The development of immunomodulatory monoclonal antibodies as a new therapeutic modality for cancer: the Bristol-Myers Squibb experience.

JPT-06742; No of Pages 22 Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

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David Berman, Alan Korman, Ronald Peck, David Feltquate, Nils Lonberg, Renzo Canetta ⁎

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Bristol-Myers Squibb, Research and Development Division, United States

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a r t i c l e

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Keywords: Immuno-oncology T-cell receptors Novel evaluation criteria Ipilimumab Nivolumab Combinations

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The discovery and increased understanding of the complex interactions regulating the immune system have contributed to the pharmacologic activation of antitumor immunity. The activity of effector cells, such as T and NK cells, is regulated by an array of activating and attenuating receptors and ligands. Agents that target these molecules can modulate immune responses by exerting antagonistic or agonistic effects. Several T- or NK-cell modulators have entered clinical trials, and two have been approved for use. Ipilimumab (Yervoy®, Bristol-Myers Squibb) and nivolumab (OPDIVO, Ono Pharmaceutical Co., Ltd./Bristol-Myers Squibb) were approved for the treatment of metastatic melanoma, in March 2011 in the United States, and in July 2014 in Japan, respectively. The clinical activity of these two antibodies has not been limited to tumor types considered sensitive to immunotherapy, and promising activity has been reported in other solid and hematologic tumors. Clinical development of ipilimumab and nivolumab has presented unique challenges in terms of safety and efficacy, requiring the establishment of new evaluation criteria for adverse events and antitumor effects. Guidelines intended to help oncologists properly manage treatment in view of these non-traditional features have been implemented. The introduction of this new modality of cancer treatment, which is meant to integrate with or replace the current standards of care, requires additional efforts in terms of optimization of treatment administration, identification of biomarkers and application of new clinical trial designs. The availability of immune modulators with different mechanisms of action offers the opportunity to establish immunological combinations as new standards of care. © 2014 Published by Elsevier Inc.

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Contents

1. Introduction. . . . . . . . . . . . . . . . . 2. Targeting the immune checkpoints . . . . . . 3. Clinical development . . . . . . . . . . . . . 4. Novel aspects of immune checkpoint modulation 5. Conclusions . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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The development of immunomodulatory monoclonal antibodies as a new therapeutic modality for cancer: The Bristol-Myers Squibb experience

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Abbreviations: AE, Adverse events; APC, Antigen-presenting cell; BMS, Bristol-Myers Squibb; CR, Complete response; CTLA-4, Cytotoxic T-lymphocyte antigen 4; DTIC, Dacarbazine; EAP, Expanded Access Program; FDA, Food and Drug Administration; GITR, Glucocorticoid-induced tumor necrosisfactor receptor; HRPC, Hormone refractory prostate cancer; LAG-3, Lymphocyte activation gene-3; NCI, National Cancer Institute; NK, Natural killer; NSCL, Non-small cell lung; OS, Overall survival; PD-1, Programmed death-1; PD-L1, Programmed death-ligand 1; PD-L2, Programmed death-ligand 2; PFS, Progression-free survival; PR, Partial response; PSA, Prostate-specific antigen; RFS, Relapse-free survival; SD, Stable disease; TIL, Tumor-infiltrating lymphocyte; TKI, Tyrosine kinase inhibitor; TNF, Tumor necrosis factor; U.S., United States; WHO, World Health Organization ⁎ Corresponding author at: Bristol-Myers Squibb, 5 Research Parkway, P.O. Box 5100, Wallingford, CT 06492-7660, United States. Tel.: 203 677 6047; fax: 203 677 7924. E-mail address: [email protected] (R. Canetta).

http://dx.doi.org/10.1016/j.pharmthera.2014.11.017 0163-7258/© 2014 Published by Elsevier Inc.

Please cite this article as: Berman, D., et al., The development of immunomodulatory monoclonal antibodies as a new therapeutic modality for cancer: The Bristol-Myers Squibb exper..., Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.11.017

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2. Targeting the immune checkpoints

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The immune response normally begins when foreign proteins and antigens are recognized, captured and processed into peptides by antigen-presenting cells (APCs), which then present such peptides in the context of a major histocompatibility complex (MHC) to T cells. Recognition of this complex occurs via a specific T-cell receptor. However, activation of the T cell typically requires an additional signal such as the

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The CTLA-4 gene was originally discovered by a French group using subtractive hybridization to identify genes enriched in cytotoxic T cells. This group showed that the gene was not constitutively expressed, but induced in activated T cells. However, initially its function was not fully understood (Brunet et al., 1987). A group of scientists at BMS first discovered that the B7 antigens, present on B-lymphocytes, are a ligand for the CD28 receptor on the T-lymphocytes (Linsley et al., 1991a). Shortly thereafter, they were able to identify CTLA-4 (also known as CD152) as a higher-affinity receptor for B7 than CD28 (Linsley et al., 1991b). This seminal work stimulated further research in the area of immunosuppression, which eventually led to the successful development of soluble CTLA-4 molecules such as abatacept (Orencia®, BMS), approved in 2005 for the treatment of rheumatoid arthritis and belatacept (Nulojix®, BMS), approved in 2011 for prophylaxis of kidney transplant rejection (Drugs@FDA, 2014). Investigators at the University of Chicago and the Dana-Farber Cancer Institute discovered that CTLA-4 can function as a negative regulator (Green et al., 1994; Walunas et al., 1994). Two groups generated CTLA-4 knock-out mice, demonstrating massive lymphoproliferation and leading to death, thereby confirming the regulatory (negative) effect of CTLA-4 on T cells (Tivol et al., 1995; Waterhouse et al., 1995).

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2.1. Cytotoxic T-lymphocyte antigen 4 (CTLA-4)

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Engaging a patient's own immune system to prevent and/or combat diseases has been adopted for many years for chronic and acute infections 53Q18 (Hotchkiss & Moldawer, 2014). Analogous approaches for cancer therapy 54 have been explored for many years, but with only a small number of 55 successes in the last century leading to regulatory approval (interferon 56 alfa 2A and 2B for hairy cell leukemia in 1986, intravesical Bacillus 57 Calmette–Guérin (BCG) for recurrent, localized bladder cancer in 58 1989, and aldesleukin for renal cancer in 1992). Additional indications 59 were subsequently granted to interferon alfa 2A for chronic myeloid 60 leukemia, to interferon alfa 2B for follicular and AIDS-related non61 Hodgkin's lymphoma and for resected melanoma, and to aldesleukin 62 Q19 for metastatic melanoma (United States [U.S.] Food and Drug Adminis63 tration [FDA], 2014). 64 Within the last few years, a deeper understanding of the immune 65 system at the molecular level has spurred a renaissance of interest 66 and resulted in successful development for therapeutic vaccines 67 (sipuleucel-T in 2010 for prostate cancer) and immunostimulatory 68 monoclonal antibodies (ipilimumab in 2011, in the U.S., and nivolumab 69 in 2014, in Japan, for metastatic melanoma). While still at the experi70 mental level, additional immunologic therapeutic modalities involving 71 the two classes mentioned above, as well as innovative approaches 72 utilizing adaptive and innate immunity and modified cellular therapies, 73 promise to further advance immuno-oncology as a mainstay in the 74 future treatment of malignancies. In this paper we will report our direct 75 experience in the contributions that Bristol-Myers Squibb (BMS) has 76 made to this area of cancer research, limited to the immunomodulatory 77 compounds that have already reached the stage of clinical development.

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co-stimulatory interaction between the CD80 (B7.1) and CD86 (B7.2) ligands expressed by the APC and the CD28 receptor expressed by the T cell. The activation of T cells is further regulated by the balance of inhibitor (i.e. checkpoint) and co-stimulatory pathways, which are critical in healthy subjects to prevent auto-immunity (producing selftolerance) and to protect normal tissues when the immune system is activated against a pathogen (Pardoll, 2012). Many immune checkpoint or co-stimulatory molecules regulating the interaction between ligands on the APC and receptors on the T cell have been identified (Fig. 1). The targeting of cell membrane receptors is possible with molecules such as monoclonal antibodies which can mimic or block the effect of a receptor or of a ligand and thereby enhance the immune response (Melero et al., 2013b).

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Fig. 1. Immune checkpoint modulators. aThe antibody has shown evidence of an antagonistic effect. bCD40 is a member of the TNF receptor family, expressed on APCs in contrast to other members of this family which are expressed on T cells; the corresponding ligand (CD40L) is expressed on the T cell. APC: antigen-presenting cell; CTLA-4: CTLA-4: Cytotoxic T-lymphocyte antigen 4; GITR: Glucocorticoid-induced tumor necrosis factor receptor; LAG-3: Lymphocyte activation gene-3; PD-1: Programmed death-1; PD-L1: Programmed death ligand 1.


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LAG-3 (also known as CD223) was discovered in 1990 as a gene that was expressed in activated, but not resting, T and NK cells (Triebel et al., 1990). LAG-3 functions as an inhibitory receptor on T cells, and by increasing the effect of regulatory T cells (Huard et al., 1995; Grosso et al., 2007). In mouse models of chronic viral disease, LAG-3 is one of the key inhibitory receptors, along with PD-1, that contribute to T-cell ‘exhaustion’ (Wherry, 2011). The ligand for LAG-3 is a MHC class II molecule. Simultaneous blockade of LAG-3 and PD-1 synergistically enhance T-cell activity and antitumor immunity in mouse models (Woo et al., 2012). Alan Korman's group at Medarex (now part of BMS) produced a monoclonal antibody directed against LAG-3 that has recently entered clinical trials: BMS-986016 (BMS) alone or in combination with nivolumab (ClinicalTrials.gov; NCT01968109).

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2.4. T-cell agonists

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Pharmacologic blockade of checkpoints inhibitors which ‘remove the brake’ imposed on the immune system, have been clinically validated. More recently, there has been also an intense research aimed at developing potential agonist compounds that would ‘press on the gas pedal’ to directly activate T cells. These agonists function only in concert with T-cell receptor signaling. Several of these compounds have reached the stage of clinical development.

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2.4.1. CD137 CD137 (also known as 4-1BB or TNFRSF9) was identified in the early 90's (Pollok et al., 1993) and has one ligand, CD137-L (or 4-1BBL), which is present on APCs. Monoclonal antibodies that directly activate CD137 can co-stimulate and enhance the effect of T cells, increasing their cytotoxicity and protecting them from programmed death, as initially reported by investigators at the BMS Pharmaceutical Research Institute in Seattle (Melero et al., 1998). An additional interesting property of this agonistic antibody is the ability to enhance NK-cell function in antibody-dependent cell-modulated cytotoxicity (ADCC), thus enhancing the antitumor effects of antibodies which target tumor cells directly, such as rituximab, cetuximab and trastuzumab (Kohrt et al., 2012). The BMS group developed a number of monoclonal antibodies directed against CD137 (Melero et al., 1997), from which urelumab (BMS-663513), a fully human IgG4, was selected for clinical development (Sznol et al., 2008). Another monoclonal antibody directed against CD137, an IgG2 humanized molecule (PF-05082566, Pfizer) has recently entered the clinic (Segal et al., 2014).

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2.4.2. Other members of the tumor necrosis factor superfamily The tumor necrosis factor (TNF) receptor family (or ‘superfamily’) contains a number of receptors which are expressed on cells of the immune system, as well as their trans-membrane ligands (Armitage, 1994). One such receptor is CD27 (also known as TNFRSF7), which is found on NK cells, as well as on T- and B-cell populations (Hendriks et al., 2000). The ligand for CD27 is CD70, which has been found on activated dendritic cells including thymic dendritic cells in humans.

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Tasuku Honjo and colleagues at Kyoto University discovered the gene encoding the PD-1 receptor (Ishida et al., 1992). PD-1 (also known as CD279) is a transmembrane protein receptor which is not detectable on resting T cells but is highly expressed by activated T cells (Pardoll, 2012). Two ligands have been identified for PD-1 (Freeman et al., 2000; Latchman et al., 2001): PD-L1 (originally referred to as B7 Homolog 1 or B7-H1) by Lieping Chen's group at the Mayo Clinic (Dong et al., 1999) and PD-L2 (originally referred to as B7-Dendritic Cell or B7-DC) by Drew Pardoll's group at Johns Hopkins (Tseng et al., 2001). In 2002, Chen's group found that that many human tumors overexpress PD-L1, and that mouse PD-L1 expression on tumor cells confers a growth advantage in immune competent syngeneic mouse tumor models (Dong et al., 2002). That same year, Honjo and colleagues reported that a monoclonal antibody to mouse PD-L1 had therapeutic activity in syngeneic mouse tumor models and that PD-1-deficient mice are more resistant to transplanted tumors that wild-type mice (Iwai et al., 2002). Medarex scientists, led by Alan Korman, developed human antibodies against human PD-1 as well as, separately, against its ligand PD-L1, and demonstrated their ability to enhance T-cell responses in vitro. Antitumor effects in the mice were also observed with surrogate antibodies to mouse PD-1 and PD-L1 (Wang et al., 2014). Nivolumab (MDX-1106, BMS-936558), a fully human, monoclonal immunoglobulin IgG4 antibody that binds to the PD-1 receptor, was selected for clinical development. Of note, nivolumab is able to block the interaction of PD-1 with both of its ligands, PD-L1 and PD-L2. A number of additional monoclonal antibodies reported to target the PD-1 receptor are currently under development. These include

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pembrolizumab (MK-3475, formerly known as lambrolizumab, Merck), a humanized IgG4 (Hamid et al., 2013), pidilizumab (CT-011, formerly known as CT-AcTibody or BAT, CureTech and, temporarily, Teva), a humanized IgG1 (Berger et al., 2008), and AMP-514 (MEDI0680, Amplimmune and MedImmune/Astra-Zeneca) which recently entered the clinic (ClinicalTrials.gov; NCT02013804). In the case of pidilizumab, its specificity for PD-1 binding has been questioned (Pardoll, 2012). Another approach to target the PD-1 receptor, which has reached the clinic, is the recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224 (Amplimmune and Glaxo Smith-Kline) (Smothers et al., 2013).

