‘To what extent is the combined use of ipilimumab and nivolumab in cancer treatment viable?'

Introduction

Immunotherapy is ‘an innovative treatment approach that empowers the human immune system to overcome cancer and other debilitating diseases’ (Fred Hutchinson Cancer Research Center, 2015). Ipilimumab and nivolumab are types of monoclonal antibodies, known by their brand names of Yervoy and Opdivo respectively. The US Food and Drug Administration (FDA) approved Yervoy on 25th March 2011 (Yervoy (ipilimumab) FDA Approval History, 2011) for treatment of late-stage melanoma. The FDA approved Opdivo for advanced melanoma on 22nd December 2014, and then on the 4th March 2015, approved use was expanded for treatment of lung cancer (Opdivo (nivolumab) FDA Approval History, 2015). It wasn’t until 22nd August 2011 (NHS Choices, 2011), that Yervoy was licensed for late-stage melanoma in Europe by the European Commission, based on the advice of the European Medicines Agency (EMA); Opdivo was licensed on 19th June 2015 for melanoma patients (Bristol-Myers Squibb Newsroom, 2015). The global biopharmaceutical company Bristol-Myers Squibb manufactures both of these immunotherapy drugs.

Brief History of Cancer Treatment

The ancient Egyptian trauma surgery textbook, Edwin Smith Papyrus, dated to around 3000 BC, holds the earliest description of cancer. Many cases of breast tumours or ulcers removed by cauterization are noted, and the lack of treatment for this disease is highlighted. (American Cancer Society, 2014)

Even then it was known that after being surgically removed, the cancer was likely to return. Ancient physicians and surgeons found no curative treatment once cancer had spread, believing that intervention, in the form of early and unsophisticated surgery, may result in more harm, such as blood loss. However, there were major advances in general and cancer surgery in the 19th and early 20th century, as well as the availability of anaesthesia in 1846. William Stewart Halsted, professor of surgery at Johns Hopkins University, developed the radical mastectomy in the late 19th century, which then became the basis of cancer surgery for almost a century. However, clinical trials in the 1970s revealed that less extensive surgery could be equally effective. The limitations of surgery were revealed as the understanding of metastasis improved, allowing more refined treatments that removed minimal amounts of normal tissue to be developed towards the end of the 20th Century. This depended on improved oncology, surgical instruments and combining surgery with other treatments such as chemotherapy and radiation. Now, modern surgery includes the use of new methods such as fibre-optic technology, cryosurgery, laparoscopic surgery and radiofrequency ablation. (ibid)

Hormone therapy was discovered in the 19th Century after Thomas Beatson discovered a relationship between the ovaries and formation of milk in the breasts of rabbits. In 1878 he removed the ovaries of rabbits and found that the production of milk was stopped. Oophorectomy was then tested on advanced breast cancer patients, and often resulted in improvement. The discovery of the stimulating effect of oestrogen on breast cancer provided a foundation for hormone therapy, and has guided research into how hormones affect the growth of cancer, in the hope of developing new drugs. (ibid)

1896 saw Roentgen presenting his new ‘X-ray’, winning the first ever Nobel Prize in physics in 1901. Radiation therapy began shortly after, and in France, it was discovered that daily doses of radiation over several weeks could improve a patient’s condition and their overall chance for a cure. However, at the start of the 20th century it was discovered that it could also be the cause of cancer; although advances in radiation physics and computer technology over the remainder of this century made it possible to aim radiation more precisely. This can be seen in therapies such as conformal radiation therapy (CRT), intensity-modulated radiation therapy (IMRT) and intraoperative radiation therapy (IORT). (ibid)

The discovery of a compound called nitrogen mustard during World War II, which was found to be effective against lymphoma, started the era of chemotherapy. This compound acted as a model and triggered development of increasingly more effective alkylating agents that damage the DNA of rapidly growing cancerous cells, destroying them. Today, chemotherapy is improved and used in several ways, including new agents and delivery techniques, such as monoclonal antibody therapy and liposomal therapy; improved ability to overcome multi-drug resistance; and drugs that reduce side effects, such as anti-emetics. A major discovery was the use of combination chemotherapy, instead of single agents. “Early in the 20th century, only cancers small and localized enough to be completely removed by surgery were curable. Later, radiation was used after surgery to control small tumor growths that were not surgically removed. Finally, chemotherapy was added to destroy small tumor growths that had spread beyond the reach of the surgeon and radiotherapist. Chemotherapy used after surgery to destroy any remaining cancer cells in the body is called ‘adjuvant therapy’.” (ibid)

Targeted therapies influence the processes controlling growth, division and spread, as well as impacting the signals that cause natural death, of cancerous cells. These work in three main ways: growth signal inhibitors, recognized in the 1960s; anti-angiogenesis agents, the concept of which surfaced in the 1970s; and apoptosis-inducing drugs. (ibid)

Immunotherapy, the treatment category ipilimumab and nivolumab fall into, works by mimicking natural signals used in the body to control cell growth, in other words, they imitate or influence the immune response. This can be done directly, affecting cancerous cell growth, or indirectly, aiding healthy cells in controlling the cancer. “One of the most exciting applications of biologic therapy has come from identifying certain tumor targets, called antigens, and aiming an antibody at these targets. This method was first used to find tumors and diagnose cancer and more recently has been used to destroy cancer cells. Using technology that was first developed during the 1970s, scientists can mass-produce monoclonal antibodies that are specifically targeted to chemical components of cancer cells. Refinements to these methods, using recombinant DNA technology, have improved the effectiveness and decreased the side effects of these treatments” (ibid). In the late 1990s, rituximab (Rituxan) and trastuzumab (Herceptin) were approved as the first therapeutic monoclonal antibodies, used to treat lymphoma and breast cancer. Now, monoclonal antibodies are often used in the treatment of specific cancers, and are at the forefront of cancer research. (ibid)

