via both direct and indirect stimulation. Furthermore, MDSCs themselves produce VEGF and therefore, could potentially be self- activating as well [28, 29].

As a consequence of C5a stimulation, MDSCs are attracted to the tumor site and enhance tumor growth by inhibiting T-cell activation and NK cell cytotoxicity, inducing the production of tumorigenic cytokines, and increasing tumor angiogenesis [30]. For example, C5a-induced release of VEGF inhibits the maturation of dendritic cells (DCs) and therefore, increases the population of MDSCs. Interleukin-1β directly stimulates the production of MDSCs [31]. C5a has been found to modulate the production of reactive oxygen and nitrogen species in MDSCs [23].

Recent studies in both tumor models and cancer patients have shown that the activity of NADPH oxidase in MDSCs is upregulated in tumor environments, which results in increased production of reactive species. The resulting reactive oxygen and nitrogen species suppress antitumor CD8+ T-cell responses [32]. In particular, the production of peroxynitrite leads to the binding of nitrate to T-cell receptors (TCRs), preventing the receptor from binding to tumor antigens. Additionally, MDSCs hinder T-cell receptor function by causing downregulation of zeta chain expression. This downregulation is achieved via high levels of arginase in MDSCs. Because arginase functions to breakdown arginine, MDSCs regulate arginine levels; as an amino acid critical for the development of the zeta chain of the TCR, arginine is vital for T-cells to function properly [31, 33].

MDSCs also inhibit T-cells by sequestering cysteine from the tumor microenvironment. T-cells lack both cystathionase, which converts methionine to cysteine, and the xC- transporter, which functions to bring cystine into the cell to be reduced into cysteine. Consequently, acquisition of cysteine by T-lymphocytes is dependent on the exportation of cysteine by antigen presenting cells (APCs). MDSCs reduce the level of cysteine in the environment by importing cystine but due to lack of the necessary ASC transporter, fail to export cysteine back into the environment, reducing the amount of available cysteine for T-lymphocytes [34] (Figure 3).

Figure 3
The interaction between C5a and myeloid-derived suppressor cells (MDSCs). C5a attracts MDSCs to tumor sites by directly binding to receptors on the MDSC surface. C5a also indirectly activates MDSCs by binding to other cell types, inducing the release ...

Studies have demonstrated that when C5aR signaling is blocked the number of CD8+ T-cells significantly increases with decrease in tumor size [23]. This is hypothesized to be due to decreased MDSC stimulation and therefore, increased antitumor immune system function.

Go to:

Stimulation of Angiogenesis

It has been observed that C5a generates a microenvironment favorable for tumor growth [24]. This microenvironment was characterized by increased angiogenesis and expression of IL-6, IL-10, LAG3, PDL1, ARG1, and CTLA-4, most of which are related to an immunosuppressive state [24]. The vascular density of a given tumor strongly correlates with the likelihood of tumor to metastasize. New vessel formation allows the tumor to access the circulation and metastasize throughout the body. The process of angiogenesis is most commonly found to be associated with two families of growth factors, basic fibroblast growth factor (bFGF or FGF1), vascular endothelial growth factor A (VEGF-A), and VEGF-C [35 – 37].

VEGF-A has been credited as the most important angiogenic cytokine and plays a critical role in tumor metastasis [36]. Not only is it commonly overexpressed in solid cancers; its concentration is directly proportional to malignancy [38–40]. VEGF-A increases vascular permeability and has 50,000 times greater potency than that of histamine [36, 41]. VEGF-A also functions to inhibit apoptosis in endothelial cells and stimulate chemotaxis of monocytes and macrophages and endothelial cell migration and division [36, 42, 43].

C5a has been found to stimulate VEGF in the spontaneously arising retinal pigment epithelia cell line “ARPE-19.” Likewise, C5a can also stimulate VEGF production in vivo by intravitreous injection in mice [44]. Accordingly, use of a C5a antagonist blocked the production of VEGF [44, 45]. One potential hypothesis for the mechanism in which C5a induces VEGF release is via the cytokines IL-1β, IL-6 and IL-8. C5a stimulates monocytes to release IL-1, IL-6, IL-8 and TNF-α [46]. IL-1, IL-6, and IL-8 are known potentiators of VEGF production [47, 48]. Although IL-8 is traditionally thought of as a chemoattractant for neutrophils that stimulates neutrophil degranulation, the expression of IL-8 has been found to be associated with tumor tumorgenicity, angiogenesis, and metastasis in vivo [49].

Corrales et al. [24] observed that C5a stimulates angiogenesis in vitro by augmenting the formation of tube-like structures and increasing cell migration. When a C5a receptor antagonist was introduced, the expression of these molecules and formation of vessels decreased significantly. Furthermore, there was also a significant decrease in tumor growth [24].

Go to:

Stimulation of Cancer Invasion and Migration

MMPs play an important role in tumor angiogenesis, invasion and metastasis by degrading the extracellular matrix, inducing of tumor resistance to apoptotic signals, and increasing tumor evasion of the host defense [50, 51]. MMP concentration can be correlated to the presence and aggressiveness of various cancers, such as renal cell carcinoma, lung, colorectal, and breast cancer [52–55]. It is thought that the stromal cells surrounding a tumor, and not the cancerous cells themselves, are responsible for releasing MMPs [56]. Transcription of MMPs is not common under normal cellular conditions but can be stimulated by cytokines, specifically IL-1 and TNF-α, oncogene products, changes in cell shape, and mechanical stress [51, 56–58].

C5a has been shown to increase cancer cell invasion and migration via the induction of cell motility and matrix metalloproteases (MMPs); cancer cells that expressed C5a receptors had 13x greater invasiveness than their controls [25]. Although C5a receptors are not normally expressed on epithelial cells, their expression can be stimulated by inflammation and expression has been seen in certain cancers [59, 60]. Nitta et al. [25] found C5aR expression on cancerous epithelium of the esophagus, stomach, colon, liver, bile duct, pancreas, bladder, prostate and mammary glands, whereas noncancerous versions of these tissues do not express C5aR [25]. It is suggested that the expression of C5a in epithelium is the result of malignant transformation. Blocking of the C5aR with the use of an antibody against C5aR inhibits cancer invasion enhanced by C5a. Furthermore, the extent of cell invasion stimulated by C5a directly correlated with the concentration of MMP-8. C5a has also been found to stimulate MMP-1 and MMP-9 in macrophages in vitro [61].

Go to:

Inhibition of Apoptosis

C5a may also play a role in tumor progression by functioning as an antiapoptotic mediator [62]. C5a has been found to prevent activation of apoptotic caspase 3 and therefore DNA fragmentation, in murine cortico-hippocampal neuronal cultures. It is hypothesized that the binding of C5a to the C5aR triggers the mitogen-activated protein kinase (MAPK) pathway, which prevents glutamate-induced apoptosis [63].

In addition to being neuroprotective, C5a has also been implicated in liver regeneration. Daveau et al. [64] found C5a stimulation of both normal and regenerating liver to be associated with increased expression of hepatocyte growth factor and c-Met mRNAs [64]. Interestingly, high levels of hepatocyte growth factor and c-Met receptor expression have both been associated with tumor metastasis and invasiveness [65].