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However, it was the work of the group led by Jim Allison, first at the University of California, Berkeley and then at Memorial Sloan-Kettering in New York that clarified that the outcome of T-cell activation was determined by the balance of CD28 (stimulatory signal) and CTLA-4 (inhibitory signal) (Krummel & Allison, 1995). Allison's group went on to demonstrate that in vivo blockade of the CTLA-4 receptor resulted in an antitumor effect in a murine model and that this effect was mediated by T-lymphocyte proliferation, thus identifying this as a potential novel mechanism for anticancer therapy (Chambers et al., 2001). In collaboration with Allison's group, scientists at Medarex (now BMS's Biologics Discovery, California) eventually demonstrated that CTLA-4 blockade produces both a direct enhancement of T-cell effector function and a concomitant inhibition of regulatory T-cell activity (Peggs et al., 2009). Shortly after the initial discovery by Allison, Alan Korman and Nils Lonberg at Medarex decided to develop a fully human anti-CTLA-4 antibody for anticancer therapy. The antibody they selected, now called ipilimumab, is a human IgG1 Kappa immunoglobulin with high binding affinity to the CTLA-4 receptor and that blocked its interaction with B7.1 and B7.2 (Keler et al., 2003). After ipilimumab had initiated clinical trials, another monoclonal antibody directed against CTLA-4, tremelimumab (Astra-Zeneca/MedImmune, originally known as ticilimumab, CP-675,206 when under the sponsorship of Pfizer first, and Debiopharm second) (Ribas et al., 2005) was also selected for clinical development. In contrast to ipilimumab, tremelimumab is an IgG2 isotype antibody. Human IgG2 isotype antibodies do not typically engage cell-mediated effector function as efficiently as human IgG1 isotype antibodies, and it is possible that this isotype difference could impact the relative activity of these two agents. Effector-mediated depletion of intratumoral regulatory T cells is an important component of the activity of anti-CTLA-4 antibodies in mouse tumor models (M.J. Selby et al., 2013; Simpson et al., 2013); however, there have been no side-by-side comparisons of the clinical activity of ipilimumab and tremelimumab, and both antibodies have shown antitumor activity and similar inflammatory adverse events in melanoma patients.

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2.5.3. B7-H3 A new member of the B7 family, B7-H3 has been recently described (Loos et al., 2010). The potential co-inhibitory T-cell receptor for this ligand has not been identified. A humanized IgG1 including Fc engineering and directed to this target has been developed and brought to the clinic, MGA271 (MacroGenics and Servier) (ClinicalTrials.gov; NCT01391143).

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3. Clinical development

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2.5.1. PD-L1 The programmed death ligand 1 (PD-L1 or B7-H1 or CD274) is a member of the B7 ligand family and is more broadly expressed than PD-L2 (Pardoll, 2012). PD-L1 is not only induced on activated T-lymphocytes, but also on other activated non-T-lymphocyte cells, including B and NK cells, as well as on many tumors. Moreover, when a prolonged presence of an antigen occurs, as in chronic viral infections, a persistent high expression of PD-1 is induced that can produce exhaustion or anergy of the immune system. Inflammatory conditions at the tissue and/or tumor level induce PD-L1 (and PD-L2) expression. Finally, PD-L1 can interact with the CD80 receptor on T cells, sending a further immunosuppressive signal. Targeting this major inhibitory ligand can thus produce complex and powerful effects. The first monoclonal antibody directed against the PD-L1 ligand to enter clinical trials was BMS-936559, developed by BMS concomitantly with the anti-PD-1 monoclonal antibody nivolumab. BMS-936559 is a high affinity, fully human, immunoglobulin IgG4 that can block the binding of PD-L1 to both PD-1 and CD80 (B7.1) (Brahmer et al., 2012). A number of additional PD-L1 targeted antibodies have rapidly followed into the clinic, including MPDL3280A (Roche/Genentech), a human monoclonal IgG1 antibody containing an engineered Fc-domain to eliminate Fc receptor binding (Gordon et al., 2013); MEDI4736 (AstraZeneca/MedImmune), a human immunoglobulin G1 kappa, also engineered to eliminate Fc-receptor binding (Lutzky et al., 2014), and MSB0010718C (EMD Serono), a human IgG1 antibody (Heery et al., 2014).

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2.5.2. CD70 CD70 is a transmembrane cell surface protein that is highly expressed in clear-cell renal carcinoma as well as in B-cell non-Hodgkin's lymphoma. CD70 is the ligand for CD27, as described before.

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Given the nature and the extent of this review, we will cover in this section only the compounds that have reached the stage of clinical development under BMS sponsorship. Other compounds that have reached this stage have been mentioned in the previous section.

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3.1. Ipilimumab (anti-CTLA-4)

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The development of ipilimumab has been previously reviewed in detail (Hoos et al., 2010a; Wolchok et al., 2013a). For the purpose of this paper, we would like to focus on the critical choices and findings that occurred during its development and update the overall results. In laboratory models, antibody blockade of CTLA-4 induced antitumor immunity mediated by CD4+ and CD8+ effector T cells. Of note, early preclinical testings were conducted which involved combinations with cytokines, vaccines and chemotherapy, posing the basis for the ensuing clinical development of novel regimens. To translate these preclinical results to the clinical setting, scientists at Medarex (now part of BMS) used transgenic mice comprising human immunoglobulin genes to generate fully human monoclonal antibodies to human CTLA-4. Ipilimumab, the antibody selected for clinical development, was initially designated as 10D1 or MDX-010 (Grosso & Jure-Kunkel, 2013). To accelerate entry into clinical testing, the material used in the initial Phase I trials was derived from the original transgenic mouse hybridoma; however all subsequent and ongoing clinical trials (and the approved version of the drug) were conducted with material derived from a transfectoma (Weber et al., 2008).

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3.1.1. Phase I The clinical development of ipilimumab began in mid-2000 with monotherapy Phase I trials conducted under the sponsorship of Medarex (Tchekemedyian et al., 2002; Hodi et al., 2003), as well as with the collaboration of the Surgery Branch of the U.S. National Cancer Institute (NCI), which focused on combinations with a peptide vaccine, gp100 (Phan et al., 2003), and with aldesleukin (Maker et al., 2005). The NCI investigators also conducted a monotherapy intra-patient dose-escalation trial (Maker et al., 2006). In the initial experience, ipilimumab presented a characteristic set of mechanism of action-related (‘on-target’) side effects (see safety section later), but without reaching a dose-related maximum tolerated level up to 20 mg/kg. In addition, although the rate of objective responses was not particularly high, some impressively durable antitumor effects, including complete (CR) and partial (PR) responses, were observed. Remarkably, objective responses were seen in each of the Phase I trials completed. The Phase I studies accrued mostly patients with heavily pretreated, metastatic malignant melanoma. Because of the early efficacy observations in this disease, a small trial of ipilimumab in combination with another vaccine (a combination of tyrosinase, gp100 and MART-1 peptides) was conducted in the adjuvant setting (Sanderson et al., 2005), a relatively novel approach to clinical development at such an early junction.

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BMS developed antibodies directed to CD70 with the intent of utilizing them for immuno-conjugate targeting. A Phase I trial of one such compound, BMS-936561 (MDX-1203), was recently reported (Owonikoko et al., 2014). A second CD70-directed monoclonal, ARGX-110 (arGEN-X) has also subsequently entered the clinic (Awada et al., 2014).

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Interaction of CD27 with its ligand has been shown to promote differentiation of CD8+ T-cells into effector cytotoxic T cells (Brown et al., 1995). An anti-CD27 human antibody, varilumab (CDX-1127, Celldex Therapeutics) has entered clinical trials, both in hematologic malignancies (Ansell et al., 2014) and in solid tumors (Infante et al., 2014). Combinations of varilumab with BMS's anti-PD-1 monoclonal nivolumab, and with Oncothyreon's tumor-associated antigen MUC1 vaccine, ONT-10, are planned for clinical development. Another co-stimulatory molecule is OX40 (also known as CD134 or TNFRSF4), and a murine anti-human OX40 monoclonal antibody has been tested in the clinic (Weinberg et al., 2006; Melero et al., 2013a). OX40 is currently the target of a second antibody in development, MEDI6383 (MedImmune) (ClinicalTrials.gov; NCT02221960). The glucocorticoid-induced TNF receptor (GITR) has been targeted by an agonistic humanized anti-GITR IgG1 antibody (TRX518, Tolerx and GITR Inc.) (Rosenzweig et al., 2010) and by another agonistic antibody, MK-4166 (Merck) (ClinicalTrials.gov; NCT02132754). Finally, a member of the TNF superfamily is CD40, which is expressed on APCs, whereas its ligand (CD40L) is expressed on the T cell (Melero et al., 2013a). Many therapeutic antibodies are directed to CD40: CP-870,893 (Pfizer and VLST) (Vonderheide et al., 2007), dacetuzumab (Seattle Genetics) (Khubchandani et al., 2009), Chi Lob 7/4 (Southampton University) (Johnson et al., 2010), and lucatumumab (Novartis) (Bensinger et al., 2012). Of note, these antibodies can actually present very different characteristics, including either antagonism or agonism, and agonist activity may be dependent on, or potentiated by, Fc receptor ligation (Li & Ravetch, 2013). In addition, the potential for depletion of regulatory T-cells using Fc regions that bind to activating receptors may provide an additional mechanism of action of these antibodies, as has also been observed for antibodies to GITR and CTLA-4 (Bulliard et al., 2013; M.J. Selby et al., 2013; Simpson et al., 2013).

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Table 1 Single-agent and combination activity of ipilimumab in patients with metastatic melanoma (Phase I and II trials).

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3.1.3. Phase III in melanoma The large experience accumulated with ipilimumab in melanoma prompted the launch of three Phase III trials. The first of these trials compared, in previously treated patients, the peptide vaccine gp100 alone, ipilimumab alone (at 3 mg/kg) and their combination and was based on the Phase 2 studies of this regimen. Indeed, the 1:1:3 randomization was originally aimed at identifying the individual contribution of the two components of the combination. Only four induction doses were administered, but reinduction with the same treatment assigned at randomization was allowed upon relapse. This trial, for the first time in the history of the treatment of metastatic melanoma and for a T-cell checkpoint inhibitor, demonstrated a significant improvement in overall survival (OS) for both of the ipilimumab-containing arms as compared with the vaccine alone (Hodi et al., 2010). A reduction of the risk of death of about one-third in the two ipilimumab arms resulted in the first regulatory approval of ipilimumab. Secondary endpoints such as objective response rates, according to World Health Organization (WHO) criteria (1.5 vs. 10.9 vs. 5.7%), and progression-free survival (PFS) (with medians of 2.76 vs. 2.86 vs. 2.76 months, respectively, for gp100 alone vs. ipilimumab alone vs. the combination but with a significant reduction of the risk of progression, as compared to the vaccine alone, of 36% with ipilimumab alone and 19% for the combination), favored both ipilimumab-containing arms over the vaccine alone. The combination regimen, however, was no better than ipilimumab alone.

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local prophylaxis with budesonide could improve the safety profile (Thompson et al., 2012), and confirmed on a large database the effectiveness of the compound (O'Day et al., 2010) when utilized as a single agent in patients with advanced disease. Of note, consistent efficacy in patients with advanced melanoma was observed in the entire Phase II experience with ipilimumab, whether the drug was administered alone or in combination, whether at lower (3 mg/kg) or higher (10 mg/kg) dosages, and whether in heavily pretreated or in previously untreated patients (Table 1). Besides the consistency of the results, the observation of durable effects producing long-term survival in about 20 to 25% of the patients was particularly encouraging. Long-term survival updates from these and other trials have been reported (Hersh et al., 2011; Prieto et al., 2012; Lebbé et al., 2014).