Immunoglobulins

In general, antibodies have a symmetrical structure composed of a pair of identical glycosylated heavy chains, and a pair of identical nonglycosylated light chains. Disulfide bonds link these heavy chains, and the light chains are connected by a disulfide bond to one heavy chain. This creates a basic subunit of two of each chain in a Y-shaped structure. Immunoglobulins are proteins that have this general structure, without having known antigen-binding properties. There are five different classes of immunoglobulins, each with their own distinctive structural and biological features: IgM, IgD, IgG, IgE and IgA. The immunoglobulin’s class depends on its heavy chain: M, D, G, E and A. (Goding, 1996)

Antibody genes exist in three groups: κ light chains, λ light chains, and heavy chains. In an organism each group of genes lie on specific chromosomes, for example, in a mouse the κ group can be found on chromosome 6, the λ genes on chromosome 16, and the heavy chain group on chromosome 12. The genes for the variable region correlate to the genes of the constant region. “The fact that the antibody gene rearrangements are orderly and monitored by the cell for productive expression ensures that the great majority of cells express a single allelic form of the heavy chain and a single allelic form of a single light chain type.” (ibid)

IgG, is the main immunoglobulin found in human blood, the second most abundant circulating protein and contains long-term protective antibodies against numerous infectious agents. There are four different types of IgG, again split into classes, in order of decreasing abundance: IgG1, IgG2, IgG3, IgG4. Each sub-class has a slightly different function in terms of the immune response, due to differences in their constant region (Immune Deficiency Foundation, 2013); specifically their hinges and upper CH2 domains which are involved in binding to IgG-Fc receptors and C1q, resulting in the sub-classes’ varied effector functions (Vidarsson, Dekkers, and Rispens, 2014). For example, IgG1 and IgG3 subclasses are rich in antibodies against proteins such as the toxins produced by the diphtheria and tetanus bacteria, as well as antibodies against viral proteins. In contrast, IgG2 antibodies are predominantly against the polysaccharide capsule of certain disease-producing bacteria (such as, Streptococcus pneumoniae and Haemophilus influenzae) (ibid). “IgG molecules are able to react with Fcγ receptors that are present on the surfaces of macrophages, neutrophils, natural killer cells, and can activate the complement system. The binding of the Fc portion of IgG to the receptor present on a phagocyte is a critical step in the opsonizing property IgG provides to the immune response. Phagocytosis of particles coated with IgG antibodies is a vital mechanism to cope with microorganisms. IgG is produced in a delayed response to an infection and can be retained in the body for a long time. The longevity in serum makes IgG most useful for passive immunization by transfer of this antibody.” (Immunoglobulin IgG Class, 2014)

Ipilimumab is a fully human anti-CTLA-4 monoclonal antibody (IgG1κ) produced in Chinese hamster ovary cells by recombinant DNA technology (YERVOY 5 mg/ml concentrate for solution for infusion, 2015). “IgG1 comprises 60-65% of the total main subclass IgG, and is predominantly responsible for the thymus mediated immune response against proteins and polypeptide antigens. IgG1 binds to the Fc-receptor of phagocytic cells and can activate the complement cascade via binding to C1 complex. IgG1 immune response can already be measured in new borns and reaches its typical concentration in infancy. A deficiency in IgG1 isotype typically is a sign of a Hypogammaglobulinemia” (Immunoglobulin IgG Class, 2014). CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) is a protein receptor that, functioning as an immune checkpoint, downregulates the immune system. CTLA4 is a member of the immunoglobulin superfamily, found on the surface of Helper T cells, and acts

as an "off" switch when bound to CD80 or CD86 on the surface of antigen presenting cells by transmitting an inhibitory signal to T cells. (Wikipedia: CTLA-4, 2015)

Nivolumab is a human immunoglobulin G4 (IgG4) monoclonal antibody (HuMAb), again produced in Chinese hamster ovary cells by recombinant DNA technology (Nivolumab BMS 10 mg/mL concentrate for solution for infusion, 2015). “Comprising usually less than 4% of total IgG, IgG4 does not bind to polysaccharides. Testing for IgG4 has been associated with food allergies in the past and recent studies have shown that elevated serum levels of IgG4 are found in patients suffering from sclerosing pancreatitis, cholangitis and interstitial pneumonia caused by infiltrating IgG4 positive plasma cells. The precise role of IgG4 is still mostly unknown” (Immunoglobulin IgG Class, 2014). However, correlation between a relief of symptoms and IgG4 induction in immunotherapy appears to be present. (Vidarsson, Dekkers, and Rispens, 2014).

Antibody Mechanisms

There are a number of mechanisms by which antibodies may act; which mainly result in stimulating and engaging other components of the immune system. They can simply block the interactions of molecules, or be involved in more complex mechanisms, such as activating the classical complement pathway (complement dependent cytotoxicity, CDC) by interaction of C1q on the C1 complex with clustered antibodies. Antibodies may also act as a link between antibody-mediated and cell-mediated immune response through engagement of Fc receptors. (Antibody Effector functions, 2015)