C5a serves as a transcriptional signal for tumor necrosis factor (TNF) and IL-1β [66]. TNFα is a stimulator of NF-κB, a transcription factor that functions in cell proliferation and is correlated to tumor growth [67]. It has also been proposed that the membrane-associated IL-1α stimulates anti-tumor immune response, whereas secreted IL-1β from the malignant cells or within the tumor microenvironment might induce pro-inflammatory effects leading to increased tumor invasiveness and tumor-mediated immune suppression [68]. In addition to TNF and IL-1β, C5a also regulates IL-6 [69]. Due to the fact that most of the genes that are regulated by IL-6 are involved in stimulation of the cell cycle and inhibition of apoptosis, IL-6 is potentially another way in which C5a plays a hand in tumor progression [70].

Go to:

Therapeutic Use of C5a

Currently, C5a has proved useful in cancer vaccine development via its role in the enhancement of NK cells, dendritic cells, and T-cell activation, all of which aid in tumor suppression. The C5a containing vaccines have been found to prevent tumor growth, specifically in melanoma, lymphoma, and E.G7 OVA tumors [71–73].

A study was conducted in which the effectiveness of vaccines was compared [73]. Among the three vaccines used in this study, one vaccine was composed of the antigen “SIINFEKL” from ovalbumin and an endogenous ligand for toll-like receptor 4 (TLR4). SIINFEKL is an epitope for cytotoxic T lymphocytes (CTLs) and TLRs function as important activators of the innate immune system. The second vaccine contained SIINFEKL, the TLR4 ligand, and C5a. Results showed that the vaccine containing C5a elicited a significantly higher CTL response than the vaccine containing only the TLR4 ligand and SIINFEKL antigen. Furthermore, the C5a containing vaccine also stimulated greater NK cell activity. These results paralleled with their findings regarding the ability of the two vaccines to prevent E.G7-OVA tumor growth; 61% of the mice vaccinated with the vaccine containing C5a, while only 35% of mice immunized with the vaccine containing only TLR4 ligand were protected against tumor challenge in regard to tumor size and death.

C5a agonists have also been used in vaccines for tumor prevention [71, 72]. Kollessery et al. [72] demonstrated that vaccines composed of either the C5a agonist EP54 or EP67 protected mice from RAW117-H10 lymphoma. Despite being injected with a lethal dose of the RAW117-H10 cells, all vaccinated mice lived over year as compared to the controls, which died within 17 days. Results suggest that the vaccines prevented visible metastasis to the liver and furthermore, induced CTLs with cytotoxic specificity to RAW117-H10.

Likewise, Floreani et al. [71] also used C5a agonists to inhibit tumor growth. By covalently linking the C5a agonist, YSKFDMP(MeL)aR, to two different melanoma antigens separately, TRP2-P2 and TYR, and injecting these two agonist-antigens into mice, melanoma tumor growth was inhibited. However, when mice were injected with only one of the agonist-antigens, tumor growth inhibition did not proceed past 17 days [71].

In addition to cancer therapy, C5a has also been utilized in the treatment of arthritis. As C5a deficiency is protective against arthritis, two different anti-C5a vaccines were developed and have been tested experimentally in murine models. Both vaccines were successful in combating different facets of the disease.

One vaccine has been generated by fusing maltose to C5a. This fusion protein vaccine was tested in three different arthritic murine models. Overall, the vaccine was successful in stimulating specific C5a antibodies and decreasing the incidence and severity of the inflammation generated in arthritis. However, the antibodies generated were unsuccessful in preventing C5a activation and formation of MAC. The vaccine also failed to inhibit the anti-collagen antibodies characteristic of collagen antibody induced arthritis (CAIA) [74].

A second vaccine was produced by substituting the amino acid “p-nitrophenylalanine (4NPA)” for a tyrosine at the 35 position within C5a. This anti-C5a vaccine was exclusively tested in the collagen-induced arthritis (CIA) model. The vaccine was successful in abating the severity of the arthritis but was only partially protective against CIA development. The vaccine itself lead to a loss of B and T-cell tolerance to C5a in mice whose cells expressed the particular receptor “class II MHC H-2(q).” Both the indigenous C5a and modified C5a induced antibodies capable of effectively neutralizing C5a. The high titers of IgG prevented disease development, but did not reverse the course of ongoing disease [75].

Lastly, C5a has been successfully utilized in antibacterial therapy. Using the CD88 agonist “EP67,” researchers were capable of limiting Staphylococcus infections via the enhancement of cytokine release and neutrophil infiltration. EP65 has also been effective in Streptococcus killing, specifically group B Streptococcus (GBS). Successful killing occurred in mice lacking both CD88 and CXCr2 (a neutrophil receptor); thus, it is hypothesized that the mechanism of action of EP65 against GBS is not due to enhancement of the immune response, like with Staphylococcus, but rather by direct bacterial killing [76].

Expert Commentary and Five year Review

The role of C5a in tumorigenesis is complex and much is still unknown; however, some key characteristics have been determined. The function of C5a in cancer is dose-dependent and whether it inhibits or supports tumor progression is contingent on the concentration of C5a at the tumor site. At low levels, C5a inhibits tumor growth via enhancing NK cell and M1 macrophage tumor infiltration and cytotoxicity. C5a potentiates tumorigenesis through a plethora of mechanisms. Possibly the most crucial function of C5a in tumor promotion is its ability to activate and attract MDSCs to the tumor site. MDSCs are responsible for inhibiting T-lymphocyte activation and NK cell cytotoxicity leading to an immunosuppressed state. C5a also stimulates tumor angiogenesis, invasion, and migration. Perhaps, it is by inducing the release of growth factors, such as VEGF, that C5a facilitates the formation of the primary tube-like structures characteristic of developing vessels. C5a promotes invasion and migration by stimulating MMP release from epithelial cells aberrantly expressing C5a receptors. Lastly, C5a has been implicated as an anti-apoptotic mediator.

Although the function of C5a has been observed to vary with its concentration, additional studies are warranted to confirm and compare the effects of C5a at various concentrations, and perhaps in diverse microenvironment, in regard to its pro- and antitumor activity. It is also important to acknowledge that each function of C5a may require a separate threshold concentration. Thus, it is critical to determine the functional levels of C5a for individual cancers. Theoretically, this information could be used to test and therapeutically manipulate C5a concentration in patients in the antitumor range. If the effect of C5a is concentration-dependent, then it is also important to determine the underlying mechanism(s) of C5a production in cancer. Once identified, the specific pathway could be therapeutically interrupted to inhibit C5a production. Additionally, the tumor microenvironment may elicit synergistic or antagonistic effects on the action of C5a.

Due to the fact that the vascular density of a tumor is directly proportional to the degree of metastasis, further knowledge as to exactly how C5a stimulates VEGF release could be highly relevant. The inflammatory cytokines, including IL-1β, IL-6, and IL-8, could be critical to serve as a potential bridge between C5a and VEGF release. C5a is known to stimulate the release of IL-1β, IL-6, and IL-8 from monocytes and these cytokines are known potentiators of VEGF production. None-the-less, additional studies are warranted to accept or refute this hypothesis.

​

Key Issues

  • If the concentration of C5a determines its anti-tumorigenic or tumorigenic effect, what is the threshold concentration?
  • Is the effect of C5a in various concentrations dependent on the tumor microenvironment and cancer type?
  • Studies are required to completely elucidate the underlying mechanisms of the anti-tumorigenic or pro-tumorigenic effects of C5a.
  • Comparative role of C5a receptors, CD88 and C5L2, in tumor invasive ness and tumor progression needs further investigation.
  • What is the mechanism by which C5a stimulates VEGF release?
  • What is the effect of pro-inflammatory cytokines in angiogenesis induced by the interaction between C5a and VEGF?
Read more

Cancer Research: Adenosine Blockade

 

STEP ONE

Be informed.  Read information below.

STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

STEP THREE

Submit "New Patient Form", current labwork, & imaging results prior to consultation.