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3.1.2. Phase II in melanoma Three Phase II trials were initiated in patients with melanoma. The first trial employed the combination of ipilimumab with gp100 (Attia et al., 2005) in a population of 56 mostly pretreated patients with metastatic disease, and achieving two CR and five PR (13% response rate), most of them of long duration. The second trial randomized 72 chemotherapy-naïve patients with metastatic disease to receive either ipilimumab monotherapy or ipilimumab in combination with dacarbazine (DTIC) (Hersh et al., 2011), achieving two PR (5%) and two CR and three PR (14%), respectively, also of long duration, suggesting that a cytotoxic chemotherapy did not inhibit ipilimumab activity. The third trial treated 75 patients in the adjuvant setting with ipilimumab and the peptide vaccine utilized in the adjuvant Phase I trial for a prolonged period of time (one year) (Sarnaik et al., 2011). In this small trial, neither median relapse-free (RFS) nor overall survival (OS) was reached after a median follow-up of 29.5 months. These Phase II trials confirmed the clinical behavior of ipilimumab, both in terms of safety and efficacy, as observed in the early Phase I experience. They all utilized an ipilimumab dosage of 3 mg/kg which, although not the maximum tolerated dose, induced both antitumor effects and manageable toxicities. With the exception of the adjuvant trial, these early trials administered only a limited number of four ‘induction’ doses of ipilimumab every 3 weeks. In 2005, Medarex initiated a collaboration with BMS to facilitate the development of ipilimumab as well as of the gp100 vaccine (MDX-1379). This collaboration was anchored by the expertise that both companies had developed on immunology in general, and on the CTLA-4 target in particular. Less than five years later, in 2009, BMS acquired Medarex and its entire pipeline of experimental products and immune modulators. At the beginning of the collaboration a number of additional Phase II trials in metastatic melanoma were launched. The goal of these trials was to expand the experience with ipilimumab, to further investigate dosing (dose–effect relationship) and duration (reinduction and maintenance) of treatment and to better understand the mechanism of action. These trials suggested the existence of a potential dose–effect for ipilimumab over the range of 0.3, 3 and 10 mg/kg for both efficacy and safety (Wolchok et al., 2010a), explored potential biomarkers in the tumor microenvironment (Hamid et al., 2011), tested whether the

5

Regimen (mg/kg)

Prior Rx

No. pts.

CR + PR

CR + PR + SD

Durability (median, months)

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79% 72% immuno Rx 52% chemo Rx 86% 76% 73% immuno Rx 38% chemo Rx 57% (51% immuno Rx) 48% (40% immuno Rx) 66% 64% immuno Rx 22% chemo Rx 94% 85% immuno Rx 55% chemo Rx 100% all arms 73% 77% 56% 36% 100%

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4 (5%)

18 (21%)

Responses: all N 6 months

14 56

3 (21%) 7 (13%)

5 (36%) N/A

Responses: 11+ to 15+ months Responses: 4 to 99+ months OS: 14

37 35 36

2 (5%) 5 (14%) 8 (22%)

8 (22%) 13 (37%) N/A

OS: 11.4 OS: 14.3 Responses: 5 to 89+ months OS: 16

85

17 (20%)

N/A

Responses: 5 to 76+ months OS: 13

73 72 72 40 42 57 58 155

– 3 (4%) 8 (11%) 3 (8%) 5 (12%) 9 (16%) 7 (12%) 9 (6%)

10 (14%) 19 (26%) 21 (29%) 16 (40%) 8 (19%) 20 (35%) 18 (31%)

OS: 8.6 OS: 8.6 OS: 14.6 OS: 12.9 PFS: 2.6 OS: 11.8 PFS: 2.6 OS: 19.3 (30.5 UT, 14.8 PT) OS: 17.4 (NR UT, 8.5 PT) OS: 10.2

U

3 + gp100 3 + gp100

3 3 + DTIC 0.1–3 + aldesleukin

t1:8 Q5

Maker et al. (2005)

t1:9 Q6

Maker et al. (2006)a

3 or 5 up to 9

t1:10 Q7

Wolchok, 2010

t1:11 Q8

Hamid, 2010

t1:12 Q9

Thompson et al. (2012)

t1:13 Q10

O'Day et al. (2010)

0.3 3 10 3 10 10 10 + budesonide 10

a

t1:14 CR: Complete response; DTIC: Dacarbazine; N/A: Not available; NR: Not reached; PR: partial response; PT: Pretreated; Pts: Patients; Rx: Therapy; SD: Stable diseases; UT: Untreated. a t1:15 Q11 Results updated in Prieto et al. (2012).


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3.1.4. Other experiences with monotherapy in melanoma When the effects of ipilimumab became evident in patients with advanced, heavily pretreated melanoma accrued to the Phase II program and before the results of the Phase III trials became available, the question of initiating an Expanded Access Program (EAP) arose. Indeed, such a program was started in early 2008, first in the U.S. and then elsewhere, utilizing the 10 mg/kg dose. Only after the results of the Phase III trial in pretreated patients became available, the program was amended to adopt the 3 mg/kg dosage. Rules on data collection for such programs vary from country to country but, when reportable, results from these programs have been published from several nations such as Italy (Altomonte et al., 2009; Queirolo et al., 2013), Spain (Berrocal et al., 2013), the United Kingdom (Ahmad et al., 2013), Brazil (Schmerling et al., 2014), France (Chasset et al., 2014), Canada (Lee-Ying et al., 2014), and the U.S. (K.A. Margolin et al., 2013). Not only have these initiatives allowed an early access to the drug, but they have also confirmed the clinical results on a very large scale and contributed useful additional information on the performance of the drug in patient subsets that might have not been accrued in sufficient numbers to the Phase II and III programs.

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reminiscent of what was observed with OS in all the other ipilimumab trials, was reported. Indeed, it is remarkable that all of the Phase III trials conducted with ipilimumab in malignant melanoma have met their primary efficacy objective (Fig. 2).

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The second Phase III trial of ipilimumab was conducted in previously untreated patients with metastatic disease (Robert et al., 2011). In this trial DTIC, the only universally accepted standard of care for the treatment of advanced melanoma until 2011, was compared to the combination of DTIC and ipilimumab at 10 mg/kg in a double-blinded, placebocontrolled study design. After the completion of the induction Phase, maintenance with blinded ipilimumab 10 mg/kg or placebo was administered every 12 weeks. Again, the primary endpoint of superiority in OS was met by the experimental arm, with a reduction of the risk of death of 28%. Whereas objective response rates (10.3% vs. 15.2% by WHO criteria, p = 0.09) did not differ significantly, PFS was significantly prolonged by 24% in the ipilimumab combination arm (p = 0.0006), despite the lack of numerical difference between the medians. Also for this trial a long-term survival update has been reported (Maio et al., 2013). The third Phase III trial was conducted in the post-surgical setting in patients with high-risk stage IIIA–C disease (Eggermont et al., 2014) and compared ipilimumab at 10 mg/kg with a placebo. As in the other preceding Phase II and III trials of ipilimumab in metastatic melanoma, an induction regimen of four doses administered every three weeks was adopted. However, this particular study also provided for the administration of maintenance therapy (or placebo) every 12 weeks for a maximum of three years. This study as well met its primary endpoint of RFS, as assessed by an Independent Review Committee, showing a significant reduction of the risk of recurrence by 25% (median 26.1 vs. 17.1 months for ipilimumab vs. placebo, p = 0.0013). Although the follow-up for the secondary endpoint of OS was not sufficiently mature at the time of reporting, a characteristic ‘leveling’ of the RFS curve,

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Fig. 2. Phase III trials of ipilimumab in melanoma. CI: Confidence interval; DTIC: Dacarbazine; HR: Hazard ratio; Ipi: Ipilimumab; OS: Overall survival; RFS: Recurrence-free survival.


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Fig. 3. Comprehensive overall survival results with ipilimumab in metastatic melanoma. CI: Confidence interval; OS: Overall survival.

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3.1.6. Other tumor types than melanoma

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3.1.6.1. Prostate cancer. Prostate cancer has provided the setting for a number of relatively unusual pilot trials with ipilimumab. The initial study was performed in patients with hormone-refractory prostate cancer (HRPC) who were given a single dose of 3 mg/kg, with the intent of evaluating safety and prostate-specific antigen (PSA) effects (Small et al., 2007). After this trial reported ≥ 50% reductions of PSA in 2/14 (14%) patients, subsequent trials pursued different avenues of development. A randomized Phase II trial tested again a single dose of ipilimumab 3 mg/kg alone or in combination with androgen ablation. At three months from initiation of therapy, 30/54 (55%) of patients receiving the combination and 21/54 (38%) of those receiving a single dose had undetectable PSA (Tollefson et al., 2010). Another randomized Phase II trial, this time conducted in the pre-surgical setting, tested the single dose of ipilimumab 3 mg/kg given early or later in combination with androgen ablation. At surgery, 5/41 (12%) of the patients receiving early combination obtained a downstaging of their tumor, as opposed to 1/32 (3%) of those who received it later (Granberg et al., 2010). A third, randomized Phase II trial in patients with HRPC tested a standard induction regimen of ipilimumab 3 mg/kg with or without a single dose

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3.1.5. Other experiences with combinations in melanoma The positive outcome of the Phase III trial of ipilimumab with DTIC demonstrated that the combination was superior to DTIC alone. However, due to its design, it did not address the issue of a potential detrimental effect of chemotherapy to immunotherapy. A supportive randomized trial was thus carried out in melanoma patients, and it did not document any relevant pharmacokinetic interactions of ipilimumab when combined with DTIC or with paclitaxel and carboplatin (J. Weber et al., 2013). A small randomized trial in patients with melanoma testing the concomitant administration of paclitaxel, carboplatin and ipilimumab vs. its sequential administration (ipilimumab delayed by one week) seemed to favor in response rate and disease control the sequential administration (Jamal et al., 2014). Additional trials have tested combinations of ipilimumab with temozolomide, a drug with similarities to DTIC (Patel et al., 2012), fotemustine, a drug that has received regulatory approval for the treatment of melanoma in certain European countries (Di Giacomo et al., 2012), and low-dose cyclophosphamide, with the intent of affecting regulatory T cells (Pavlick et al., 2014). The effect of cytokines in potentiating cellular immunity has been described. Sargramostim (GM-CSF) is one such commercially available cytokine. A large randomized Phase II trial was conducted by the Eastern

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Cooperative Oncology Group (ECOG) comparing ipilimumab alone at 10 mg/kg or in combination with standard GM-CSF doses. This trial accrued 250 patients and did not show a difference between arms in response rate or in PFS. However, OS favored the combination arm (median of 17.5 vs. 12.7 months, HR 0.64, p = 0.014). Even more interestingly, the combination arm appeared to be associated with a lower incidence of high-grade adverse events (AEs). These unusual characteristics could warrant further studies (Hodi et al., 2013a). Other pilot experiences with cytokine combinations include peginterferon alfa-2b (Kudchadkar et al., 2014), which is approved for the adjuvant treatment of melanoma, and recombinant interleukin-21 (BMS-982470) (Bhatia et al., 2013), an experimental compound developed by BMS. Initial promising results have been reported in a Phase I trial combining ipilimumab with bevacizumab with durable partial responses (PRs) in 36% of the patients treated (Hodi et al., 2011). Given the dramatic effect of tyrosine kinase inhibitors (TKIs) in patients with melanoma and BRAF and/or MEK mutations, the idea to combine such agents with ipilimumab is very appealing. With their different kinetics of antitumor effect (rapid vs. slower immediate tumor shrinkage, limited vs. long-term therapeutic effects for TKIs and ipilimumab, respectively) a Phase I trial of the combination of ipilimumab and vemurafenib, the first of the BRAF-V600 mutation inhibitors, was jointly initiated by BMS and Roche/Genentech, the two sponsors. Unfortunately, the concomitant administration of the two compounds produced excessive, although reversible, liver toxicity and the study was stopped (Ribas et al., 2013). Given the availability of both compounds, as the trial started in November 2011, a rapid communication of the toxicity encountered was generated, including letters to the investigators and publications. It is unclear whether a molecular basis for this interaction exists. Three other TKIs have been tested in the clinic concomitantly with ipilimumab: dabrafenib alone and also in a’triplet’ combination with trametinib (Puzanov et al., 2014a) and BMS-098662, a now-abandoned pan-BRAF inhibitor by BMS (Callahan et al., 2012), and no unacceptable liver toxicity has been reported to date. Another new and exciting chapter in the treatment of melanoma consists of the combination of ipilimumab with other vaccines and/or checkpoint inhibitors. Whereas pilot trials in combination with talimogene laherparepvec (T-VEC), a vaccine that has already reported positive results as a single agent in a Phase III trial in melanoma (Puzanov et al., 2014b) and with INCB024360, a small molecule inhibitor of indoleamine 2,3-dioxygenase (IDO) (Gibney et al., 2014), we will cover the combination of checkpoint inhibitors later in this paper.