Fc receptors (FcRs) are key immune regulatory receptors, connecting the humoral immune response to cellular effector functions. Immunoglobulins have specific receptors, depending on class: FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM) and FcδR (IgD). Human IgG has three classes of receptor found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb and FcγRIIc) and CD16 (FcγRIIIa and FcγRIIIb). FcγR1 is classes as a high affinity receptor, nanomolar range KD, whereas the remaining receptors are low to intermediate affinity, micromolar range KD. In antibody dependent cellular cytotoxicity (ADCC), FcvRs on the surface of effector cells (including natural killer cells, macrophages, monocytes and eosinophils) are able to bind to the Fc region of an IgG, which is bound to a target cell; a signaling pathway is then triggered, resulting in the secretion of various substances, such as lytic enzymes, perforin and tumour necrosis factor, which are involved in destroying the target cell. The level of ADCC effector function varies for human IgG subtypes. This is dependent on the allotype and specific FcvR, although the ADCC effector functions tend to be high for IgG1 and IgG3, and low for IgG2 and IgG4. FcγRs bind to IgG asymmetrically across the hinge and upper CH2 region; knowledge of this has lead to engineering efforts to manipulate IgG effector functions. (ibid)

“The ability of antibodies to bind an almost unlimited number of target proteins with high specificity always meant they were destined to be used as therapeutics. As early as 1900 Paul Ehrlich coined the term ‘magic bullets’ in reference to antibodies.” (Antibodies as Tools, 2015)

This includes increasing effector functions through Fc engineering. Therapeutic antibodies are mainly used in oncology, with over 200 antibodies passing through clinical testing. A key mechanism of action for these antibodies is the targeted killing of cancerous cells through encouragement and recruitment of the immune system, achieved through interactions of the

Fc domain with the complement component C1q or Fcγ receptors. Many therapeutic antibodies resulted in unsuccessful clinical trials due to insufficient efficacy. In response to this, efforts have been made to increase the potency of antibodies through enhancement of their ability to mediate cellular cytotoxicity functions such as ADCC. There has also been a specific focus on increasing the affinity of the Fc domain for the low affinity receptor FcγIIIa. A number of mutations within the Fc region have been identified which enhance binding of Fc receptors, either directly or indirectly, and significantly intensifying cellular cytotoxicity. Focusing on the glycosylation of the Fc domain is an alternative approach; Fcγrs interact with carbohydrates on the CH2 region, and the composition of these effects effector function activity. An example of this includes afucosylated antibodies, which exhibit substantially enhanced ADCC activity through increased binding to FcγRIIIa. (Fc Engineering, 2015)

In contrast, Fc engineering can lead to decreasing effector functions. Circumstances exist in which an antibody unable to activate specific effector functions is preferred. Usually IgG4 has often been used in these circumstances, but has fallen out of favour in recent years due to its unique ability to undergo Fab-arm exchange, where heavy chains may be swapped between IgG4 in vivo. Engineering approaches have determined key interaction sites for the Fc domain with Fcγ receptors and C1q, before mutating these positions to decrease or prevent binding. For example, through alanine scanning Duncan and Winter first isolated the site covering the hinge and upper CH2 of the Fc domain, at which C1q binds. Researchers at Genmab then identified mutants K322A, L234A and L235a, which are sufficient to almost completely abolish FcγR and C1q binding, when used in combination. Modification of the glycosylation on asparagine 297 of the Fc domain, known to be required for optimal FcR interaction, can lead to a decrease in binding; as well as in enzymatically deglycosylated Fc domains, recombinantly expressed antibodies in the presence of a glycosylation inhibitor and the expression of Fc domains in bacteria. (ibid)

Futhermore, Fc engineering can enhance the serum half-life of IgG, which naturally persists for an extended period in the serum due to FcRn-mediated recycling, resulting in a typical half-life of approximately 21 days. There have been multiple efforts to engineer the pH dependent interactions of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. PDL BioPharma researchers identified the mutations T250Q/M428L, resulting in an approximate 2-fold increase in IgG half-life in rhesus monkeys. These enhancements are yet to be shown in humans, but significantly increased half-lives may lead to a decrease in administration frequency, whilst maintaining or improving efficacy. (ibid)

Mechanism of CTLA-4 and Ipilimumab

On the surface of T-cells, two proteins, CD28 and CTLA-4, play key roles in regulating immune activation and tolerance. CD28 provides positive modulatory signals during the early stages of an immune response; meanwhile, signals from CTLA-4 inhibit the activation of T-cells, particularly during strong T-cell responses. “CTLA-4 blockade using anti – CTLA-4 monoclonal antibody therapy has great appeal because suppression of inhibitory signals results in the generation of an antitumour T-cell response” (Wolchok and Saenger, 2008)

A series of complex interactions is involved in the normal functioning of the immune system. Tumours express antigens that can be recognized by the immune system, however, antigen presentation alone is not sufficient to trigger an effective immune response to any pathological entity, including cancer. T-cell activation is modulated by stimulatory signals

and inhibitory signals; CD28 provides positive signals whereas CTLA-4 provides negative signals in the early stages, which work together to coordinate a response to a threat. CD28 initiates and maintains a T-cell response, partly through increase cytokine expressions mediated by interaction with CD80 (B7-1) and CD86 (B7-2), its primary ligands, on the surface of the antigen-presenting cell (APC). CTLA-4 essentially halts T-cell activation by triggering an inhibitory signal. If CTLA-4 were to be inhibited, the immune system balance would shift in favour of T cell activation, resulting in rejection of tumours by the host. (ibid)