STEP FOUR

Establish if you are a candidate for this therapy

in accordance with the Georgia

"Access to Medical Treatment Act".

 

----------------------------------------------------------------------------------------------------------------------------------------------------------

*** SPECIAL NOTE FROM DR. WICHMAN ***

The following excellent article was reproduced from the Computational and Structural Biotechnology Journal at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415113/:

 

Comput Struct Biotechnol J. 2015; 13: 265–272.
Published online 2015 Apr 8. doi:  10.1016/j.csbj.2015.03.008
PMCID: PMC4415113

A2aR antagonists: Next generation checkpoint blockade for cancer immunotherapy

Robert D. Leone, Ying-Chun Lo, and Jonathan D. Powell⁎
Author information ► Article notes ► Copyright and License information ►
 
Go to:

1. Introduction

The immune system has evolved an array of regulatory mechanisms to protect against tissue damage from autoimmunity or during active response to pathogen. Both central mechanisms (negative selection in the thymus) and peripheral mechanisms (e.g., deletion, anergy, and regulatory T cells (Tregs)) contribute to establishing self-tolerance. Nonetheless, inherent in active immune responses against pathogens are inhibitory and negative feedback pathways which prevent collateral damage. Included in these protective mechanisms are a broad array of inhibitory receptors that are upregulated on lymphocytes during an active immune response. These inhibitory receptors and their related signaling networks, known as “immune checkpoint pathways,” provide a negative feedback mechanism that is crucial for immunoregulation and protection of tissues from an overexuberant inflammatory response.

While the negative feedback loops created by checkpoint pathways are critical in modulating excessive inflammation, they are also subject to dysregulation in the presence of cancer and provide tumors with a means of immune evasion. Recently, clinical trials have confirmed that blockade of immune checkpoint pathways mediated by the CTLA-4 and PD-1 receptors can unleash an endogenous immune attack, leading to significant responses and long-term remissions in multiple solid tumor types [1–3]. In fact, antibody-mediated blockade of CTLA-4 and PD-1, alone or in combination, have led to unprecedented responses in refractory, metastatic melanoma, as well as in renal cell carcinoma and non-small cell lung cancer. The success of checkpoint blockade in these trials has been a major step forward in the development of immunotherapy for the treatment of cancer, confirming the clinical importance of tumor immune evasion through usurping fundamental pathways of immune regulation. With the success of CTLA-4 and PD-1 inhibition in clinical trials, significant effort has focused on uncovering other targetable checkpoint pathways active in the tumor microenvironment. In this regard, adenosine signaling through the A2a receptor has been found to function as one such promising negative feedback loop [4–7]. As we shall discuss, while the effects of A2a receptor inhibition in antitumor therapy can behave as a double-edged sword (depending on the degree and, likely, the duration of signaling blockade), preclinical studies have confirmed that blockade of A2a receptor activation has the ability to markedly enhance anti-tumor immunity. As such, A2a receptor blockade represents the potential next generation of immune checkpoint inhibition in cancer immunotherapy.

Go to:

2. CTLA-4 and PD-1 and the arrival of cancer immunotherapy

Immune checkpoint pathways such as those mediated by CTLA-4 and PD-1 receptors are critical aspects of normal physiologic function. The hallmark of these pathways is the generation of a negative feedback loop that preserves self-tolerance and prevents excessive tissue damage in the setting of immune response. The pathways regulated by CTLA-4 and PD-1 receptors have somewhat distinct modes of action on the immune response [5,8]. CTLA-4 is upregulated during initial activation of effector T cells and is thought to counteract the activity of the co-stimulatory receptor CD28 by two mechanisms. By out-competing the lower affinity CD28 for engagement of shared, cognate ligands B7.1 and B7.2 on antigen presenting cells (APCs), as well as by providing a direct inhibitory signal, the CTLA-4 receptor dampens the effector T cell activation sequence [5,9–11]. CTLA-4 is also strongly expressed on regulatory T cells and enhances immunosuppression through enhancing Treg activity and proliferation [12]. Like CTLA-4, PD-1 is induced upon effector T cell activation and is also highly expressed on Tregs [13–15]. Cognate ligands for PD-1 include PD-L1 and PD-L2. These are constitutively expressed on APCs and are induced in peripheral tissues during inflammatory responses or on the surface of tumor cells [13,16,17]. Among other inflammatory cytokines, interferon-gamma secreted during an immune response is a potent inducer of PD-L1 expression [18–20]. In contrast to CTLA-4, PD-1 is expressed on a broader range of immune cells (e.g., B lymphocytes and monocytes) [5,20–22]. And while PD-1 signaling is initiated during T cell activation, its primary effects of inducing CD8 + T cell anergy and, conversely, regulatory T cell activity and proliferation appear to be more pronounced during effector function in the peripheral tissues [5]. The critical roles of PD-1 and CTLA-4 in immune modulation were demonstrated in early studies that showed severe autoimmune pathologies in PD-1 and CTLA-4 knockout strains [22–24].

Although crucial in moderating inflammatory responses and preventing autoimmunity, checkpoint pathways can provide an immune evasion mechanism for tumors, allowing unchecked growth and progression. Preclinical studies have shown that PD-1 is expressed on a broad range of tumor infiltrating lymphocytes and is especially prominent on infiltrating Tregs and CD8 effector cells [25,26]. The PD-1 ligands, PDL1 and PDL2, are upregulated on a variety of tumor cells, and are also expressed by myeloid cells in the tumor microenvironment [27]. In studies by Dong et al., tumors expressing high levels of PD-L1 were found to promote apoptosis of tumor antigen-specific T cells in vitro as well as in mouse tumor models [17]. Early studies of antibody-mediated CTLA-4 blockade in a variety of transplantable tumor models (e.g., colon carcinoma, fibrosarcoma, ovarian cancer, and prostate cancer) demonstrated significant tumor response. An especially important finding in these studies was that once mice had experienced a response to CTLA-4 blockade, they were resistant to tumor rechallenge. These results demonstrated that, in addition to promoting regression of primary tumors, checkpoint inhibition facilitates the generation of an immunologic memory response that is associated with durable tumor remission [28,29].

These preclinical findings were validated in clinical trials of several immune checkpoint inhibitor antibodies [20]. In two large initial phase III trials, the anti-CTLA-4 monoclonal antibody ipilimumab significantly prolonged survival and produced durable responses in patients with advanced melanoma [1,30]. CTLA-4 blockade has also been shown to be active in patients with renal cell carcinoma and in patients with NSCLC [2,3]. Clinical studies of anti-PD-1 mAbs have also shown improvement in overall survival with durable responses in a variety of heavily pre-treated tumor types, including melanoma, NSCLC, and renal cell carcinoma [31]. Anti-PD1 mAbs have shown activity in hematologic malignancies as well, demonstrating a 66% ORR when combined with rituximab for follicular lymphoma [32], and a 51% ORR in patients with diffuse large B-cell lymphoma (DLBCL) who have progressed after autologous stem cell transplant [33]. Blockade of the PD-1 ligand PD-L1 has also shown activity in melanoma, renal cell cancer, and NSCLC, with overall response rates of 10–17% [34]. Importantly, a recent phase I trial demonstrated that the combination of PD-1 and CTLA-4 blockade produces greater than additive response rates in melanoma patients, with an ORR of 42% [35].