F

Together with additional purpose-specific Phase II trials and retrospective subsets analyses, these data have contributed to the knowledge 499 of the effects of ipilimumab in patients with stabilized brain metastases 500 (Weber et al., 2011), active brain metastases (Margolin et al., 2012), in 501 previously untreated patients given the 3 mg/kg dose (K. Margolin 502 et al., 2013; Patt et al., 2013), in patients with and without HLA-A2 503 mutations (Wolchok et al., 2010), with and without BRAF and NRAS 504 mutations (Shahabi et al., 2012; Queirolo et al., 2013) and in patients 505 with uveal (Danielli et al., 2012; Luke et al., 2013; Moser et al., 2014; 506 Q21 Piulats Rodriguez et al., 2014) and acral melanoma (Deo, 2014; 507 Johnson et al., 2014a; Zimmer et al., 2014). 508 Finally, a number of other analyses of ipilimumab effects according 509 to baseline characteristics such as age (Chiarion-Sileni et al., 2013), 510 baseline lactate dehydrogenase (LDH) levels (Smylie et al., 2009), 511 good and poor prognostic factors (Schadendorf et al., 2009) and out512 comes of prior targeted or immunological therapy (Joseph et al., 2011; 513 Ackerman et al., 2012) have also been reported. 514 Long-term follow-up was recently collected and analyzed from all 515 completed Phase II and Phase III trials in metastatic disease, as well as 516 from the ongoing follow-up of patients accrued to the EAP (Fig. 3). 517 With a large database comprising more than 4800 patients, and a 518 follow-up which currently extends up to 10 years from initiation of 519 ipilimumab, it is remarkable that the OS Kaplan–Meier curve appears 520 to show a sustained plateau after two to three years from the beginning 521 of therapy, with about one-fifth of the patients presenting prolonged OS 522 results.

7


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of therapeutic vaccines have failed to demonstrate a significant impact upon this disease (Shepherd et al., 2011). However, increased understanding of the clinical relevance of tumor-infiltrating lymphocytes (TILs) in lung cancer has contributed to renewed interest in this therapeutic modality (Ruffini et al., 2009). In the case of pulmonary malignancies such as small cell and non-small cell lung (NSCL) cancer, where the aggressiveness of metastatic disease might require the rapid administration of platinum-containing chemotherapy or of targeted therapies to control tumor growth, the concern was how to integrate the use of immunotherapy with the existing standard of care, rather than demonstrating the single-agent role of ipilimumab. Thus, at the preclinical level, a number of cytotoxic agents usually utilized in lung cancer were tested in combination with ipilimumab against ipilimumab alone in a number of experimental solid tumors (Masters et al., 2009). Then, two large, prospectively randomized, double-blind, three-arm Phase II trials were conducted testing a platinum doublet alone, a platinum doublet concomitant with ipilimumab, and a platinum doublet where ipilimumab was started sequentially (after two cycles) to chemotherapy. These trials were conducted in small cell (Reck et al., 2013) and in NSCL cancer (Lynch et al., 2012). Both studies met their primary endpoint of prolongation of PFS according to immune response criteria when the sequential regimens were compared with standard chemotherapy. In the NSCL trial, the improvement appeared to be greater in the subset of patients with squamous cell histology. These results, together with the conduct of a Phase I trial with the phased regimen in Japanese lung cancer patients (Nokihara et al., 2013) and with an analysis correlating ipilimumab exposure, tumor shrinkage and survival (Y. Feng et al., 2012) supported the launching of two large Phase III trials for previously untreated patients with small cell and with squamous cell lung carcinoma.

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of docetaxel 75 mg/m2. A PSA reduction by ≥ 50% was seen in 3/20 (15%) of the patients receiving the combination and 3/24 (12.5%) of those receiving ipilimumab alone (Small et al., 2006). The observation in animal models that radiation therapy can induce responses at a distance from the irradiation portal (the so-called abscopal effect) has spurred interest in exploring this combination (Demaria et al., 2005). A Phase I/II trial tested the addition of escalating doses of ipilimumab (3, 5 or 10 mg/kg) alone or in combination with 8 Gy/lesion (3 or 10 mg/kg). A total of 75 patients with metastatic HRPC were treated with manageable AEs. Activity by PSA decline and tumor response was observed, without a clear dose–effect or a clear advantage by the addition of irradiation in the small cohorts treated (Slovin et al., 2013). Following this pilot experience, a double-blinded Phase III trial was conducted in metastatic HRPC who had received prior docetaxel, with the intent to assess the efficacy of ipilimumab when added to radiation therapy vs. radiation therapy alone (8 Gy) within a few days prior to initiation of study therapy. A total of 799 patients were randomized, and the trial missed the primary endpoint of demonstration of survival prolongation in the combination arm. Median OS was 10.0 months in the placebo arm and 11.2 months in the ipilimumab arm (HR 0.85, p-value 0.0530). Despite this, prespecified secondary (PFS) and exploratory (PSA response rate) endpoints favored the ipilimumab arm. Moreover, a trend in favor of OS benefit was observed also in the prespecified subgroup analysis where only patients with visceral metastases appeared not to benefit from immunotherapy (Kwon et al., 2014). Notwithstanding the fact that this trial was not positive, the results observed in patients with less advanced disease increased the expectations for the results of a second, ongoing Phase III trial that has recently completed accrual in patients without visceral disease or prior chemotherapy exposure (Beer et al., 2013). Considerable interest in the utilization of immunotherapy in prostate cancer has been spurred by the approval of sipuleucel-T, the first therapeutic cancer vaccine to reach the general public (Cheever & Higano, 2011). Indeed, the combination of vaccines with CTLA-4 blockade is supported by in vivo preclinical data (Hurwitz et al., 2000). The first vaccine to be studied together with ipilimumab was GVAX, a cellular vaccine consisting of two prostate cancer cell lines, transduced to secrete GM-CSF (van den Eertwegh et al., 2012). A total of 28 patients were treated with fixed doses of GVAX and escalating doses of ipilimumab (0.3, 1, 3 and 5 mg/kg), and late onset of PSA response was seen in five patients (17.8%), lasting in excess of six months. Unfortunately, two separate Phase III trials of GVAX alone in prostate cancer, one in symptomatic (Small et al., 2009) and one in asymptomatic patients (Higano et al., 2009) have failed to demonstrate a survival advantage over standard therapy, harming the future prospect of this vaccine in this disease. A second, somewhat simpler alternative was to administer directly GM-CSF, which is commercially available, and ipilimumab. A Phase I trial of this combination has been completed. A total of 36 patients with HRPC were treated with standard doses of GM-CSF and escalating doses (0.5 to 10 mg/kg) of ipilimumab. Four (11%) of the patients (three receiving 3 mg/kg, one receiving 10 mg/kg) had a PSA response (Harzstark et al., 2010). Another vaccine that is currently in Phase III trials for prostate cancer is PSA-tricom. This is a poxviral, vector-based vaccine which expresses transgenes for PSA and T-cell co-stimulator molecules and has been initially developed by the U.S. NCI. Because of its characteristics, a pilot combination trial with ipilimumab has been conducted. A total of 30 patients were treated with fixed doses of the vaccine and escalating doses of ipilimumab (1, 3, 5 and 10 mg/kg). One of the six (17%) chemotherapypretreated patients had a PSA response, as opposed to 14 of the 24 (58%) who were chemotherapy-naïve. The combination did not appear to exacerbate the known side effects of PSA-tricom (Madan et al., 2012).

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3.1.6.2. Lung cancer. Lung cancer has not traditionally been thought of as an immunotherapy sensitive malignancy. Indeed, several clinical trials

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3.1.6.3. Additional tumor types. Single-agent trials of ipilimumab have been reported in renal cell cancer (Yang et al., 2005), pancreatic cancer (Royal et al., 2009), non-Hodgkin lymphoma (Ansell et al., 2006) and recurrent glioblastoma (Hu et al., 2014; Schaff et al., 2014). A combination modality trial with TKIs in gastrointestinal stromal tumors (GIST) and sarcoma (Shoushtari et al., 2014) has also been reported. A number of relatively unusual combined-modality trials have also been reported in the neo-adjuvant setting in bladder cancer (Carthon et al., 2010) and in breast cancer patients undergoing cryoablation (Diab et al., 2014), concomitantly to radiotherapy and cetuximab in head and neck cancer (Bauman et al., 2014), to stereotactic radiosurgery in melanoma (Shoukat et al., 2013), to isolated limb perfusion in melanoma (Ariyan et al., 2014) as well as in patients relapsing after hemopoietic stem cell transplantation (Bashey et al., 2005). Two additional ongoing randomized Phase II studies are currently addressing the role of ipilimumab administered as maintenance therapy in patients with gastric or with ovarian cancer who had been initially treated with standard chemotherapy (ClinicalTrials.gov; NCT01585987; NCT01611558). A Phase I trial of ipilimumab has been undertaken in children with solid tumors, exploring escalating doses of 1, 3, 5 and 10 mg/kg (Merchant et al., 2012).

705 706

3.2. Nivolumab (anti-PD-1)

727

PD-1 is predominantly involved in regulating the effector phase of T-cell responses as well as the non-responsiveness of T cells resulting from long-term antigen exposure. The fact that cancer cells may directly inhibit an antitumor T-cell response provides tumors an opportunity to exploit T cells. Expression of PD-1 by tumor-reactive T cells present at the tumor site can provide for potentially rapid and efficient response by PD-1 blockade (Pardoll, 2012). Nivolumab is a fully human IgG4 PD-1 immune checkpoint inhibitor antibody which is directed to the PD-1 receptor. A comprehensive

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3.2.2. Phase II Whereas, technically speaking, an initial Phase II trial of nivolumab was not performed, the size of the populations accrued in the Phase I trials provided suitable information on its efficacy in the tumor types tested, albeit at different doses. 3.2.2.1. Melanoma. The results of the cohorts of patients with previously treated, metastatic melanoma have been published separately (Topalian et al., 2014) and updated with longer follow-up (Hodi et al., 2014). In this relatively large group of ipilimumab-naïve patients (5% of which had received a prior BRAF inhibitor), an objective response rate of 32% was observed, with long durability of responses. Of note, late onset of responses was less frequent than with ipilimumab, occurring in about 5% of the patients. Additional patients had long-term disease stabilization and, in this entire group, OS rates of 63% at one year and of 48% at two years were observed

846

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3.2.2.2. NSCL cancer. The observation of objective responses in the Phase I trial of nivolumab and their rapid onset provided the rational for a large cohort expansion for patients with NSCL cancer. The results obtained in this group of patients have been recently updated with longer follow-up (Brahmer et al., 2014). As in the case of melanoma, no clear-cut dose–effect relationship was observed in patients with lung cancer with the possible exception of the lowest dose cohort. A total of 129 previously treated patients were accrued (128 had received a platinum-based regimen, 36 a TKI). There were 54 patients with squamous and 74 patients with non-squamous histology (histology unknown in one case). The objective response rate was 17% across all doses (24% at the 3 mg/kg every two weeks level) with an additional 10% of SD. There was no difference in response rates between histologies, and the vast majority of responses occurred rapidly (within 16 weeks) and had a median duration of 74 weeks. Median PFS was 2.3 months and median survival 9.9 months. Objective responses were seen in 11% (4/36) of patients previously treated with TKIs, and in 17% (2/12) of patients with a known EGFR mutant status.

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3.2.1. Phase I A Phase I clinical trial began in October 2006, and 39 patients with various solid tumors received a dose of nivolumab at 0.3, 1, 3 or 10 mg/kg (Brahmer et al., 2010). Remarkably, one patient with colorectal cancer achieved a complete response (CR) to a 3 mg/kg dose, which lasted in excess of 21 months, and two other patients, one with melanoma and one with renal cancer, achieved a PR with doses of nivolumab of 10 mg/kg. Twelve additional patients presented mixed responses or stable disease (SD). Benefitting patients received multiple doses of therapy. The drug was well tolerated, with only one patient developing a grade III colitis, responsive to steroids and infliximab (Lipson et al., 2013). A second Phase I trial was begun in October 2008, which provided for treatment to be repeated at two-week intervals, and grew in size by the addition of multiple subsequent cohorts to a total of 296 patients (Topalian et al., 2012). Doses of 0.1, 0.3, 1, 3 and 10 mg/kg were administered and, as in the case of the first Phase I trial, no dose-limiting toxicity or maximum tolerated dose was determined. As significant and durable antitumor effects were being observed, cohorts of patients with melanoma (N = 104), renal (N = 34) and NSCL cancer (N = 122) were added and expanded. No objective responses were however reported in 19 patients with colorectal or in 17 patients with prostate cancer. Cohort expansion was possible also because of the relatively favorable safety profile, resulting only in 18 (6%) cases of grade III or IV AEs. However, nine patients developed pneumonitis, and this was severe in three (1%) of them. Patients received treatment, in the absence of progression or toxicity, for a maximum of two years. Baseline biopsy specimens were available in a subset of 42 patients, and only in those patients whose tumor expressed PD-L1 positivity by a murine anti-human antibody 5H1 in the tumor and/or at the infiltrating T-lymphocytes, objective responses were observed. The clinical pharmacokinetics was assessed in both Phase I studies and described as linear, doseproportional with a modest (20–44%) inter-subject variability and an elimination of half-life of 17 to 25 days (Agrawal et al., 2012). Another Phase I trial, initially limited to patients with previously treated metastatic melanoma, combined nivolumab at doses of 1, 3 and 10 mg/kg with a multi-peptide (MART-1/gp100/ NY-ESO-1) vaccine (Kudchadkar et al., 2012; J.S. Weber et al., 2013). This trial accrued 30 patients, and objective, durable responses were observed at all dose levels studied. Of note, two of six patients who progressed on nivolumab responded to subsequent ipilimumab. The relatively good tolerance to treatment was confirmed, with one case of optic neuritis and two cases of grade III interstitial pneumonitis observed with the vaccine combination, and two cases of grade III–IV skin toxicity observed with nivolumab alone.