Interactions between APCs and antitumour T-cells is key to developing antitumour T-cell immunity, which is modulated through the influences of the competing stimulatory and inhibitory molecules. The first signal in T cell activation is provided by the binding of the T cell receptor (TCR) to its cognate antigen, however, a second stimulating signal is required for T-cell proliferation. This signal is provided by CD28. CTLA-4 and CD28 are homologous, and both found on the surface of T-cells, where they compete to bind to B7 costimulatory molecules on APCs. CTLA-4 has a significantly higher affinity for binding to these molecules, giving CTLA-4 a competitive advantage over CD28. Not only are the roles of these two proteins essential for the functioning of the immune system, but also determine the fate of T-cells: activation or anergy. Preclinical studies have shown that CTLA-4 is necessary to the downregulation of autoreactive and potentially destruction peripheral T-cell responses; blockade of CD28 inhibits antitumour immunity whereas blockade of CTLA-4 stimulates antitumour immunity; and CD28 stimulates the production of cytokines, such as interleukin-2 (IL-2) and upregulates antapoptotic genes, contrasting to the binding of CTLA-4 to B7 molecules which results in inhibition of IL-2. It has been proposed that CTLA-4 not only limits the body’s response to autoantigens, but also helps diversify the T-cell population, meaning that during an immune response, T-cells specific to one epitope would not necessarily dominate; therefore facilitating the targeting and destruction of pathogens. (ibid)

So how can T-cells proliferate and the immune system function at all when the affinity of CTLA-4 for the B7 family of ligands is significantly greater than that of CD28, especially noting that CTLA-4 is capable of forming a lattice of extensive and intricate protein networks, effectively excluding CD28 and B7 ligands from interacting? There are several details that allow CD28 an advantage over CTLA-4. For example, CD28 is expressed on the surface of naïve and activated T-cells, and is present in 90% of CD4+ and 50% of CD8+ T-cells. In contrast, CTLA-4 expression is only induced by the activation of T-cells and its upregulation reaches a maximum 2-3 days after the start of a response. Furthermore, CD28 localises to the T-cells plasma membrane, evenly distributed and intimately involved in any T-cell and APC interactions, whereas CTLA-4 is located in the endosomal compartment, where surface expression of this protein is highly restricted, which could be a regulatory point for controlling its inhibitory influence. (ibid)

CTLA-4’s ability to inhibit the activation of any T-cell depends on numerous factors, including the strength of the T-cell receptor (TCR) signal and the activation state of the APC. CTLA-4 signals through an immunoreceptor tyrosine-based inhibitory motif to inhibit CD4+ and CD8+ T-cell responses. Research suggests that the localization of CTLA-4 to the immunological synapse is preferred to conditions of stronger TCR signaling. Therefore CTLA-4 is more likely to inhibit strong T-cell responses; this has significant implications for the roles of CD28 and CTLA-4 in the coordination and regulation the T cell response to antigens. “The preferential restriction of cells bearing higher affinity TCRs for any given antigen may allow for greater representation of cells bearing lower affinity TCRs and thus diversify the T-cell response to a threat. This diversified population of T cells may have

greater crossreactivity to similar antigenic epitopes and may be important in the development of a protective T-cell response.” (ibid)

Data from preclinical and clinical trials show that anti-CTLA-4 monoclonal antibody therapy results in direct activation of CD4+ and CD8+ effector cells; it does not considerably affect the suppressive capacitiy of regulatory T-cells, meaning that CTLA-4 blockade does not inhibit CD4+ nor CD25+ cells with the enhancement of effector T-cell activity secondary to reduction in regulation. However, it does result in an altered ratio of effector cells to regulatory cells within the tumour, with an increase in both CD4+ and CD8+ effector cells, in mice. (ibid)

“Evidence from numerous studies indicates that CTLA-4 provides a braking mechanism on T-cell activation and serves a critical role in immune response. CTLA-4 competes for the B7 family of ligands with CD28, a key costimulatory molecule that is essential for the effective activation of T-cell–mediated immunity. CTLA-4 blockade results in enhanced antitumor immunity, most likely through the direct activation of T cells. Anti–CTLA-4 monoclonal antibody therapy, either as a monotherapy or in combination with a vaccine, may potentially allow for a more specific immune response against tumor targets.” (ibid) Ipilimumab is a CTLA-4 immune checkpoint inhibitor which blocks T-cell inhibitory signals induced by the CTLA-4 pathway. This results in an increase in the population of reactive T-effector cells, which mobilize to mount a direct T-cell immune attack against tumour cells. CTLA-4 blockade may also reduce the function of T-regulatory cells, contributing to an anti-tumour response. Ipilimumab is capable of selectively depleting T-regulatory at the site of the tumour, to increase the intratumoural T-effector/T-regulatory cell ratio, driving tumour cell death. (YERVOY 5 mg/ml concentrate for solution for infusion, 2015)

Mechanism of PD-1 and Nivolumab

Evaluation of past immunotherapeutic approaches to treating cancer found limited success. Increased understanding of the checkpoint signaling pathway involving the programmed death 1 (PD-1) receptor and its ligands: PD-L1 (B7-H1) and PD-L2 (B7-DC), has clarified the role of these approaches in tumour-induced immune suppression; this has led to critical advancement in the development of immunotherapeutic drugs. (Dolan and Gupta, 2014)

The interaction between PD-1 and its ligand PD-L1/2, is a key pathway that is hijacked by tumours in order to restrict immune control. Reversing the inhibition of adaptive immunity can actively stimulate the immune system, utilizing antagonistic antibodies to block checkpoint pathways, releasing tumour inhibition. These antibodies target CTLA-4, the PD-1 receptor and PD-L1 and facilitate antitumour activity. These agents are unique due to the characteristic of targeting lymphocyte receptors or their ligands. (ibid)