Go to:

3. Adenosine-A2aR signaling: the emergence of a novel immune checkpoint pathway

While the clinical importance of immune checkpoints mediated by CTLA-4 and PD-1 has become clear, there are a number of other pathways active in the immune microenvironment that also appear to be important contributors to tumor immune evasion. While several of these pathways—like PD-1 and CTLA-4—are triggered by membrane-bound ligands (most notably LAG-3 and TIM-3 pathways), there are also soluble ligands found in the immune microenvironment that can function as triggers for checkpoint pathways [5]. Such soluble checkpoint ligands include tumor metabolites and cytokines such as IL-10 and TGF-beta [36]. Studies over the last two decades have also identified extracellular adenosine as a critical element in immune regulation [37–40].

3.1. Adenosine signaling through A2aR protects against exuberant immunologic response

Like CTLA-4 and PD-1, adenosine signaling in the inflammatory setting serves to dampen immunologic response and protect tissues from associated injury. While extracellular adenosine levels are typically very low, tissue breakdown and hypoxia (common to inflammatory and tumor microenvironments) generate high levels of extracellular adenosine [41,42]. Extracellular adenosine can signal through a set of four G-protein-coupled receptors: A1, A2a, A2b, and A3 [43]. Adenosine signaling through A2a and A2b receptors—expressed on a variety of immune cell subsets and endothelial cells—has been established as having an important role in protecting tissues during inflammatory responses [44–46]. Because of its distribution and dynamic expression pattern on a broader array of immune cells, most of this protective effect is thought to be secondary to signaling through the high-affinity A2a adenosine receptor. In a set of seminal experiments, Sitkovsky et al. showed that under physiologic conditions tissue injury is accompanied by A2aR-mediated accumulation of intracellular cAMP in immune cells [40]. These studies also noted a concomitant decrease in the release of pro-inflammatory cytokines (e.g., INF-gamma, TNF-alpha, IL-6). Genetic or pharmacologic blockade of the A2aR had profound effects on tissue inflammation, allowing for uncontrolled inflammatory response and tissue injury in mouse models of hepatitis and sepsis. A2aR-null mice experienced extensive tissue injury and death in inflammatory models that cause only minor, transient injury in wild type animals. Importantly, alternate inflammatory control mechanisms were unable to effectively compensate for the tissue damage resulting from the absence of A2aR signaling, thus establishing the adenosinergic pathway as a critical and non-redundant negative feedback control mechanism of inflammatory responses [40].

Subsequent experiments in our lab and others have confirmed the critical role of A2aR signaling in modulating tissue inflammation. In a mouse model of T cell mediated pneumonitis, A2aR signaling was found to significantly reduce tissue inflammation and prolong survival [7]. In these studies, a normally non-fatal pneumonitis caused by T cell transfer targeted to antigen-expressing lung tissue was found to be 80% fatal if the transferred T cells were from A2aR-null mice. Conversely, the effects of a normally lethal dose of A2aR-competent T-cells were almost completely abrogated by pharmacologic treatment with the A2aR-specific agonist CGS-21680. Thus, A2aR engagement provides an important tolerizing signal, which moderates tissue destruction and prolongs survival in the setting of T-cell mediated inflammation. Other studies have confirmed the non-redundant role for A2aR inflammatory modulation in a variety of other mouse models of inflammation, including sepsis, inflammatory bowel disease, and rheumatoid arthritis [38,47–49].

Through these and other studies a picture has emerged of adenosinergic signaling through A2aR as a negative feedback loop that regulates local and systemic inflammatory response. Under normal physiologic conditions extracellular release of adenosine is balanced by rapid cellular uptake that prevents a significant increase in extracellular levels [50,51]. In contrast, inflammatory environments and tumors produce high levels of extracellular ATP and adenosine [41,42,52]. As tissues are subjected to immune attack, increased cellular turnover and hypoxia trigger release of ATP and adenosine. While build-up of extracellular adenosine is partly a result of direct liberation of intracellular adenosine formed from increased ATP metabolism during cellular stress, levels are also increased by the catabolism of extracellular ATP and ADP by the tandem activity of the ectonucleotidases CD39 and CD73 (Fig. 1). In response to hypoxia-induced Hif1-alpha generation in tumors and inflamed tissues, CD39 and CD73 are upregulated on endothelial cells, stromal cells, some solid tumor cells and, importantly, on several subsets of immune cells, including Tregs, CD8 + T cells, B cells, and others [6,46,53,54]. Elevated levels of extracellular adenosine activate specific purinergic receptors such as A2a (high affinity) and A2b (low affinity), which, as mentioned, have broad expression on immune cells and endothelial cells—the A2a receptor being a particular focus of attention given its higher affinity and wide distribution. A2a and A2b are Gs protein linked and trigger the accumulation of intracellular cAMP through stimulation of intracellular adenylyl cyclase [43,55,56]. The rise in intracellular cAMP—acting primarily through protein kinase A—has a broad range of immunosuppressive effects [57], including increased production of immunosuppressive cytokines (e.g., TGF-beta, IL-10) [7,58], upregulation of alternate immune checkpoint pathway receptors (e.g., PD-1, LAG-3) [7,59], increased FOXP3 expression in CD4 T cells driving a regulatory T cell phenotype, and induction of effector T cell anergy [7]. As in CTLA-4 and PD-1 pathways, significant influence of A2aR signaling on Tregs and effector T cells is likely the fundamental driving force of its immunosuppressive effect (though A2aR signaling on myeloid cells and NK cells likely also plays an important role). Since Tregs express high levels of CD39 and CD73, as CD4 + T cells are driven toward a Treg phenotype by A2aR-mediated FOXP3 expression, an immunosuppressive amplification circuit generating increasing amounts of adenosine is created and quickly dampens the inflammatory response [60]. CD8 + effector cells, on the other hand, become less cytotoxic with decreased TCR signaling and increasingly anergic under the influence of A2aR signaling [7].

Fig. 1
A2aR blockade in the tumor microenvironment. With increasing tumor cell breakdown in the setting of hypoxia, increased cellular stress, and chemotherapy, ATP, adenosine, and tumor associated antigens (TAA) are released into the tumor microenvironment ...

Given the importance of adenosinergic signaling in mediating negative feedback loops of immune responses, the effect of A2aR blockade on enhancing immunologic response has been investigated. In vivo studies in our lab utilizing A2aR knockout mice as well as studies using pharmacologic A2aR blockade, consistently demonstrate increased proliferative capacity and effector function of CD4 + and CD8 + T cells in response to activating antigen [61]. In fact, transient pharmacologic A2aR blockade in these studies was found to enhance immunologic memory, improving effector function several weeks after initial antigen challenge. Notably, this is not the case in A2aR-null mice, however, wherein persistent A2aR blockade eventually leads to an exhausted phenotype and disrupts transition to a memory phenotype (Waickman and Powell, unpublished findings). The difficulty in transitioning to a memory phenotype was also demonstrated in recent work by Cekic et al. [62]. In these studies, absence of A2aR signaling on A2aR-null lymphocytes hinders the accumulation of CD8 + effector-memory T cells in tumors in mouse models of melanoma and bladder cancer. In an earlier study by the same group, the absence of A2aR signaling was also shown to disrupt the homeostatic maintenance of the naïve T cell compartment, although it did not diminish the number of memory T cells in (non-tumor bearing) mice [63]. In this regard, A2aR signaling appears to attenuate the downregulation of the IL-7 receptor in response to TCR signaling through the PI3K-AKT pathway. Such signaling is important in both naïve T cell maintenance as well as transitioning to longer-lived phenotypes after initial T cell activation. It is important to note that these studies have examined the absence of A2aR signaling in knockout models and in the setting of irreversible A2aR blockade. As such, great care will be needed to optimize the dose and schedule of A2aR blockade within immunotherapeutic regimens.