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D

739

The ONO Pharmaceuticals Company, located in Japan, had collaborated with BMS from the early days of development of nivolumab. They conducted a Phase II trial in Japan in melanoma patients who had been previously treated with DTIC. Patients received 2 mg/kg every three weeks, and an objective response rate of 23% was observed, with an additional 49% of patients achieving SD. A median PFS of 5.7 months was reached (Yamazaki et al., 2013). In this trial a relatively good level of tolerance was reported, with a 17% incidence of grade III–IV AEs, mostly limited to liver function disorders. These results were pivotal to the regulatory approval of the drug, granted by the Japanese Health Authorities on July 4, 2014. In the context of two separate Phase I trials which tested nivolumab in combination with a multipeptide vaccine consisting of MART-1, gp100 and NY-ESO-1 peptides (Kudchadkar et al., 2012; J.S. Weber et al., 2013) or with ipilimumab (Wolchok et al., 2013b), two cohorts of ipilimumab-pretreated patients were treated with single-agent nivolumab at 3 mg/kg or 1 or 3 mg/kg every two weeks, respectively. Even in these series the efficacy observed was remarkable in terms of response and their duration. Although cross-trial comparisons are always to be considered with caution, these results suggest that PD-1 blockade by nivolumab might represent one of the most active single-agent treatments for melanoma (Table 2). In the trial that combined nivolumab with the multipeptide vaccine (J.S. Weber et al., 2013), objective responses were seen at all dosage levels. In 34 patients who had received no prior ipilimumab, eight (23.5%) objective responses were seen, lasting from 24 to 140+ weeks and seven additional SD exceeding 24 weeks were observed. In 15 patients who had received prior ipilimumab, four (26.6%) responses and four SD were seen. As for ipilimumab, a small pilot trial of nivolumab was conducted in the adjuvant setting in patients with resected stages IIIc or IV (Gibney et al., 2013). The same multipeptide vaccine studied in the Phase I was combined with nivolumab at doses of 1, 3 or 10 mg/kg, with nivolumab alone continuing as maintenance every three months after completing 12 doses (24 weeks) of vaccination and nivolumab. Of the 33 patients enrolled, seven relapsed after a median follow-up of 14 months, including a patient who had a spontaneous disease regression after a biopsyproven relapse. Altogether, these results have prompted the launch of several Phase III trials of nivolumab, alone or in combination with ipilimumab, in previously treated and in previously untreated melanoma patients. One such trial, comparing nivolumab vs. DTIC in previously untreated patients in countries where ipilimumab for first-line therapy had not yet been approved, has been very recently reported to have met its primary endpoint of prolongation of survival (Bristol-Myers Squibb, 2014). The results, however, have not yet been presented at a scientific meeting.

E

preclinical characterization of nivolumab was conducted by scientists at BMS (Wang et al., 2014).

T

737 738

9


801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 Q22 845

847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863

10 t2:1 t2:2


Table 2 Single-agent activity of nivolumab in previously treated patients with metastatic melanoma (Phase I and II trials).

t2:3

Author

Dosage (mg/kg)

Prior Rx

No. Pts.

CR + PR

CR + PR + SD

Durability (median, months)

t2:4

Hodi

0.1-10 q 2 weeks

107

34 (32%)

41 (38%)

t2:5

Yamazaki

35

8 (23%)

25 (73%)

Response: 24.9 PFS: 3.7 OS: 17.3 PFS: 5.7

t2:6

Weber

38

10 (26%)

17 (45%)

t2:7

Wolchok

2 q 3 weeks 3 q 2 weeks 1–3 q 2 weeks

100% 65% immuno Rx 5% BRAF-Ia 100% 100% DTIC 100% 100% ipilimumab 100% 100% ipilimumab 6% BRAF-I

30

6 (20%)

13 (43%)

BRAF-I: BRAF inhibitor; CR: Complete response; DTIC: Dacarbazine; PR: Partial response; Pts: Patients; RX: Therapy; SD: Stable disease. a BRAF-I.

864 865

A second trial of nivolumab as a single agent was conducted in previously untreated patients with NSCL cancer (Gettinger et al., 2014). A total of 20 patients were given a dose of 3 mg/kg every two weeks. Six of them had received prior adjuvant or neo-adjuvant systemic treatment. Half of them had a non-squamous histology. None had EGFR mutations. Two CRs and four PRs were obtained (response rate: 30%) and the median duration of response was not reached at the time of reporting, ranging from 23.7 to 71.4+ weeks. Similar response rates were seen in squamous and non-squamous histologies. Again, the majority of the responses occurred rapidly, within 12 weeks. In both studies of nivolumab monotherapy in lung cancer the tolerability was good: grade III–IV AEs occurred in 14% of the pretreated and in 10% of the untreated patients. Of note, three treatment-related deaths, all involving pneumonitis, occurred in the 129 pretreated patients. In this population, altogether, there were seven cases (9%) of pneumonitis. In the previously untreated study, no pneumonitis occurred. Given the level of activity observed, combination trials of nivolumab with standards of care for lung cancer have been initiated. Platinum (cisplatin or carboplatin) doublets with gemcitabine or pemetrexed or paclitaxel have been concomitantly administered to nivolumab at 10 mg/kg, given every three weeks to parallel the chemotherapy regimens (S.J. Antonia et al., 2014). A total of 56 previously untreated patients entered the trial, and objective responses ranging from 33 to 47% were observed with the various combinations. Medial duration of response ranged from 23.9 weeks to not reached. Additional SD was seen in 27 to 58% of the patients. Of note, the combination with carboplatin and paclitaxel required a reduction of the nivolumab dose to 5 mg/kg due to toxicity. Median PFS ranged from 21 to 31 weeks, and median survival from 50.5 weeks to not reached. Grade III–IV pneumonitis was seen in four patients (7%). The combination with erlotinib is very appealing, as preclinical data indicate that blocking the PD-1 pathway in EGFR mutant tumors produces in vivo tumor regressions and survival prolongation (Akbay et al., 2013). The clinical trial added nivolumab 3 mg/kg every two weeks to erlotinib 150 mg/day and reported four objective responses (19%) and nine SD (43%) in 21 patients, 20 of whom were erlotinibpretreated. Responses lasted from 60.1 to 72.3+ weeks. Median PFS was 29.4 weeks (Rizvi et al., 2014). Of importance, the combination of nivolumab with a TKI such as erlotinib did not produce the same type of liver toxicity that had been previously reported with the ipilimumab and vemurafenib concomitant administration. Because of the remarkable results observed in these early experiences, Phase III trials of single-agent nivolumab vs. standard chemotherapy have been initiated both in previously treated and in previously untreated patients, and in both NSCL cancer histologic subtypes.

884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913

E

D

P

R O

O

responding and nine additional patients achieving SD. Median duration of response was 12.9 months, and OS, in this group of pretreated patients, was 70% at one year. Six percent of the patients presented grade III–IV respiratory disorders and 6% hypophosphatemia. Two randomized dose-ranging Phase II trials of single-agent nivolumab were subsequently carried out. Both utilized an every three weeks dosing regimen. The first of these trials accrued previously treated patients with at least one antiangiogenic therapy, and randomized to three arms at 0.3, 2 or 10 mg/kg (Motzer et al., 2014). A total of 168 patients were accrued, and the primary endpoint of the study, PFS, appeared to favor the two higher-dose arms (medians 2.7, 4.0 and 4.2 months, respectively, p = 0.9). Response rates (20, 22 and 20%) were similar; however, OS also appeared to favor the two higher-dose arms (medians 18.2, 25.5 and 24.7 months, respectively). Grade III–IV AEs were 5%, 17% and 13%, with no severe pulmonary toxicity observed. Whereas this study did not conclusively demonstrate a dose effect for nivolumab, the survival results observed in each arm were very promising for a population of heavily pretreated patients. The second randomized Phase II trial also accrued 67 previously treated patients who were given either 0.3, 2 or 10 mg/kg every three weeks. An additional, non-randomized cohort of 23 previously untreated patients was given nivolumab at 10 mg/kg (Choueiri et al., 2014). This trial required the availability of tumor specimens for biomarker assessment. Once again, the higher-dose arms presented numerically superior results in terms of PFS (18, 32 and 49%, respectively, at 24 weeks) and of objective response rates (9, 23 and 22%, respectively). In the treatment-naïve arm, PFS at 24 weeks was 45% and the response rate 13%. Grade III–IV AEs were 18% in the pretreated and 8% for the untreated cohort. A single case of grade III–IV pneumonitis was observed, in the pretreated group. An additional trial for patients with renal cancer explored the combination of nivolumab with two TKIs with activity in this disease, either sunitinib or pazopanib (Amin et al., 2014). Patients who had received prior treatment with pazopanib were assigned to be given sunitinib at standard doses plus nivolumab at 2 mg/kg every three weeks, escalated to 5 mg/kg. A total of 33 patients were accrued, with one CR and 16 PR (52%) with a median duration of 37.1 weeks. An additional 10 patients had SD. Median PFS was 48.9 weeks. Grade III–IV hepatic AEs were 18% for ALT and 9.1% for AST increases, three patients presented renal function abnormalities. One patient had a grade III–IV pneumonitis. Patients who had received prior treatment with sunitinib were assigned to be given pazopanib at standard doses plus nivolumab at 2 mg/kg every three weeks. No dose escalation occurred in this group, due to gastrointestinal (grade III–IV in 20%) and hepatic (grade III–IV elevation of ALT and AST in 20%) AEs. In the 20 patients treated, nine (45%) PR and seven additional SD were observed. Median duration of response was 30.1 weeks and median PFS 31.4 weeks. In consideration of these results, a Phase III trial was initiated in renal cell cancer patients who had been previously treated with ≤ 2 prior antiangiogenic therapies and ≤ 3 prior regimens. They are being randomized to nivolumab 3 mg/kg every two weeks or oral daily everolimus at standard doses.

T

882 883

C

880 881

E

878 879

R

876 877

R

874 875

O

872 873

C

870 871

N

868 869

U

866 867

F

t2:8 t2:9

Response: not reached (from 12+ to 48+ weeks) –

3.2.2.3. Renal cell carcinoma. The small cohort of patients with renal cell cancer accrued to the Phase I trial of nivolumab consisted of 34 patients treated with doses of either 1 or 10 mg/kg every two weeks (Drake et al., 2013). Objective responses were seen at both doses, with 10 (29%)


914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 Q23 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964


988 989 990

3.3.1. Combination in melanoma With this foundation, a clinical trial combining the two antibodies in patients with melanoma was initiated in December 2009 (Wolchok

T

987

C

985 986

E

983 984

R

981 982

R

979 980

N C O

977 978

U

975 976

F

Although both ipilimumab and nivolumab disrupt the inhibitory interaction of CTLA-4 and PD-1 with their respective ligands, their biological activity appears to be complementary. While the anti-CTLA-4 effect of ipilimumab may be more prominent in the early stages of the T-cell activation perhaps at the lymph node, other activities of CTLA-4 blockade may complement the effect of PD-1 blockade. Regulatory T-cell depletion at the tumor site may occur as well as the removal of the suppressive role of CTLA-4 in TILs (Ahmadzadeh et al., 2009). The anti-PD-1 effect of nivolumab is likely to be more prominent in the tumor microenvironment, where PD-1 expression is very pronounced in TILs (Fig. 4). In vivo experiments carried out at BMS and elsewhere have confirmed the enhanced antitumor effect of the dual CTLA-4 and PD-1 blockade in mouse models (Korman et al., 2007; Curran et al., 2010; M. Selby et al., 2013).