PD-1 is an immunoinhibitory receptor, belonging to the CD28 family, and is expressed on T-cells, B-cells, monocytes, natural killer cells and many tumour-infiltrating lymphocytes (TILs). PD-L1 is expressed on many cells including resting T-cells, B-cells and macrophages, whereas PD-L2 expression is only found on macrophages and dendritic cells alone. Some tumours may have a higher expression of PD-L1. These ligands inhibit T-cell proliferation, cytokine production and cell adhesion. PD-L2 controls T-cell activation in lymphoid organs; PD-L1 seems to decrease T-cell function in peripheral tissues. The induction of PD-1 on activated T-cells takes place in response to the engagement of PD-L1/2, which limits effector

T-cell activity in peripheral organs and tissues during inflammation, preventing autoimmunity; this is crucial in preventing tissue damage when the immune system is responding to infection. An antitumour immune response may be triggered if this pathway is blocked. The PD-1 pathway is similar to CTLA-4, in downregulating the response of T-cells by overlapping signaling proteins that form part of the immune checkpoint pathway; they function differently however, CTLA-4 concentrates on regulating the activation of T-cells, whereas PD-1 regulates effector t-cell activity in peripheral tissues in response to infection or the progression of a tumour. High levels of both of these molecules are expressed on regulatory T-cells, which have shown to have immune inhibitory activity, essential for self-tolerance. “The role of the PD-1 pathway in the interaction of tumour cells with the host immune response and the PD-L1 tumour cell expression may provide the basis for enhancing immune response through a blockade of this pathway. Drugs targeting the PD-1 pathway may provide antitumor immunity, especially in PD-L1 positive tumours.”(ibid). Nivolumab, a monoclonal antibody that binds to the PD-1 receptor, potentiates T-cell and antitumour responses, via blockade of PD-1 binding to its ligands. This resulted in decreased tumour growth in a study on mice. (Nivolumab BMS 10 mg/mL concentrate for solution for infusion, 2015)

Potential for the Combination of these Drugs

Preclinical evidence suggests that the roles of CTLA-4 and PD-1 in the regulation of adaptive immunity are complementary, providing rationale for combing drugs that will target these pathways. New data reveals that cytotoxic agents are able to antagonize immunosuppression in the microenvironment of the tumour, promoting immunity based on the concept that tumour cells die in multiple ways, and that some types of apoptosis can result in an enhanced immune response. “For example, nivolumab was combined with ipilimumab in a phase 1 trial of patients with advanced melanoma. The combination had a manageable safety profile and produced clinical activity in the majority of patients, with rapid and deep tumour regression seen in a large proportion of patients. Based on the results of this study, a phase 3 study is being under- taken to evaluate whether this combination is better than nivolumab alone in melanoma.” (Dolan and Gupta, 2014).

Clinical Trials

Since its development, many clinical trials have been designed to compare the use of anti-CTLA-4 monotherapy, ipilimumab, with the combination therapy of ipilimumab and nivolumab. Metastatic melanoma, metastatic renal cell carcinoma (MRCC) and small cell lung cancer (SCLC) have been tested with the use of this immunotherapeutic approach.

Melanoma is a prototype of immunogenic tumour that has been known to respond to immunotherapeutic approaches with interferon alfa and interleukin 2 (ibid). This has featured in many clinical trials.A double-blind study that involved 142 metastatic melanoma patients who had not previously received treatment, was used to compare ipilimumab with ipilimumab and nivolumab. Patients were assigned in a 2:1 ratio to receive ipilimumab (3 mg per kilogram of body weight) combined with either nivolumab (1 mg per kilogram) or placebo, once every 3 weeks for four doses. This was then followed by nivolumab (3 mg per kilogram) or placebo every fortnight until the disease progressed or unacceptable toxic effects occurred. The primary end point was the rate of investigator-assessed, confirmed objective response among patients with BRAF V600 wild-type tumours. Among patients with BRAF wild-type tumours, the rate of

conformed objective response was 61% (44 of 72 patients) in the combination group versus 11% (4 of 37 patients) in the ipilimumab-monotherapy group (P<0.001). 16 patients (22%) of the combination group reported complete responses versus none in the monotherapy group. The median duration of response was not reached in either group and the median progression-free survival was not reached with the combination therapy, but was 4.4 months with monotherapy (hazard ratio associated with combination therapy compared with monotherapy for disease progression or death, 0.40; 95% confidence interval [CI], 0.23 to 0.68; P<0.001). The response rate and progression-free survival results were similar for the 33 patients with BRAF mutation-positive tumours. The objective-response rate and progression-free survival among advanced melanoma patients were significantly greater with ipilimumab combined with nivolumab, rather than ipilimumab monotherapy. The combination therapy had an acceptable safety profile. (Postow et al., 2015)

In a similar study, a 1:1:1 ratio was assigned to 945 previously untreated patients with unrespectable stage III or IV melanoma to nivolumab alone, nivolumab plus ipilimumab, or ipilimumab alone. Progression-free survival and overall survival were co-primary end points. The median progression-free survival was 11.5 months (95% CI, 8.9 to 16.7) with combination therapy, compared with 2.9 months (95% CI, 2.8 to 3.4) with ipilimumab monotherapy (hazard ratio for death or disease progression, 0.42; 99.5% CI, 0.31 to 0.57; P<0.001), and 6.9 months (95% CI, 4.3 to 9.5) with nivolumab monotherapy (hazard ratio for the comparison with ipilimumab, 0.57; 99.5% CI, 0.43 to 0.76; P<0.001). In patients with PD-L1 positive tumours, the median progression-free survival was 14.0 months in the combined therapy group and in the nivolumab group; in patients with PD-L1 negative tumours, progression-free survival was longer with combination therapy than with nivolumab monotherapy (11.2 months [95% CI, 8.0 to not reached] versus 5.3 months [95% CI, 2.8 to 7.1]). Among patients who had not previously been treated for metastatic melanoma, nivolumab monotherapy or combination therapy resulted in significantly longer progression-free survival than ipilimumab monotherapy. Patients with PD-L1 negative tumours responded more effectively to combination therapy of PD-1 and CTLA-4 blockade than either agent alone (Larkin et al., 2015). Dr James Larkin, a consultant at the Royal Marsden hospital and one of the UK’s lead investigators, told the BBC: “For immunotherapies, we’ve never seen tumour shrinkage rates over 50% so that’s very significant to see. This is a treatment modality that I think is going to have a big future for the treatment of cancer.” (The Guardian, 2015)

Similar to melanoma, kidney cancer has also been a prototype of immunogenic tumour that responds to immunotherapy (Dolan and Gupta, 2014).

A Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (MRCC) took place where patients were randomly split into two groups. In one group of 21 patients, they received 3mg/kg of nivolumab and 1mg/kg of ipilimumab (arm N3 +I1); in the other of 23 patients, they received 1mg/kg of nivolumab and 3mg/kg (arm N1+I3). This was administered intravenously every three weeks for four doses, and then followed by 3mg/kg of nivolumab every fortnight until progression (protocol-defined that post-progression treatment was allowed). The primary objective was to assess safety; the secondary objective was to assess efficacy. 80% of patients (35 in total, 17 in N3+I1, 18 in N1+I3) had prior systemic therapy. Objective response rate was 43% (N3+I1) and 48% (N1+I3). The median duration of response was 31.1 weeks (7 ongoing) in N3+I1 and not reached (9 ongoing) in N1+13. Responses occurred by the first tumour assessment (week 6) in 44% of patient in the N3+I1 arm and in 55% of patients in the N1+I3 arm. Stable disease

as the best overall response was seen in 5 (24%; N3+I1) and 8 (35%; N1+I3) patients. Nivolumab and ipilimumab showed an acceptable safety profile and encouraging antitumour activity in MRCC, with most responses ongoing. Studies are ongoing to explore this combination in a Phase III trial. (Hammers et al., 2014)

There is also an ongoing trial comparing the combination of nivolumab and ipilimumab with sunitinib (another biological therapy, also known as Sutent). The aims of this trial are to see the efficiacy of nivolumab and ipilimumab in renal cell cancer and to see any possible side effects. (A trial of nivolumab combined with ipilimumab for kidney cancer (CA209214), 2015)

A Phase I/II study of nivolumab with or without ipilimumab for the treatment of recurrent small cell lung cancer (SCLC) hoped to see improvement in patients who had responded well to initial platinum (PLT) based chemotherapy (CT), but the disease had rapidly progressed afterwards. Combined blockade of PD-1 and CTLA-4 immune checkpoint pathways was known to have a manageable safety profile with anti-tumour activity. Patients who were PLT sensitive or refractory and had progressive disease were enrolled regardless of tumour PD-L1 status or the number of prior CT regimens. Patients were randomised to a group that administered 3mg/kg of nivolumab intravenously every fortnight, or to other groups that involved administration of 1mg/kg of nivolumab and 1mg/kg of ipilimumab, 1mg/kg of nivolumab and 3mg/kg of ipilimumab or 3mg/kg of nivolumab, intravenously every 3 weeks. These treatments lasted for four cycles and were followed by 3 mg/kg of nivolumab every fortnight. The primary objective was the overall response rate. Other objective included safety, progression-free survival, observational study and biomarker analysis. 75 patients were enrolled (40 into the nivolumab monotherapy group, 35 into the combination therapy groups) of which 59% had less than or equal to 2 prior regimens. Of the 40 evaluable monotherapy patients, partial responses was seen in 6 (15%), the duration of ongoing responses was 80-251+ days; stable disease occurred in 9 (22.5%); and progressive disease in 25 (62.5%). In the 20 evaluable combined therapy patients, 1 had a complete response (5%), the duration of response was 322+ days; 4 had a partial response (20%), duration of response was 41-83+ days); 6 experienced stable disease (30%); and 9 had progressive disease (45%). In the combined group, 12 patients had not reached the first tumour assessment and 3 were not evaluable. 9 patients (23%) have continued treatment with nivolumab monotherapy and 19 (54%) have continued the combined therapy. In this PD-L1 unselected SCLC population with progression post-PLT, nivolumab alone or combined with ipilimumab was tolerable. Overall response rate was 15% (nivolumab) and 25% (nivolumab and ipilimumab) for evaluable patients; durable responses were noted. (Antonia et al., 2015)

An ongoing trial is taking place of nivolumab and ipilimumab for people with solid tumours that have spread and have most recently recruited patients with SCLC. The aims of this trial are to compare nivolumab monotherapy with combined therapy and to test the safety of this treatment. (A trial of nivolumab and ipilimumab for people with solid tumours that have spread (CA209032), 2015)

Adverse Reactions

Perhaps one of the most important disadvantages of any medical treatment is the side effects. As individual drugs, both ipilimumab and nivolumab have many adverse reactions.

Ipilimumab has some mild to moderate effects such as moderate diarrhoea or colitis, adverse reactions in the endocrine glands such as hypophysitis, and unexplained motor neuropathy or muscle weakness. More severe or life-threatening effects include gastrointestinal haemorrhage, symptoms of hepatotoxicity, severe motor neuropathy, pancreatitis and toxic epidermal necrolysis. In a clinical trial of around 10,000 patients to evaluate its use with various doses and types of tumours, therapy was discontinued for adverse reactions in 10% of patients (YERVOY 5 mg/ml concentrate for solution for infusion, 2015).