3.2. A2aR blockade for immunotherapy in cancer

Analogous to CTLA-4 and PD-1, the immunologic dampening triggered by adenosine at sites of inflammation is mirrored by its effect in the tumor microenvironment. Several pioneering studies by Blay et al. allowed generalization of the idea of adenosine-mediated immunosuppression to the tumor microenvironment. In publications from the 1990s, this group theorized that supraphysiologic extracellular adenosine levels—driven by high cell turnover and hypoxia—could be responsible for observed immunosuppression in patients with solid tumors. In studies using a microdialysis probe it was demonstrated that extracellular adenosine levels in solid tumors were 10–20 times higher than adjacent tissues and reached levels sufficient to disrupt function of activated Cytotoxic T Lymphocytes (CTLs) [42]. During the same period, pioneering studies by Sitkovsky et al., began to uncover the critical interactions between extracellular ATP, adenosine and distinct subsets of immune cells [64–66]. Since that time, it has been found that, in addition to hypoxia and increased cell turnover, many cells in the tumor microenvironment (e.g., tumor cells, infiltrating immune cells, stromal cells, and endothelial cells) undergo ectopic expression of CD39 and CD73, further contributing to the buildup of extracellular adenosine [67,68]. In addition to dampening the effect of CTLs, increased extracellular adenosine has been found to down-modulate the activity of a range of immune functions in the tumor microenvironment, including the activity of macrophages, NK cells, neutrophils, and dendritic cells [69–73].

Given the similarities between adenosine-mediated immune modulation and established checkpoint pathways such as CTLA-4 and PD-1, the application of A2aR blockade to tumor immunotherapy is particularly exciting. In pioneering studies in 2006, Ohta et al. showed the complete rejection of two distinct tumor lines, CL8-1 melanoma and RMA T cell lymphoma, in a majority of A2aR null mice [4]. Notably, each of these tumor lines was 100% fatal in wild type mice. Responses in these models were dependent solely on CD8 + T cell activity. In another experiment, pharmacologic blockade of A2aR significantly augmented the tumor rejecting capacity of adoptively transferred, tumor-specific CD8 + T cells in a sarcoma model in mice [4]. They also showed the capacity for A2aR antagonism to strongly enhance CD8 + T cell-mediated destruction of the poorly immunogenic LL-LCMV tumor line. In studies by Beavis et al., A2aR antagonism was effective in reducing metastasis in CD73-expressing tumors in mouse models [74]. Included in these studies were investigations of the metastatic potential of the CD73-expressing murine breast cancer line, 4T1.2, as well as the melanoma line B16F10, which had been transduced to express CD73. Notably, in these studies NK cells were found to play a dominant role in limiting metastatic growth in these models.

Studies from our group have confirmed the increased capacity of A2aR−/− mice to reject tumor cells in a variety of settings. In our initial tumor studies, A2aR−/− mice showed significantly better tumor rejection and survival in a subcutaneous tumor model using the EL4 lymphoma cell line [61]. Interestingly, subcutaneous inoculation with a low-dose of EL4 lymphoma cells, which were readily rejected by both A2aR−/− as well as wild type mice, allowed A2aR null mice to reject a subsequent challenge (on day 60) with an otherwise lethal dose of the same EL4 tumor line. Wild type mice in these experiments were unable to reject this re-challenge with tumor cells. This enhanced responsiveness was also elicited by vaccination with a 1:1 mixture of GMSF-secreting, irradiated melanoma cells (GVAX) and irradiated OVA peptide producing EL4 cells. In this case, the population of OVA-specific CD8 T cells in draining lymph nodes 7 days post inoculation was significantly elevated in A2aR null mice over wild type mice. In another experiment, GVAX inoculation was significantly more effective in protecting A2aR null mice from forming pulmonary lesions following subsequent (60 days after GVAX vaccine) tail vein injection of B16 melanoma cells.

An additional finding from our initial studies in A2aR null mice was the ability of A2aR blockade to synergize with inhibition of other checkpoint pathways [61]. Again using a subcutaneous EL4 model, A2aR-null mice exhibited longer tumor-free survival (TFS) and overall survival (OS) when treated with a soluble B7-DC/Fc fusion protein starting on the first day of tumor inoculation and continued for the length of the experiment. (B7-DC/Fc fusion protein acts as a ligand that specifically targets the PD-1 receptor expressed on dendritic cells and triggers profound T cell activation.) Improvement in TFS and OS were significant when compared to both untreated A2aR null mice as well as wild type mice with and without B7-DC/Fc. The increased effectiveness of A2aR blockade and concomitant PD-1 inhibition over either treatment alone was also seen in studies by Mittal et al., wherein metastases of CD73 + tumors was significantly decreased by combination therapy [75]. In these studies, Mittal et al. also demonstrated that A2aR blockade increases the activity of CTLA-4 and TIM-3 inhibition in controlling metastatic growth of CD73 + melanoma. Again, this group demonstrated a primary, though not exclusive, role for NK cells in metastatic control. In other studies of combination strategies, Iannone et al. showed that pharmacologic A2aR blockade can improve the efficacy of CTLA-4 therapy in mouse melanoma models. Of note, the efficacy of CTLA-4 inhibition in these studies was also enhanced by blockade of adenosine production upstream to A2aR by pharmacologic inhibition of CD73 activity [76].

As discussed above, CD73 and CD39 are ectonucleotidases that work in tandem to catabolize extracellular ATP to adenosine in the immune microenvironment. Though a complete review is beyond the scope of this article, investigations of CD73 blockade have shown significant effect on tumor control in mouse models and have also been especially effective in combination with both CTLA-4 inhibition and PD-1 blockade [53,77]. In studies of human tissue, CD73 expression on tumor cells was associated with chemotherapy resistance and poor overall prognosis in patients with triple-negative breast cancer [78]. A similar association has also been uncovered in several other types of cancer, including rectal carcinoma, gastric cancer, colorectal cancer, gallbladder cancer, chronic lymphoblastic leukemia, and prostate cancer [79]. These translational studies offer evidence of the importance of adenosine signaling in the tumor microenvironment in tumor progression. This idea has been bolstered by preclinical studies showing anti-CD73 mAb-induced reduction of primary tumors and metastases in two mouse models (4T1.2 and E0771) of breast cancer [80].

Go to:

4. Translating A2aR blockade to tumor immunotherapy

With the clinical success of CTLA-4 and PD-1 checkpoint blockade in producing long-term responses in several distinct tumor types, there has been growing interest in understanding the specific determinants of host response during immunotherapy. As such, the critical parameters regarding immunologic response are being closely investigated, and it is becoming clear that future study of immunotherapeutic strategies will require assessment in a multitude of therapeutic and immunologic contexts. A single pathway, such as that triggered by extracellular adenosine, typically has multiple receptors, intra- and extracellular targets, and a range of distinct effects, all of which may depend on the specific developmental stage of a given target cell. As a case in point, a recent study found that A2a receptor blockade has distinct effects on T cell activation vs. effector-memory cell generation in a mouse melanoma model [62]. As mentioned, recent studies by Cekic, et al. have elucidated the importance of intact A2aR signaling for both maintenance of the naïve T cell compartment, as well as the transition to memory cell phenotypes in tumor-bearing mice. In these studies it was shown that persistent absence of A2aR signaling can actually stimulate tumor growth in some models [74,81]. Unpublished work from our lab confirms that, while transient blockade of A2aR signaling early in the immune response can drastically enhance the potency of a late recall response, complete elimination of A2aR signaling in knockout models appears to hinder efficient transition of CD4 + and CD8 + T cells to a memory phenotype. Further investigation of the importance of A2aR signaling in establishing, maintaining, or ameliorating anergy, exhaustion, and senescence of effector T cells will be informative avenues of inquiry.