O

974

971

R O

3.3. Combination of ipilimumab and nivolumab

969 970

P

973

967 968

et al., 2013b). This was, to our knowledge, the first clinical experiment in combining two checkpoint inhibitory molecules. The initial dose-escalation design planned for fixed doses of ipilimumab (3 mg/kg) to be combined with increasing doses of nivolumab (0.3 mg/kg in 14 patients, 1 mg/kg in 17 patients, and 3 mg/kg in 6 patients). At the highest dose level, 3 of 6 patients had asymptomatic, but persistent, grade III–IV serum lipase elevations. Thus a de-escalated dose-level group (ipilimumab 1 mg/kg and nivolumab 3 mg/kg) of 16 patients was added. The lower-dose cohorts presented grade III–IV AEs in 46% of the patients, with hepatic (15%), gastrointestinal (9%) and renal (6%) events being most common. A single case of pneumonitis and one of uveitis were also reported. The antitumor effects observed were impressive: in the 53 patients treated (38% had prior immunotherapy, 6% prior BRAF inhibitors), five CR and 16 PRs were observed (39.6%). Additional, unconfirmed PR (two patients), immune-related PR (four patients) or SD lasting ≥ 24 weeks (four patients) were also observed. Equally impressive was the rapidity of the tumors shrinkage (with the vast majority of responses occurring before 24 weeks from treatment initiation) and the depth of the effect, with 16 patients (including the five CRs) experiencing a reduction of ≥ 80% of their initial tumor burden within 12 weeks. As minimal residual nodules were not systematically biopsied, it is not clear whether there were additional CRs among the PRs who had a N 80% tumor shrinkage. What is clear is that these tumor shrinkages were sustained for a long time. The long-term results of this trial have been recently updated (Sznol et al., 2014) with the addition of yet another cohort of 40 evaluable patients given nivolumab at 1 mg/kg plus ipilimumab at 3 mg/kg. Unlike for the initial cohorts, for this additional cohort ipilimumab was discontinued after four induction courses. In this added cohort, 51% of

D

972

3.2.2.4. Other tumors. Results for nivolumab have been reported so far only in one other solid tumor type, ovarian cancer (Hamanishi et al., 2014). A total of 13 platinum-resistant patients have been accrued to a dose-escalation trial in Japan. Doses of 1 and 3 mg/kg every two weeks were being studied. A total of three PR (23%) with duration of 4+, 5 and 10+ months were observed, and an additional SD occurred in four patients. A single case of severe AE was seen, with fever, disorientation and gait disturbance.

E

965 966

11

Fig. 4. Rationale for combining ipilimumab and nivolumab. CTLA-4: Cytotoxic T-lymphocyte antigen 4; MHC: Major histocompatibility complex; PD-1: programmed death-1; TCR: T-cell receptor.


991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020

1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084

3.3.2. Combination in NSCL cancer In lung cancer, both squamous and non-squamous histologies were accrued, and patients had received no prior treatment (S.A. Antonia et al., 2014). Two regimens were explored, one with 3 mg/kg ipilimumab plus 1 mg/kg of nivolumab (N = 24) and the other with 1 mg/kg of ipilimumab plus 3 mg/kg of nivolumab (N = 25). Objective responses were seen with the first regimen in three (13%) of the patients, with seven additional SD. Median PFS was 16.1 weeks. With the second regimen, objective responses were five (20%), with additional nine SD. Median PFS was 14.3 weeks. Grade III–IV treatment-related AEs occurred in 49% of the patients across treatment arms. Grade III–IV pneumonitis occurred in three (6%) patients and was reversible with steroid treatment and discontinuation. With the small number of patients available, no major differences in safety or efficacy were apparent across treatment arms. Additional experience with this combination in the treatment of lung cancer needs to be gathered.

3.5. Urelumab (anti-CD137)

1104

O

F

1086 1087

R O

1042 1043

The theme of early combination of immune checkpoint inhibitors is being pursued also in the case of the clinical development of BMS986016, an anti-LAG-3 monoclonal antibody. As previously discussed for CTLA-4 and PD-1, LAG-3 and PD-1 can exert functionally distinct effects on CD8+ T-cells. Both PD-1 and LAG-3 have been observed to be expressed on CD8+ T cells which have become chronically activated but non-functional in the presence of a viral infection (Grosso et al., 2009; Wherry, 2011). In addition, CD8+ effector cells in TIL in murine models have also been observed to express both PD-1 and LAG-3 (Woo et al., 2012). Blockade of PD-1 and LAG-3 can revert this condition, which has been referred to as ‘lymphocyte exhaustion.’ The recently initiated Phase I clinical trial will first administer BMS-986016 alone, at escalating doses, in order to obtain a pharmacological and clinical characterization of the drug. Subsequently nivolumab will be added, including patients with solid tumors who had been previously exposed to checkpoint inhibition (ClinicalTrials.gov; NCT01968109).

P

1040 1041

1085

3.3.3. Combination in renal cancer In renal cancer, both untreated and pretreated patients were randomized to receive either one of the two regimens described above (Hammers et al., 2014). Again, nivolumab single-agent 3 mg/kg every two weeks was administered after the completion of the four induction courses with the combination. In the arm with 3 mg/kg ipilimumab and 1 mg/kg nivolumab, 23 patients were accrued (18 pretreated). One CR and 10 PRs (47%) were observed, with an additional eight SD. Median duration of response was not reached, and median PFS was 38.3 weeks. In the arm with 1 mg/kg ipilimumab and 3 mg/kg nivolumab, 21 patients were accrued (17 pretreated). Nine PR (43%), with additional five SDs were observed. Median duration of response was 31.1 weeks, and median PFS was 36.6 weeks. Also in this trial, responses appeared to occur rapidly, within six weeks in half of the responders, and only four relapses have occurred. Grade III–IV AEs occurred in 61% of the patients with the first regimen and 29% with the second. Hepatic (26%) and gastrointestinal (17%) grade III–IV AEs were seen in the latter; whereas no hepatic and a single case of gastrointestinal (5%) grade III–IV AEs were seen in the former. Even if preliminary, these results offer the opportunity to study a novel immunotherapy regimen highly effective in this disease.

1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103

Urelumab was the first agonistic checkpoint antibody that entered clinical trials (Logan et al., 2008; Sznol et al., 2008). Dosages of 0.3, 1, 3, 6, 10 and 15 mg/kg every three weeks were studied in the doseescalation phase (24 patients), and then in a randomized phase of three doses (1, 3, 10 mg/kg) in 97 additional patients. Four PRs and five SD were observed in 47 patients with melanoma, five SD in 26 patients with renal cell carcinoma, and no activity in 24 patients with ovarian cancer. However, the program was stopped because of the emergence of excessive liver toxicities at the higher doses (Li & Liu, 2013). This liver toxicity may be a class effect since it has also been described in mouse models of CD137 agonism (Dubrot et al., 2010). Clinical trials have been reinitiated at lower doses and are currently ongoing. A single-agent trial (Melero et al., 2013b) and a combination trial with rituximab (Kohrt et al., 2013) are being conducted, with special emphasis on lymphomas because of the demonstrated ADCC enhancement property of urelumab (Kohrt et al., 2012).

1105

3.6. BMS-936559 (anti-PD-L1)

1122

The monoclonal antibody BMS-936559 was the first compound directed to an immunomodulatory ligand which entered the clinic (Brahmer et al., 2012). Of note, this compound was studied by BMS in parallel with nivolumab, the PD-1 receptor directed monoclonal. Beginning in April 2009, 207 patients were accrued to a Phase I trial that studied escalating doses of 0.3 (three patients), 1 (37 patients), 3 (42 patients) and 10 (125 patients) mg/kg. No dose–effect was demonstrated, with rare grade III–IV AEs observed in 5% of the patients at each dose (except for 0.3 mg/kg). Doses were repeated every two weeks. Objective responses were seen in melanoma, with 9/52 (17%) PRs and 14 SD; in NSCL cancer, with 5/49 (10%) PRs and six additional SD; in renal cell carcinoma with 2/17 (12%) PRs and seven additional SD; and in ovarian cancer, with 1/17 (6%) PR and three additional SD. No activity was seen in 18 patients with colorectal, 14 with pancreatic, seven with gastric and four with breast cancer. Responses and disease stabilization were durable, and PFS rates at 24 weeks ranged from 12 to 41%. On the basis of the results observed in the parallel Phase I trial of nivolumab the authors indicated, with the limitation of the lack of a direct randomized comparison between the two compounds, that the observed frequency of objective responses appeared somewhat lower with the anti-PD-L1 monoclonal than with nivolumab.

1123

D

1038 1039

3.4. BMS-986015 (anti-LAG-3)

T

1036 1037

C

1034 1035

E

1032 1033

R

1030 1031

R

1028 1029

O

1027

C

1025 1026

N

1023 1024

the patients were pretreated and 7% had had a BRAF inhibitor. In this cohort, four CR and 13 PRs (42.5%) were observed, with four additional SD. Tumor reduction by N 80% was seen in 28% of the patients. Safety-wise, grade III–IV AEs occurred in 61% of the patients in the new cohort, with gastrointestinal (20%), skin (15%) and hepatic (12%) AEs being the most frequent. Two cases each of severe pneumonitis and uveitis were also seen. In the combined initial series and the added cohort, the durability of the effect was remarkable: median duration of response was not achieved, with only five relapses observed. Even more remarkable, the updated OS results of the initial cohorts indicated that 85% at one year and 79% at two years of the patients survived, with a median survival of 40 months and a median PFS of 27 weeks. OS data are not yet mature in the added cohort, but median PFS was 37 weeks. The results of this study have prompted the initiation of two randomized trials of the combination in previously untreated patients with metastatic melanoma. In the first trial, the combination is being compared to single-agent ipilimumab, with response rate as the primary endpoint. In the second, larger Phase III trial, each individual agent is being compared with the combination, with OS as the primary endpoint. The regimen selected for both trials utilizes a dosage of 3 mg/kg of ipilimumab and of 1 mg/kg of nivolumab given every three weeks, with ipilimumab discontinuation after four induction doses. At the time of this writing, both trials have been fully accrued (ClinicalTrials.gov; NCT01927419 [CheckMate 069]; NCT01844505 [CheckMate 067]).

U

1021 1022


E

12


1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121

1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143


1144

4. Novel aspects of immune checkpoint modulation

1145 1146

Exploring new areas of research offers the challenge of facing novel and/or unexpected aspects. The development of immuno-oncology 1147 treatments presented clinical characteristics that sometimes deviated 1148 from the known paths of more traditional chemotherapy and even 1149 targeted therapy, both in terms of safety and efficacy. 1150

4.1. Safety

1151

1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205

1206 1207

4.1.2. Management of immune-related AEs (ipilimumab) Given the nature of the AEs observed in the early trials of ipilimumab, a number of specific guidelines aimed at the prevention and/or treatment of these AEs were progressively introduced in the clinical development program (Chin et al., 2008). The majority of the algorithms introduced provided for careful monitoring at the beginning of treatment, early introduction of low-dose steroids in the presence of low-grade, but persisting toxicity, increase of steroid dosage in the case of no improvement and introduction of alternative immuno-suppressive therapy (such as infliximab) in the case of further resistance to steroid treatment. This type of approach requires a particular level of education and familiarity by both physicians and nurses who might be used to different

1258 1259

O

R O

P

D

E

1168 1169

T

1166 1167

C

1164 1165

E

1162 1163

R

1160 1161

R

1158 1159

N C O

1156 1157

4.1.1. Immune-mediated and immune-related AEs (ipilimumab) The fact that the stimulation of the immune system could produce both antitumor and ‘auto-immune’ events was recognized soon after the beginning of the clinical trials with ipilimumab. These AEs have been defined with various terms, with the one adopted in the current label of Yervoy® (ipilimumab) (Drugs@FDA, 2014) being ‘immunemediated adverse reactions’ and the most frequently used term in the literature being ‘immune-related AEs.’ The difference between the two definitions consists of the fact that ‘immune-mediated adverse reactions’ refers to a retrospective assessment where the inflammatory nature of the events is documented, directly or indirectly by the consideration of adjacent events and information (e.g., recovery after anti-inflammatory treatment) and can thus collapse multiple events representing the evolution of the same episode. On the contrary, ‘immune-related AEs’ refers to a prospective data collection approach that covers events that are presumably related to the mechanism of action and thus can prompt the investigator to institute anti-inflammatory guidelines early in the course of the event. Whereas the former approach is more accurate, the latter might be more practical, even if it could overestimate the nature of the event. Regardless of the terminology, these events present the common denominator of a proliferation of activated T-cell and pro-inflammatory reactions in normal organs and tissues. The most common severe AEs observed with ipilimumab (so far the most largely studied immune checkpoint inhibitor) are enterocolitis, hepatitis, dermatitis, endocrinopathy, and neuropathy. Enterocolitis, often starting with diarrhea symptoms, if not properly recognized and managed, could worsen and lead to bowel perforation and, indeed, these severe toxicity episodes occurred in the early clinical trials with ipilimumab (Beck et al., 2006). Histopathologic examination of colonic biopsies in patients with grade II or more diarrhea documented the presence of mixed inflammatory cell infiltrates in the lamina propria and foci of cryptitis, crypt abscesses, glandular destruction, and erosion of the mucosal surface (Berman et al., 2010). A comprehensive review of completed trials of ipilimumab at various doses in melanoma (14 trials with 1498 patients) reported an incidence of immune-related gastrointestinal AEs, grouped from different terms, of 33% (any grade) and 9% (grade III–IV), the latter including five cases of perforation (two lethal). One additional case of lethal gastrointestinal toxicity was reported, without perforation (Ibrahim et al., 2011). Another comprehensive review of the time of onset and resolution of AEs (limited to Phase II trials of ipilimumab in melanoma) reported a median time to onset of any gastrointestinal AE (except for grade I) of 6.6 weeks, duration of 2.1 weeks and resolution of 2.3 weeks (Lebbe et al., 2008). Hepatitis, consisting of liver function abnormalities potentially leading to hepatic failure, could also be related to an immunological activation. Also for the liver, biopsies documented an inflammatory pattern of injury that was non-specific and similar to that observed in acute viral and autoimmune hepatitis (Kleiner & Berman, 2012). The incidence of immune-related hepatic AEs was 1.6% (any grade) and 1.1% (grade III–IV) with two lethal cases of hepatic failure. Three additional cases of lethal hepatic failure could not be attributed to an immune-