Immunotherapy with ipilimumab is associated with inflammatory adverse reactions resulting from increased immune activity (known as immune-related adverse reactions), which is likely to be related to its mechanism of action. Most of these surface during the induction period of therapy, although some reactions appear months after the final dose. Life-threatening complications can be minimised with early diagnosis and appropriate management. Systemic high-dose coriticosteroid may be required for managing severe immune-related adverse reactions, although use at baseline, before starting ipilimumab, should be avoided due to the risk of potential interference with pharmacodynamic activity and efficacy; it does not appear to affect efficacy after treatment has commenced (ibid).

As a response to any adverse reactions, there are four main levels of action. First, the dose of ipilimumab is withheld until the reaction resolves to Grade 1, 0 or returns to baseline; then, if resolution occurs, therapy is resumed. However, in response to no resolution, doses continue to be withheld until reaction resolves before continuing therapy. Discontinued use of ipilimumab is implemented if a resolution to Grade 1, 0 or baseline is not reached (ibid).

In clinical trials of patients with unresectable or metastatic melanoma, the most common adverse reactions associated with ipilimumab 3 mg/kg in 5% or more patients were fatigue, diarrhoea, pruritus, rash and colitis. 11 of 1,024 evaluable patients tested positive for binding antibodies in an electrochemiluminescent (ECL)-based assay. However, infusion-related or peri-infusional reactions that correspond with hypersensitivity or anaphylaxis were not reported in these patients; neutralising antibodies against ipilimumab were not detected. (Fellner, 2012)

Ipilimumab is not recommended for use during pregnancy, because there is no data and the potential risk of treatment to the developing foetus is currently unknown. However, animal reproductions studies have shown reproductive toxicity and it is known that human IgG1 crosses the placental barrier. It should only be considered if the clinical benefit outweighs the risk (YERVOY 5 mg/ml concentrate for solution for infusion, 2015). Ipilimumab has been shown to be present at low levels of concentration in milk from cynomolgus monkeys that were treated during pregnancy. It is not known if it is secreted in human milk, although secretion of IgGs is generally limited in breast milk. No effects are anticipated for systemically breastfed infants, although due to the potential risk, a decision must be made as to whether to continue or discontinue breastfeeding or Yervoy treatment, balancing the benefits of breastfeeding for the child and of the therapy for the woman. Studies evaluating the effect on fertility have not been carried out and so are currently unknown (ibid).

Yervoy has a minor influence on the ability to operate machinery and to drive due to potential side effects such as fatigue. Therefore, patients should be advised to use caution when carrying out these activities, until they are sure that ipilimumab does not adversely impact them (ibid).

Nivolumab is also associated with immune-related adverse reactions, and patients should be carefully monitored at least up to 5 months after the last dose. If any severe or life-threatening adverse reactions occur, use of Opdivo must be permanently discontinued. The most frequent adverse reactions in two studies of squamous non-small cell lung cancer (NSCLC) reported in more than 10% of patients, included fatigue, decreased appetite and nausea. The majority of these reactions were mild to moderate (Nivolumab BMS 10 mg/mL concentrate for solution for infusion, 2015). Many of the side effects experienced are similar to those seen in ipilimumab.

In the event of moderate to severe immune-related adverse reactions, nivolumab should be withheld and the administration of corticosteroids should commence. A taper of at least one month must be respected upon improvement before recommencing therapy, if not reactions may worsen rapidly. If no improvement occurs, non-corticosteroid immunosuppressive therapy should be used. Opdivo should not be resumed while the patient is receiving either of these alternatives (ibid).

Similar to ipilimumab, little or no data is available regarding use during pregnancy, breast-feeding and effect on fertility. Therefore risk to infants cannot be excluded and nivolumab should only be used in exceptional circumstances where the benefit outweighs the risk. Additionally, effective contraception should be used for at least 5 months after the last dose, to ensure the risk is minimised (ibid).

Operating machinery and driving should also be treated in the same way as ipilimumab (ibid).

Some adverse effects regarding the combined therapy of these drugs have been seen in the results of clinical trials. A comparison of combined therapy and ipilimumab monotherapy found that drug-related adverse events of grade 3 or 4 were reported in 54% of patients who received combined therapy, and only 24% in the monotherapy group. Most of these events were resolved with immune-modulating medication (Postow et al., 2015). Another trial, comparing combined therapy to ipilimumab monotherapy and nivolumab monotherapy, found that adverse reactions of grade 3 or 4 occurred in 16.3% of the patients in the nivolumab group, 55% of the combined group and 27.3% of the ipilimumab group (Larkin et al., 2015). On one trial looking at nivolumab monotherapy or combined therapy in SCLC, one patient experienced myasthenia gravis during the study, which was fatal (Antonia et al., 2015).

All cancer treatments may result in side effects. For example, chemotherapy, using a drug such as doxorubicin, may also damage normal cells as well as cancerous cells. These can include flu-like symptoms and a high temperature of over 38°C (can be signs of infection), hair loss, vomiting, diarrhoea and difficulty with breathing (Royal Marsden, 2014).

Use of doxorubicin can also have serious effects on cardiac muscle, which could be devastating (Doxorubicin hydrochloride 2mg/ml solution for infusion - Summary of Product Characteristics (SPC) - (eMC), 2014). Sunitinab (Sutent), another biological therapy mentioned earlier for experimental use in combination with ipilimumab and nivolumab in an ongoing trial, also can have severe adverse reactions including renal failure, anaemia, fatigue, diarrhoea and hypertension. Sudden death and multi-system organ failure also had a possible link to this treatment (SUTENT 12.5mg, 25mg, 37.5mg and 50mg Hard Capsules - Summary of Product Characteristics (SPC) - (eMC), 2015).