Though there is certainly much work to be done in understanding the nuances of adenosinergic signaling on tumor immune response, the findings outlined in this review have a number of implications for clinical studies. Chief among these findings is the identification of adenosine-A2aR signaling as a critical and non-redundant negative regulator of inflammatory response that can be co-opted by tumors and function as a means of immune evasion. Signaling through this pathway has effects on activation, early expansion, and effector phases of T cell response. Furthermore, several preclinical studies have demonstrated the efficacy of A2a receptor inhibition in promoting tumor regression. In a number of studies A2aR blockade has been combined with other approaches to immunotherapy to potentiate additive effects on tumor control (Table 1).

Table 1
A2aR blockade in murine models of cancer.

As we move closer toward application of A2aR blockade in clinical trials, it is important to note that several A2a receptor antagonists have already gone through phase III trials for Parkinson Disease. These agents have generally been very well tolerated, without severe immune-related toxicities associated with CTLA-4 and PD-1 antagonism [82]. Recently reviewed by Pinna, these agents include Istradefylline, which has been approved for Parkinson Disease in Japan, as well as several agents presently in clinical trials (PBS-509, ST1535, ST4206, Tozadenant, V81444). Preladenant is an A2a receptor antagonist which has been discontinued after demonstrating poor efficacy in late phase clinical trials. Despite promising efficacy and a low incidence of adverse events, another A2aR antagonist, Vipadenant, was also discontinued after phase II studies [82].

4.1. Optimizing the immunotherapeutic effects of A2aR inhibition

While data from our lab and others show that A2aR blockade during initial T cell activation can greatly enhance T cell expansion and generation of memory phenotypes, studies by Ohta et al. show that A2aR blockade during adoptive T cell therapy in sarcoma models has a role in enhancing T cell effector function as well [4]. In addition, recent studies have shown that long-term A2aR blockade may interfere with the generation of immunologic memory [62]. Integrating these findings to achieve clinically effective A2aR inhibition will require careful consideration of the timing of blockade, as well as combination schemes using a range of other therapeutic approaches. In considering the importance of dosing, scheduling, and combination therapy, it is instructive to note that of the two initial CTLA-4 inhibitors, ipilimumab succeeded in phase III trials and garnered FDA approval whereas tremelimumab failed. This was despite the fact that these two agents showed equivalent intrinsic activity and phase II response rates [5]. The failure of tremelimumab in phase III studies is generally attributed to suboptimal dosing and scheduling, as well as other trial design flaws [1,5,83].

4.2. A2aR blockade during early immune response: combination therapy with vaccines and chemotherapy

Adenosine signaling has significant effects on several distinct cell types involved in the early stages of immune response. Specifically, while A2aR signaling on effector T cells decreases early post-activation proliferative capacity, A2aR signaling is also important in myeloid cells, polarizing professional APCs toward a more tolerogenic or suppressive phenotype and inhibiting the activation of effector cells (Fig. 1) [84–86]. The ability of A2aR blockade to reproducibly enhance vaccination strategies in a variety of tumor models confirms the robust effect of this pathway on T cell activation and early expansion. As many cancer vaccines have historically met with only limited success, adjunctive therapy with A2aR blockade may offer an important potentiating strategy (Table 2).

Table 2
Potential therapeutic applications of A2a receptor blockade.

Largely through the work of Kroemer and Zitvogel, it has become increasingly clear that there is an immunologic component associated with the action of many cytotoxic chemotherapeutic agents [87–89]. In this regard, several chemotherapeutic agents, including anthracyclines, oxaliplatin, cyclophosphamide, gemcitabine, and bortezomib appear to produce an in situ vaccination as a consequence of their initial cytotoxic effect. In so doing, these agents appear to facilitate an immunogenic cell death, which has several important attributes [87–91]. Immunogenic cell death, as defined by Kroemer et al., is a process that stimulates an immune response against dead cell antigens (tumor antigens) through the timed release of soluble mediators as well as early changes on the surface of cancer cells. Interestingly, the release of ATP has been identified as a critical mediator in this process. While ATP acts as immunostimulant, facilitating the recruitment of dendritic cells into the tumor bed, it is eventually catabolized to adenosine by ectonucleotidases CD39 and CD73 that are often highly expressed in the tumor microenvironment. As such, the immunostimulatory effects of ATP give way to the immunosuppressive effects of adenosine. This presents an excellent opportunity for concomitant A2aR blockade. A2aR antagonism during chemotherapy may allow the expansion of tumor-specific T cells, and simultaneously repress the induction of tumor-specific regulatory T cells, thus helping to kindle the immunologic response. To this end, the work of Zitvogel and Kroemer, as well as the work by Stagg and others, have shown the effectiveness of combining adenosinergic signaling blockade in the context of cytotoxic chemotherapy [90,91]. Stagg et al. demonstrated the success of this approach by inhibiting adenosine production (upstream of A2aR) with CD73 blockade in combination with doxorubicin chemotherapy in a murine breast cancer model [78]. CD73 blockade in these experiments enhanced antitumor immune response, especially when given in combination with doxorubicin, prolonging survival in mice with established metastatic breast cancer compared with either agent given as monotherapy. In this work, a similar effect was also observed when a specific A2aR blocking agent, SCH58261, is used in combination with doxorubicin. Similar studies examining pharmacologic blockade of A2aR in combination with chemotherapy are ongoing in our lab.

4.3. A2aR blockade in the context of multiple checkpoint pathway inhibition

The ability of A2aR pathway blockade to produce additive effects in combination with targeting of other checkpoint pathways has mechanistic as well as clinical implications. Mechanistically, studies showing an additive response underline the independence of the adenosinergic-A2aR pathway from established checkpoint pathways. Clinically, the non-redundant nature of these pathways implies that combination checkpoint pathway inhibition, including adenosinergic blockade, can have potentially dramatic effects on response rates. To that end, recent trials combining CTLA-4 and PD-1 blockade reported initial findings of an overall response rate of 42%—significantly higher than either agent used alone [35]. While CTLA-4 blockade appears to be most effective in enhancing the activation phase of cellular immune response, whereas PD-1 inhibition is most profound during the effector phase [5], the addition of A2aR blockade has the potential to further lower the threshold for each of these critical immune events (Table 1). In this regard, it is possible that concomitant use of A2a receptor antagonism with CTLA-4 or PD-1 may allow for dose reductions of either agent, thereby reducing the incidence and severity of immune related toxicities.

4.4. A2aR blockade during effector phase of the immune response: combination therapy with adoptive T cell therapy

The ability of A2aR blockade to enhance effector function is an important aspect of its mode of action. Adenosine signaling through A2aR has suppressive effects on both CD4 + and CD8 + effector T cell compartments, including: polarization of CD4 + cells away from the Th1 phenotype; decreased production of IFN-gamma, IL-2, and TNF-alpha; reduced cytoxicity of CTLs; reduced TCR signaling; and reduced CTL activity leading to increased anergy [4,7,57,59,92]. This has been confirmed in preclinical studies in which A2aR inhibition has demonstrated the ability to enhance effector function during an immune response (Fig. 1) [4,7,40]. Given these properties, we expect that the combination of A2aR blockade with adoptive T cell therapy will generate enhanced T cell function and extended duration of cytotoxic response (Table 2). As mentioned, early studies by Ohta et al. specifically demonstrated the benefit of A2aR blockade in mouse tumor models using adoptive T cell therapy [4].