U

1154 1155

mediated causality. Median time to onset was 6.7 weeks, median duration 3.6 weeks and median time to resolution was 4.0 weeks. Dermatitis included skin rash and pruritus, with a course beginning usually with a discrete pruritic and erythematous papular aspect and progressing to coalescing, scaled plaques (Jaber et al., 2006). The incidence of immune-related dermatologic AEs was 44.9% (any grade) and 2.6% (grade III–IV). Time to onset was 3.6 weeks, duration 3.3 weeks and time to resolution 6.1 weeks. Alterations of endocrine function have been reported after treatment with ipilimumab, most often consisting of hypopituitarism, hypothyroidism and adrenal insufficiency, in order of frequency. Indeed, the expression of the CTLA-4 receptor in the pituitary gland has been reported (Iwama et al., 2014). Clinical symptoms of hypopituitarism can include headaches, asthenia and decreased libido. Swelling of the gland has been documented by MRI (Dillard et al., 2010; Carre et al., 2012). Hypothyroidism and adrenal insufficiency can also present a subtle onset, but cannot be readily documented by radiologic testing. The incidence of endocrine immune-related AEs was 4.5% for any grade and 2.3% for grade III–IV. Time to onset was slower and duration longer than with other immune-mediated AEs: median of 9.2 weeks to onset, mediation duration of 20.6 weeks. Time to recovery has been reported to be 20.1 weeks; however, it must be realized that endocrine abnormalities, whereas having the tendency to be controlled by replacement therapy, could often require continuation of such supportive treatment. Neurologic AEs have been grouped under the terms of peripheral neuropathy, sensory and/or motor. However, given the extensive prior treatment administered to the patients in the safety database, it is difficult to attribute them to a clear immune-mediated mechanism. Thus, whereas an overall 6% incidence of neuropathy has been reported, three cases could be definitely attributed to an immunological mechanism. Two of these cases were low-grade, but one lethal case of Guillain–Barré syndrome has been reported (Wilgenhof & Neyns, 2011). Immune-mediated adverse reactions can also affect almost every organ. Thus, rare cases of uveitis, non-infectious pericarditis (Ibrahim et al., 2011), sarcoidosis (Vogel et al., 2012), lupus nephritis (Fadel et al., 2009), hemophilia (Delyon et al., 2011) and myasthenia gravis (Johnson et al., 2014b) have been reported in the literature. Whereas this is not intended to be a comprehensive list of potential AEs, it does underline that unusual immune-mediated events can always occur, and that they will demand a specific management. Of note, in the case of ipilimumab, treatment-related AEs and immune-related AEs observed in patients with advanced melanoma were generally similar to those accrued to the Phase III trial in prostate cancer, notwithstanding the particularly heavily pretreated characteristics of the patients accrued to the latter trial and the high ipilimumab dosage (10 mg/kg) utilized along with maintenance therapy (Beer et al., 2014) (Table 3). Elderly patients with melanoma (Chandra et al., 2013) also do not seem to present a significant difference in the safety profile as compared to younger patients.

F

The vast majority of the AEs appearing during immune modulation 1152 can be directly related to the mechanism of action of these agents, 1153 and, thus, referred to as ‘on target’ events.

13


1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 Q24 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257

1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270

14

Table 3 Immune-related or selected AEs reported with ipilimumab or nivolumab (%).

Nivolumab (Various, n = 306)

Any grade

Grade. III–IV

Grade V

Any grade

Grade III–IV

Grade V

Any grade

Grade III–IV

Grade V

64 45 33 5 1.6 1.3 [2] – –

18 3 9 2 1.1 0.4 – – –

[9]a – [3] – [2] – [1] – –

63 35 41 5 10 – 2 – –

26 1.0 18 2 5 – – – –

[2] – [2] – – – – – –

46 25 14 10 6 – – 6 2

6 [1] 1.0 1.0 1.3 – – 2 [1]

[3] – – – – – – [3] –

AEs: adverse events; irAEs: immune-related adverse events. a In brackets: absolute numbers.

1271

types of toxicity management measures for standard antitumor therapy. Thus, effectiveness of these guidelines was also monitored and reported (Richards et al., 2009). The analysis concluded that a reduction of the rate of gastrointestinal perforation was achieved with the implementation of these guidelines. The experience accumulated during the course of the clinical development program was utilized in implementing a Risk Evaluation and Mitigation Strategies (REMS) program after the regulatory approval of ipilimumab. Another specific trial was carried out in the attempt of reducing gastrointestinal tract inflammation by administering a topically active, non-reabsorbable steroid, budesonide (Weber et al., 2009). Unfortunately that trial failed to show a protective effect in the budesonide arm. In the initial clinical trials, concern was raised from investigators over whether corticosteroids would dampen antitumor immunity. Three analyses conducted in patients with metastatic melanoma accrued to Phase II trials (Amin et al., 2009) and to the two Phase III trials in previously treated (Lorigan et al., 2010) and in previously untreated patients (Baurain et al., 2012) help dispel this notion, reporting similar long-term outcomes in the steroid-treated and steroid-untreated groups. Based on an initial report linking toxicity to response (Downey et al., 2007), BMS has explored whether there is an association between OS and immune mediated toxicity. Analyses conducted in two different series of patients with metastatic melanoma showed no significant correlation between OS and the occurrence of immune-related AEs (Lutzky et al., 2009; Di Giacomo et al., 2013).

1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317

C

1286 1287

E

1284 1285

R

1282 1283

R

1280 1281

O

1278 1279

4.1.3. Long-term safety (ipilimumab) The activation of the immune system achieved with inhibitory checkpoint blockade poses also the question of potential long-term effects. This consideration is compounded by the fact that long-term survivorship can be attained with this form of treatment. Analyses of the long-term survivors in the two metastatic melanoma Phase III trials of ipilimumab have thus been conducted. In the trial in previously treated patients, 94 patients who survived ≥ 2 years were identified (16 with placebo, 24 with ipilimumab alone and 54 with the combination). In the placebo arm, a single patient presented grade I vitiligo during long-term follow-up. In the ipilimumab arm, a single patient presented grade I vitiligo and grade II hypothyroidism. In the combination arm, three patients presented grade I vitiligo, one patient grade II hypogonadism, and one patient grade III colitis. This patient also presented grade I diarrhea and grade II proctitis, and the AEs recovered partially after 147 days (McDermott et al., 2013). In the trial of previously untreated patients, 112 survivors for ≥ 2 years were identified (44 randomized to DTIC, 68 to DTIC plus ipilimumab). In the DTIC arm, cases of amylase increase, lipase increase, and low-grade diarrhea (one each) were seen. In the combination arm, three cases of pruritus (one severe), two of skin rash (one severe), and

C

1276 1277

N

1274 1275

U

1272 1273

one of mild skin hypopigmentation were observed. In addition, there was one case of low-grade liver function test elevation. Of note, all of these events, except for one low-grade pruritus and one with skin hypopigmentation, occurred in patients still receiving ipilimumab maintenance treatment (Thomas et al., 2012). Altogether, and in a fairly sizeable group of patients, these analyses reassure on the long-term safety of the compound. Interestingly, the observation of vitiligo is related to the persisting presence of an activated immunologic system.

1318 1319 1320 1321 1322 1323 1324 1325

4.1.4. Selected AEs (nivolumab) A somewhat different approach to the one adopted to assess AEs in patients given ipilimumab was adopted for nivolumab. Clinically important AEs were captured prospectively, independent of causality, and organized into organ-defined categories (i.e., gastrointestinal tract, skin, liver, etc.). Certainly the mechanism of action of the two compounds points to the potential for similar toxicity-inducing mechanisms. In the case of nivolumab, however, the incidence of selected AEs and their severity appeared inferior to that reported with ipilimumab (Table 3) even with long-term safety assessment of the Phase I group of patients who, contrary to the ipilimumab patients, could continue to receive nivolumab for two years (Hodi et al., 2013b). Whereas the data presented in Table 3 do not intend to compare the three series of patients (which address different populations of patients and utilize different doses and regimens), their display allows for an initial quantitative and qualitative early assessment of the results that will be ultimately resolved by the ongoing randomized Phase III trials. In general, the type of AEs observed with nivolumab are consistent with those observed with ipilimumab, with the sole exception of pneumonitis and nephritis. Pneumonitis has been observed in most trials of nivolumab, with incidences ranging from 3 to 6% (any grade) and from 0 to 2% (grade III–IV). Higher incidences of 14% and 7%, however, were observed in the pilot trial combining nivolumab with chemotherapy in lung cancer. Nephritis, mostly documented by increases of serum creatinine, occurred in 2% of the cases (any grade) and in a single patient (grade III–IV) during long-term follow-up of the Phase I trial (Hodi et al., 2013b). The AEs that occur during nivolumab treatment can be managed by utilizing the same algorithm established for ipilimumab, and reversibility has been achieved.

1326

4.1.5. Other safety aspects of immune-checkpoint modulation The occurrence of dose-limiting hepatic toxicity when administering ipilimumab in combination with vemurafenib and when administering single-agent urelumab at high doses has been previously discussed. Of note, also the combination of ipilimumab and DTIC appears to produce a marked increase of hepatic, but not of gastrointestinal, toxicity as compared to the historical experience of single-agent ipilimumab at the same dose (F.Y. Feng et al., 2012).

1355

T

t3:15 t3:16

F

Any irAE or selected AEs Dermatologic Gastrointestinal Endocrine Hepatic Ocular Neurologic Pulmonary Renal

Hodi (2013)

Ipilimumab (Prostate, n = 393)

O

t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14

Beer et al. (2014)

Ipilimumab (Melanoma, n = 1498)

R O

t3:5

Q12

Ibrahim et al. (2011)

P

t3:4

D

Q14t3:3 Q13

E

t3:1 t3:2



1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354

1356 1357 1358 1359 1360 1361 1362


1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383

1387 1388 1389

t4:1 t4:2

Table 4 AEs reported with ipilimumab and nivolumab combinations (%) (all cohorts combined).