Cost of Treatment

Immunotherapy drugs, though effective, come with a hefty price tag. Ipilimumab was approved by the National Institute for Health and Care Excellence (NICE) in December 2012, despite its high cost of approximately £75,000 for a four-dose treatment course, as Bristol-Myers Squibb agreed on an acceptable discount for the Department of Health (The Guardian, 2015), after previously being denied in 2011 as the long-term benefits were unclear and the cost too high for the NHS (Fellner, 2012). Pharmaceutical companies can make enormous profits by controlling knowledge of drug manufacture, and as the market of immunotherapy treatments has been estimated to be worth up to £26bn a year in sales (The Guardian, 2015), this has become the new focus. The price of the combined treatment is likely to be much higher than this figure, due to the use of the two drugs, ipilimumab and nivolumab.

This can put pressure on health systems all over the world, as well as richer economies like the UK, because there are huge differences in cost; the British Generic Manufacturers Association says "The average cost to the NHS of a generic medicine is £3.79, whilst the average cost of a branded medicine is £19.73." The huge profit is the reward, funding and incentive for the research and development of new drugs (The Guardian, 2014). But retail prices aren’t reflective of the production costs, which are a tiny fraction of the overall price tag; they are “set according to the maximum amount that a market will bear in the absence of price-lowering competition” (ibid).

However, all cancer treatment comes at a cost. As said by Dr Annabel Bentley, medical director of Bupa Health and Wellbeing, "A single course of chemotherapy for cancer can cost between £25,000 and £30,000, and someone may need several courses” (The Telegraph, 2011). An average six-week cycle of Sutent costs around £3,139 and Pfizer, the manufacturer, provides the first cycle of treatment to NHS patients for free (The Telegraph, 2009). A herceptin-style drug called Kadcyla (trastuzumab emtansine), manufactured by Roche, that had the ability to offer some advanced breast cancer patients nearly 6 months of extra life was turned down for use in the NHS in 2014, for its high price of £90,000 per patient. However it is being funded through the special Cancer Drugs Fund, “We are very aware of the importance that people place on life-extending cancer drugs and a decision not to recommend a cancer treatment for routine NHS funding is never taken lightly.” (The Guardian, 2014)

Conclusion

Immunotherapy has unsurprisingly become the forefront of cancer research in recent years (American Cancer Society, 2014). It has shown real promise of improving the lives of many cancer patients, including complete responses.

The mechanism of this therapy, effectively ‘rewiring’ the immune system to create an anti-tumour response, is innovative and the use of antibodies which are able to bind to specific target proteins with high specificity, means their application in oncology is obvious (Antibodies as Tools, 2015).

The new combined therapy of ipilimumab and nivolumab has shown encouraging results in clinical trials, including tumour shrinkage rates of over 50% that have never been seen before (The Guardian, 2015). This is an incredible feat, worked on by oncologists and researchers

for thousands of years, since before the ancient Edwin Smith Papyrus, when there was almost no treatment for cancer (American Cancer Society, 2014). In one trial, the median progression-free survival was increased by 8.6 months by combined therapy instead of ipilimumab monotherapy (Larkin et al., 2015). These results are extremely exciting for the medical industry and there are increasing numbers of ongoing clinical trials, researching the effects this therapy will have on different types of cancers, including metastatic melanoma, renal cell carcinoma and small cell lung cancer. There is potential of this therapy being able to treat a diverse range of cancers, and therefore improve the lives of millions of patients.

Although there are cases of increased adverse reactions in the combined therapy, including a 30% increase in a trial comparing monotherapy and combined therapy (Postow et al., 2015), I believe that the benefit far outweighs the risk. For many patients, if they received monotherapy or no treatment at all, their most realistic median-progression free survival was just 2.9 months (Larkin et al., 2015), and many mortalities would have occurred much sooner. In some cases, patients were able to make a full recovery, seen in a clinical trial comparing ipilimumab monotherapy with combined therapy where 22% of the combination group reported complete responses versus none in the monotherapy group (Postow et al., 2015).

In addition to this, all cancer treatments have adverse reactions, such as flu-like symptoms in chemotherapy (Royal Marsden, 2014), and renal failure using Sunitinab (SUTENT 12.5mg, 25mg, 37.5mg and 50mg Hard Capsules - Summary of Product Characteristics (SPC) - (eMC), 2015). If the results of this combined therapy are more promising than existing treatments, which in the case of ongoing trials look optimistic (The Guardian, 2015), then a similar risk of adverse reactions is a small compromise that patients and professionals alike may be willing to make.

The high price of this treatment has caused many to question its economic viability, especially during this economic climate, where there is already strain on the NHS in the UK. Its high price reflects the results of the therapy, but also comfortably profits the pharmaceutical companies (The Guardian, 2014). However, all cancer treatment will come at a cost. It raises an important but difficult ethical question – how much is a human life worth? In reality it is impossible to fund numerous treatments for every disease; the most cost-effective treatments must be funded, without compromising the health of the population. The most difficult part is finding out which treatments these are, ongoing clinical trials will help provide evidence for making these important decisions. In my opinion, if the combined therapy of ipilimumab and nivolumab can significantly improve patients’ health on a variety of different cancers, it would be a viable option. If not however, efforts may be better off invested in developing a more cost-effective alternative.

After all my research that I’ve carried out in respect to the combined therapy of ipilimumab and nivolumab, I believe that it is an extremely promising and exciting example of immunotherapy, hence it’s high price tag. The fact that it is such a current topic, with new resources being published constantly, means that it is hard to make an absolute, well-informed decision about it viability. More evidence needs to be produced to back up the use of this treatment before it can be deemed as completely viable, however, this combined therapy has certainly had an enthusiastic introduction into the medical industry.

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