4.5. A2aR blockade in combination with other targets in the adenosinergic pathway

Lastly, while the A2a adenosine receptor is an attractive target for tumor immunotherapy, inhibition of other targets in the adenosinergic pathway has also yielded encouraging results. Of particular interest has been the upstream ectonucleotidase CD73. Several groups have shown that CD73 blockade can have dramatic effects on both primary tumor response as well as metastatic processes [93–95]. As mentioned, the level of CD73 in triple-negative breast cancer tissues was found to be negatively correlated with prognosis and response to chemotherapy [78]. Recently, CD73 expression in tumor tissue has also been correlated with poor prognosis in rectal adenocarcinoma [96]. Several other studies have also found an association between CD73 expression in tumor tissue and more aggressive clinical behavior, including studies in gastric cancer, colorectal cancer, gallbladder cancer, chronic lymphoblastic leukemia, and prostate cancer [79]. Studies in mouse models using CD73-null mice have shown increased tumor immunity in a variety of tumor types, including MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, ID8 ovarian tumors, and B16F10 melanoma [77,94,97]. CD73 blockade with both small molecules and anti-CD73 mAb has shown specific responses in mouse models of B16 melanoma and 4T1.2 breast cancer [77,97]. Also, inhibition of another adenosine receptor, A2bR, has been shown to inhibit growth of prostate cancer cell lines (though not through an immunologic mechanism) [98]. In this regard, Stagg et al.showed that A2bR activation promoted metastatic cancer cell phenotype in a 4T1.2 mouse model of breast cancer [80]. Furthermore, Cekic et al. demonstrated enhanced activation of dendritic cells and improved CXCR3-dependent T cell tumor infiltration in the setting of pharmacologic A2bR blockade [99]. In these studies, specific A2bR blockade with ATL801 slowed tumor growth in mouse models of bladder and breast cancer. It remains to be seen if simultaneous inhibition of several members of the adenosinergic pathway can produce non-redundant effects on tumor response.

Go to:

5. Conclusion

With the recent clinical success in applying CTLA-4 and PD-1 blockade to the treatment of a variety of tumors, the promise of cancer immunotherapy has begun to be realized. In targeting the maladaptive appropriation of immune checkpoints in the tumor microenvironment and not the cancer directly, these treatments may represent a sea change in the approach to treatment of many cancers. In addition to providing significant response rates in patients with highly pretreated and refractory tumors, the establishment of immunologic memory has generated durable responses in many of these patients. Given the impressive results of CTLA-4 and PD-1 inhibition in cancer patients, other checkpoint pathways operating within the tumor environment demand thorough investigation. Clearly, a challenge for the future will be to determine the most effective integration of A2aR inhibitors in terms of dosing and timing within various combination regimens. Additionally, while this review has mostly focused on the role of A2aR signaling on T cells, it is clear that A2aR blockade will also promote tumor immunotherapy through its effect of NK cells, as well as myeloid derived suppressor cells, and tumor-associated macrophages.

Articles from Computational and Structural Biotechnology Journal are provided here courtesy of Research Network of Computational and Structural Biotechnology
Read more

Cancer Research: Parafibrin / Selenite

 

STEP ONE

Be informed.  Read information below.

STEP TWO

Schedule consultation with R. Douglas Wichman, MD.

STEP THREE

Submit "New Patient Form", current labwork, & imaging results prior to consultation.

STEP FOUR

Establish if you are a candidate for this therapy

in accordance with the Georgia

"Access to Medical Treatment Act".

 

 

An external file that holds a picture, illustration, etc. Object name is fnhum-07-00735-g001.jpg

 

----------------------------------------------------------------------------------------------------------------------------------------------------------

*** SPECIAL NOTE FROM DR. WICHMAN ***

The following excellent article was reproduced from OncoImmunology at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063136/:

 

Oncoimmunology. 2014; 3: e28539.
Published online 2014 Apr 25. doi:  10.4161/onci.28539
PMCID: PMC4063136

Iron-induced parafibrin formation in tumors fosters immune evasion

Boguslaw Lipinski*
Author information ► Article notes ► Copyright and License information ►
 
Go to:

Introduction

Placke et al.1 have recently presented a view that blood platelets impair natural killer (NK) cells ability to recognize cancer cells by forming a physical barrier around the tumor cells. In connection with this it should be remembered that platelet require for their proper functions the presence of fibrinogen, an essential component of blood coagulation cascade.2 Fibrinogen (FBG) is a large molecular weight (340 kDa) multi-chain plasma protein that is converted with thrombin to fibrin monomer(s) (FM), an essential component of the clotting assembly that spontaneously polymerizes to form fibrin clots. However, prior to the formation of solid clots FMs remain soluble by forming complexes with the intact fibrinogen molecules. Due to the increased hydrophobicity of fibrin monomers3 such complexes readily interact with cellular membranes of cells in the blood system. Thus, in addition to inducing platelet aggregation, FM complexes have been shown to cause red blood cell (RBC) agglutination and increased sedimentation,4 as well as cell aggregation of the bacterium Staphylococci.5 Under normal hemostatic conditions fibrin clots are susceptible to degradation by the blood fibrinolytic system that secures proper wound healing and growth of connective tissue at the site of vessel wall injury. This system is activated by the release of tissue plasminogen activator (tPA) resulting in the generation of active plasmin that under normal conditions effectively degrades fibrin into soluble fragments. However, it is not understood why degradation of fibrin in certain types of cancer is incomplete, despite increased tPA production as in the case of prostate cancer.6

Go to:

Mechanism of Parafibrin Formation

We have previously reported that the exposure of plasma proteins to disulfide-reducing agents resulted in the formation of huge insoluble aggregates, which when adhered to tumor cell membranes have been proposed to act as a barrier to tumor recognition by the innate immune system.7 Subsequently, it was further documented that similar aggregates could be induced with another non-enzymatic agent, free iron ions.8 In contrast to thrombin-generated fibers, those formed in the presence of iron ions exhibit dramatically different physicochemical properties. The first step in this conversion involves the interaction of trivalent iron ions (Fe3+) with the hydroxyl groups of water to form highly reactive hydroxyl radicals (HO.) according to the following reaction:

HO + Fe3+ → HO. + Fe2+

Of note, our first observance of this reaction took place without the involvement of hydrogen peroxide required for the classic Fenton reaction. The hydroxyl radicals generated by the interaction of hydroxyl and iron cleaves intra-molecular disulfide bonds in the fibrinogen molecules causing their unfolding with the exposure of hydrophobic groups normally buried inside the tridimensional structure of the polypeptide chains. In the absence of specific chaperons the exposed hydrophobic epitopes form scrambled intermolecular linkages resulting in the formation of fibrin-like fibrils (parafibrin). The most important feature of parafibrin is its hydrophobicity and total resistance to proteolytic degradation. This unusual phenomenon is due to the fact that the hydrophobic forces holding together the polypeptide chains are purely physical and do not involve peptide bonds.9 Consequently, once attached to the surfaces of various cells, parafibrin induces a permanent state of inflammation by eliciting the release of cytokines and proteases from macrophages that impairs their normal functions.

The damaging effect of parafibrin has recently been extended to the pathogenesis of cardiovascular10 and Alzheimer disease.11 These pathological conditions share a common mechanism by which native FBG molecules become hydrophobic by a single event of the conversion from the folded to the unfolded state. This phenomenon is very similar to that occurring when native prion protein PrP becomes converted to its toxic form PrPSc simply by unfolding, without any alteration in its primary structure.12 Interestingly enough, this concept has been met for many years with great skepticism from mainstream protein scientists, until its author was awarded with a Nobel Prize.

