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

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

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)

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