Go to:

Iron and Cancer

Accumulating evidence suggests that a correlation exists between increased blood concentration of unbound iron and the incidence of cancer in humans.13,14 Furthermore, iron level reduction may prevent cancer morbidity and mortality.15-17 It should be noted that it is only trivalent iron (Fe3+) and not divalent (Fe2+) which participate in the generation of hydroxyl radicals and subsequent formation of insoluble parafibrin from soluble plasma fibrinogen.8 However, when hemoglobin is released from the hemolyzed erythrocytes, the divalent ferrous ions are enzymatically converted to ferric ions. Thus, any pathologic condition in which erythrocyte membranes are damaged, e.g., in response to infections and/or exposure to environmental toxins, may contribute to the excessive body storage of trivalent iron. It should be noted that this form of iron accumulates with age due to the fact that there is no mechanism for its physiologic elimination, and may therefore underlie prior observations of associations between cancer incidence and aging.

Go to:

Current Issues of Cancer Immunotherapies

It is of extreme importance to remember that the presence of fibrin-like deposits, identified as thrombosis, has been reported for almost a century.18-20 However, no explanations have been offered why such deposits are not being removed by the fibrinolytic enzymes frequently found to be dramatically activated in tumors, particularly in prostate cancer.6 Apparently, there must be some post-translational modifications of fibrinogen/fibrin structure that render the tumor thrombi resistant the proteolytic degradation. The same phenomenon may also be responsible for the disappointing results of chemotherapy for the most types of solid tumors. In the past decade, attempts have being made to develop alternative methods using adaptive and/or adoptive immunotherapies.21 It is of interest to note that while some the adoptive modalities work perfectly well in artificially contrived systems in vitro they lose their efficacies in the physiologically relevant environment in vivo. Obviously the difference is in the exposure of tumor and immune cells to blood and its components as emphasized by Placke et al.1 As mentioned before another important hemostatic factor is plasma fibrinogen that, in the presence of free iron, is converted to parafibrin. We propose that this insoluble polymer forms a protective barrier around tumor cells by means of the hydrophobic interaction, similar to those operating in the phenomena of erythrocyte and bacterial cells aggregations. The role of parafibrin in tumor immune evasion can be compared with that ascribed to the desmoplastic stroma in pancreatic cancer.21It should be born in mind, however, that even the most active NK cells generated ex vivo cannot degrade this barrier because hydrophobic forces holding it together do not represent a substrate for the action of proteolytic enzymes.9

The dual role of parafibrin in the tumor evasion is graphically presented in Figure 1. First, because the antigenic properties of this polymer are almost identical to plasma fibrinogen and/or fibrin, parafibrin is seen by the innate immune system as “self.” Second, and more importantly, even if parafibrin attracts immune cells, it cannot be removed due to its remarkable resistance to degradative proteases normally liberated from activated NK cells.22

figure onci-3-e28539-g1
Figure 1. Conceptual scenarios of the interaction between natural killer cells and cancer cells. Natural killer (NK) cells attack those tumor cells that appear to as “non-self,” but spare the “self” identified cancer ...
Go to:

The Role of Polyphenols and Other Dietary Agents

In view of the known cancer protective effect of Mediterranean diet23,24 it is quite possible that certain polyphenolic substances present in this diet, such as EGCG,25 ferulic acid,26 and curcumin27 may exert their preventive and/or therapeutic effects by helping remove the protective barrier from the tumor membranes. The proposed mechanism of the action of polyphenols is based on their amphiphilic character that allows parafibrin to be displaced in a zipper-like manner (Fig. 2).

figure onci-3-e28539-g2
Figure 2. Displacement of parafibrin protective coat from the cancer cell membrane by an amphiphilic substance. The hydrophobic groups of the amphiphilate form strong complexes with those of cancer cell membrane, thus diplacing the polypeptide ...

This mechanism is similar to that operating in the removal of fat by detergents, in which the hydrophobic groups of a detergent react with the non-polar regions of the fat with the exposure of hydrophilic sites to the aqueous milieu. In addition to these organic substances suggested here to displace parafibrin from the tumor cell surfaces, there are two minerals that may also play an important role in this phenomenon. Thus, magnesium ions contained in green leaves and vegetables may exert their anticancer properties28 by inhibiting the intrinsic blood coagulation and in this way limiting the formation of parafibrin as indicated in the pathogenesis of Alzheimer disease11 The other essential, albeit not generally recognized mineral, is selenium that in a specific chemical form as sodium selenite blocks the protein thiol groups and subsequently inhibit protein unfolding and scrambled refolding.29 Finally, it should be emphasized that the present concept of parafibrin formation and its role in tumor evasion would not be possible without the pioneering work on the protein structure-function relationship elucidated by the great American protein chemist, Chris Anfinsen.30

Go to:

Conclusions

Our perspective is that a singular electron transfer event from the redox iron ion into the hydroxyl group of water can initiate a chain of events that can explain the enigmatic phenomenon of immune evasion. This novel concept offers an additional scenario accounting for improper tumor immunoediting, as well as fostering a defective cellular immune system. However, a philosophical question arises whether such a simple idea can find its place in this day-and-age of the enormous complexity and technical sophistication of the current cancer research.

Articles from Oncoimmunology are provided here courtesy of Taylor & Francis
 
 

Sodium Selenite is a salt version of Selenium, a natural mineral found in the soil. Many people take selenium supplements in response to studies showing evidence of its antioxidant properties and may reduce the risk of cancer. Epidemiological studies have linked low levels of selenium to the risk of cancer. Also, selenium has been used to treat several ailments including asthma, skin conditions, lung cancer, prostate cancer, leukemia, Crohn’s disease, and infertility.

The typical cancer treatment using Selenium uses the inorganic version Sodium Selenite. Under the supervision of a doctor, an IV treatment is given to the patient on a weekly basis or on a schedule decided by the doctor. The doctor will follow up with blood tests to determine the efficacy of the treatment. Also, the doctor may prescribe oral supplements.

Sources and Research:

Longe, J., ed. The Gale Encyclopedia of Alternative Medicine, second edition, 2004.

Selenium: a double-edged sword for defense and offence in cancer

Read more

Hyperbaric Oxygen Therapy

 

Hyperbaric Oxygen Therapy (HBOT) helps all kinds of people. It’s used to help patients suffering from sports injuries, Chronic Fatigue Syndrome, infections, arthritis and a huge range of other medical conditions. As we all know, oxygen is vital for life, and life cannot exist without it. It follows that oxygen is essential for effective healing and recovery.

read more

Book Your Appointment NOW

Call (770) 232-7883

 

Click to E-Mail Us Now!

 

Our Address:

4488 N. Shallowford Road, Suite 201

Dunwoody, Georgia 30338

 

We Serve the Following Areas:

Atlanta, Georgia, Acworth, Alpharetta, Berkeley Lake, Braselton, Brookhaven, Buckhead, Buford, Canton, Chamblee, Conyers, Cumming, Dacula, Decatur, Doraville, Duluth, Dunwoody, Georgia, Flowery Branch, Gainesville, Grayson, Hoschton, Johns Creek, Kennesaw, Lawrenceville, Lilburn, Lithonia, Loganville, Marietta, Milton, Norcross, Roswell, Sandy Springs, Smyrna, Snellville, Stone Mountain, Sugar Hill, Suwanee, Tucker, Vinings, Woodstock, Georgia