1399 1400 1401 1402 1403 1404 1405 1406

C

E

R

1397 1398

R

1395 1396

N C O

1394

U

1392 1393

T

1407

4.2.1. Immune-related response criteria Early in the clinical development of ipilimumab it became evident that certain patients would not present the usual pattern of tumor shrinkage familiar to the oncologists when administering cytotoxic, hormonal or targeted therapy. Not only did certain patients require longer time to mount an immunological reaction, and therefore an antitumor effect, but also, in certain cases, tumor lesion growth and even the appearance of new lesions would precede the antitumor response. Thus, patients who would be classified as having progressive disease by standard WHO criteria or Response Evaluation Criteria In Solid Tumors (RECIST) could, instead, be receiving benefit from treatment. Although the resection of certain tumor nodules increasing in size was carried out, and it revealed that such an increase was in fact often due to TIL infarction, this could not account for every single case of pseudo-progression. Spurred by the experience of patients returning to the clinic with shrinking or disappearing tumors after an initial evaluation of progression, protocols were amended to provide for additional tumor evaluation after progression and a systematic review of the kinetics of response was carried out. The large database of ipilimumab was

1390 1391

Q17t4:3 Q16 Q15

Sznol et al. (2014)

Antonia (2014)

Hammers et al. (2014)

t4:4

Melanoma (n = 94)

NSCLC (n = 49)

Renal (n = 44)

t4:5

Any grade

Grade III–IV

Any grade

Grade III–IV

Any grade

Grade III–IV

77 39 19 22 4 4 3 21 17

9 14 3 14 3 2 3 15 6

24 41 6 10 – 12 – 14 12

4 18 4 8 – 6 – 8 4

39 23 25 15 – 7 11 – –

– 14 – 5 – – – – –

t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14

Dermatologic Gastrointestinal Endocrine Hepatic Ocular Pulmonary Renal Lipase Amylase

4.2.2. Long-term effects and their prediction The kinetics of survival also appears to differ between immunologic and traditional therapies (Hoos et al., 2010b). The long-adopted tenet by which events in a time-to-event analysis occur by constant proportional hazard is being challenged by the shape of survival curves presented in this review in Figs. 2 and 3. One of the practical consequences of this situation is that a classical event-driven study design might require very long times to reach its maturity. Thus, the question of identifying proper predictors and/or surrogates for long-term survival has become very important. Two different exploratory approaches have been proposed by BMS. First, an analysis has been conducted of the association of tumor shrinkage and OS (Feng et al., 2014). Although this methodology had been previously utilized for standard treatments in lung and colorectal cancer (Wang et al., 2009; Suzuki et al., 2012; Bruno et al., 2014), it had not been tested with immunological therapies. Databases of single-agent ipilimumab and nivolumab in various tumor types were utilized, and tumor assessments at week 8 or 12 were analyzed, together with the relative pharmacokinetics results, in order to develop a tumor growth dynamics model to be used to predict survival outcomes. In the case of ipilimumab in melanoma, the ECOG performance status, baseline LDH, drug clearance and percent tumor shrinkage at week 8 predicted for survival outcomes. Of note, drug exposure did not. In the case of nivolumab in melanoma and NSCL cancer, again the percent tumor shrinkage at 8 weeks predicted for survival. This type of modeling will need a validation from a prospective experiment, but it could help refining the possibility of an early prediction of the ultimate patient benefit. A second approach has been taken by modeling an analysis of OS in metastatic melanoma patients which takes into consideration the ‘stabilization’ of the survival curve after two to three years of follow-up, as seen in Figs. 2 and 3. A total of 10,000 simulations were carried out, showing that, based on a standard approach with a proportional (exponential) hazard model, a much longer trial would be needed and/or the study power will be reduced (Chen, 2013a). A proposed ‘milestone’ analysis,

1438

F

When the tumor cell is not directly targeted by anticancer therapy, as in the case of immunomodulatory interventions, the kinetics of efficacy can be expected to be different.

1370 1371

O

1386

1369

R O

4.2. Efficacy

1367 1368

1408 1409

P

1385

1365 1366

utilized, and this resulted in the proposal for establishing new immunerelated response criteria (Wolchok et al., 2009). Different patterns or kinetics of tumor response were described, and this allowed the possibility of identifying a group of about 10% of patients who would have not met standard criteria for clinical benefit (CR or PR or SD) but who presented long-term survival outcomes similar to those assessed as benefitting by standard criteria. It is not clear whether all immunological interventions will produce the same behavior in tumor response and, perhaps, the very mechanism of T-cell activation that ipilimumab produces might have magnified this effect. However, the same type of phenomenon has been observed with single-agent nivolumab, although with a lower frequency (5% according to Hodi et al., 2013b), and less commonly with the combination of nivolumab and ipilimumab (Sznol et al., 2014). Certainly, in the case of the two randomized Phase II trials of ipilimumab and chemotherapy in NSCL and small cell lung cancer, it was the adoption of immunerelated PFS criteria that helped in the selection of the sequential regimens that were brought into Phase III. The immune-related response criteria were initially developed as a tool to help the oncologist and the patient to better decide if continuation of treatment could be warranted even in the presence of a tumor progression. This goes against the criteria by which oncologists have been historically trained to operate and can provide a sense of the difficulties that innovation brings about. The immune-related response criteria still need prospective validation before becoming accepted as regulatory endpoints. However, regulatory health authorities have already accepted the concept of “treating beyond progression” in selected cases of anticancer drug development (FDA/Center for Biologics Evaluation & Research [CBER], 2011; European Medicines Agency [EMA], 2012).

D

1384

It is too early to present a definitive safety assessment of the compounds at the beginning of clinical development such as BMS-986016 (anti-LAG-3) and of low-dose urelumab (anti-CD137). However, in the case of BMS-936559 (anti-PD-L1), the safety profile initially observed in the Phase I trial confirms the suggestion of a low level of toxicity when blocking the PD-1/PD-L1 pathway. The interest emerging on the combination of ipilimumab and nivolumab is intensely scrutinizing both the efficacy and the safety characteristics of this innovative approach in various tumor types. The current information available is summarized in Table 4. Again, whereas the data are still somewhat early, it is conceivable that this synergistic combination could elicit both increased efficacy and toxicity. It would appear that, at least qualitatively, immune-related AEs are in line with what was previously observed with each individual checkpoint inhibitor. The possible exception is represented by increases of serum lipase and amylase that, albeit asymptomatic, had not been previously reported with either agent. The pilot nature of these trials, utilizing different dosages and regimens of the combination, is still a work in progress. However, it is also conceivable that different tumor types and/or different stages of disease might require different dosing approaches. This is likely to hold true also for other future combinations of other immune checkpoint modulators.

E

1363 1364

15


1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437

1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472

1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537

F

O

R O

1492 1493

P

1490 1491

1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564

D

1488 1489

opposed to the classical concept of tumor resistance, can be addressed by reinduction treatment. In the case of ipilimumab, reinduction was studied in metastatic melanoma patients who had disease progression after completing their induction, with or without maintenance. With ipilimumab, in a series of six Phase II trials whose patients received reinduction at 10 mg/kg, a total of seven CR and 21 PRs (23%) with 31 additional SD were observed in 122 reinduced patients (Neyns et al., 2013). In the Phase III trial for previously treated patients, patients were reinducted with ipilimumab 3 mg/kg (and gp100 if they had been randomized to the combination arm). A total of one CR and six PRs (18%) with 14 SD were observed in 38 patients. Neither of the patients reinduced with gp100 alone responded (Robert et al., 2010). Finally, reinduction was allowed also for patients who were accrued to the EAP. In the EAP that adopted 3 mg/kg, reinduction was administered at the same dosage. Whereas objective response data were not collected, median survival in 108 reinduced patients was 21.1 months, with one- and two-year survivals of 81% and 43%, respectively (K.A. Margolin et al., 2013). Of note, the incidence of grade III–IV immune-related AEs during reinduction was 14% at 10 mg/kg and 5% in both series at 3 mg/kg. The published experience of reinduction therapy with nivolumab is, so far, limited to a single, previously-treated melanoma patient who had been accrued to the first Phase I trial (Brahmer et al., 2010) and obtained, after receiving multiple doses of 10 mg/kg, a PR, discontinued treatment after several months of therapy and remained in response for an additional 22 months without therapy. Upon relapsing, the patient was reinduced with nivolumab and obtained a PR lasting for 16+ months (Lipson et al., 2013).

1565

4.3. Biomarkers

1566

4.3.1. Prognostic factors (ipilimumab) In discussing the key question of the role of biomarkers in cancer treatment, it is important to take into consideration both clinical and mechanistic (biological) factors. For instance, in the case of ipilimumab, the vast majority of the predefined clinical subsets of potential prognostic value in all of the Phase III trials completed pointed to a positive effect of treatment. These included age, gender, disease stage, ulceration of primary LDH, liver function tests, prior treatment, Gleason score, pain and geography (Hodi et al., 2010; Robert et al., 2011; Eggermont et al., 2014; Kwon et al., 2014). The few exceptions were women with age ≥ 50 years in the Robert trial, unknown status of tumor ulceration in the Eggermont trial, and presence of visceral metastases in the Kwon trial. Given the usually small number of patients in subsets and the fact that confidence intervals are quite wide, these results must be viewed in the proper context. It would appear that these clinical factors might have more of a prognostic than a predictive role as an effect was observed also for the less favorable subsets.

1567

4.3.2. Predictive factors (ipilimumab) Understanding the mechanism of pharmacologic immune modulation becomes quite complex, as both the biomarkers that are inherent to the tumor cell and those that involve the T-cell activation are relevant. Moreover, the variations that might occur over time in the tumor microenvironment also need to be considered, as the so-called ‘plasticity’ of the immune system response might become apparent only after the beginning of treatment (Ascierto et al., 2013). The next step in the search of potential predictive biomarkers might involve the genomic characterization associated with outcome. Initial reports have indicated that the CT60 G/A genotype may be associated with both better response and OS in melanoma (Queirolo et al., 2011). However, the results of genetic polymorphism have not been confirmed (Hamid et al., 2011). Somatic diversification of the T-cell repertoire may also provide a characteristic which may define the antitumor immune response. A decline in the number of diversified T-cell clonotypes

1584

T

1486 1487

C

1484 1485

4.2.3. Dosing regimens Whereas traditional cytotoxic therapy generally presents dosedependent effects resulting in the definition of dose-limiting toxicities and of a maximum tolerated dose, recently introduced novel targeted therapies do not always follow this pattern. The same consideration applies to immune checkpoint-directed therapy, compounded by the fact that the therapeutic target is not located at the tumor level. In the case of ipilimumab, Phase I trials were unable to attain a maximum tolerated dose up to a dose of 20 mg/kg (Weber et al., 2008). Based on an in vitro cell-based competition assay measuring the blockade of the CTLA-4 binding to the B7-1 ligand, it was determined that a maximum effect was sustained at concentrations of 20 μg/ml of ipilimumab. These concentrations could not be achieved in patients given a dose of 0.3 mg/kg but were achieved in about 30% of patients given 3 mg/kg and in 95% of patients given 10 mg/kg. Of note, steady-state was reached in most patients after three induction doses of ipilimumab (Dai et al., 2008). These analyses reported also an association between exposure and the occurrence of immune-related AEs. These data supported the conduct of many ipilimumab studies at 10 mg/kg, which began before the results of the first Phase III trial of ipilimumab at 3 mg/kg became available. A randomized trial (Wolchok et al., 2010a) suggested the existence of a potential clinically relevant dose effect over the range of 0.3, 3 and 10 mg/kg, reaching statistical significance for response rates. However, as patients who experienced tumor progression in the two lower-dose arms were re-challenged with ipilimumab at 10 mg/kg, long-term survival effects are not informative. Of note, a dose–effect appeared to exist also for the safety aspect of the equation. The question of optimal dosing of ipilimumab is being addressed by two large Phase III trials in melanoma, one being conducted in metastatic and one being conducted in the adjuvant setting. At the time of this writing, both trials have completed their accrual (ClinicalTrials.gov; NCT01515189; NCT01274338). Although initial trials of ipilimumab adopted only the four induction doses regimen, subsequent trials have proposed the administration of maintenance therapy. In each of the Phase III trials in first-line metastatic melanoma (Robert et al., 2011), in adjuvant melanoma (Eggermont et al., 2014) and in prostate cancer (Kwon et al., 2014) a maintenance regimen of 10 mg/kg given every 12 weeks was adopted. Patients who received maintenance in the ipilimumab arms were 17%, 42% and 24%, respectively. Whereas it is clear that less heavily pretreated patients (in the adjuvant trial) had a higher rate of compliance, the role of maintenance therapy and its tolerability still needs to be fully elucidated. In the case of nivolumab, a clear dose–effect was not established with a possible exception of efficacy at the lowest dosage tested in NSCL cancer. Whereas the pharmacokinetics of the antibody were linear, PD-1 receptor occupancy was assessed by antibody binding of circulating CD3+ T-cells and it ranged from 64 to 72% at all doses tested in the Phase I trials (Brahmer et al., 2010; Topalian et al., 2012). Serum half-life of nivolumab was 12 days at doses of 3 mg/kg and below, and 20 days at doses of 10 mg/kg. On the basis of these data, and also by virtue of the favorable safety profile observed, the regimen selected for further development of nivolumab was 3 mg/kg every two weeks or equivalent regimens administered every three weeks. For BMS-936559 (anti-PD-L1), data were similar, with lack of a dose–effect, linear pharmacokinetics, half-life of 15 days and median receptor occupancy exceeding 65% in all dose cohorts treated (Brahmer et al., 2012). The issue of lack of responsiveness to immunotherapy, as

E

1482 1483

R

1481

R

1480

O

1479

C

1477 1478

N

1475 1476

to be carried out after the time of the curve stabilization is expected, could result in savings in the size of the trial as well as in the time of study analysis, preserving statistical power even in the presence of delayed effects. Moreover, this type of analysis could maintain the integrity of the study if there were a desire to maintain the standard log-rank analysis to characterize final survival results. And, obviously, this analysis could be performed only in the presence of a robust supporting prior evidence of outcome stabilization (Chen, 2013b).

U

1473 1474


E

16


1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583

1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599

17

O

F


1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644

4.3.4. Predictive factors (sequence and combination) Unlike biomarkers that reflect mutations in tumor DNA, those of checkpoint inhibitors at the tumor and microenvironment level can be more dynamic, depending upon changes in time and in prior exposure to agents which can enhance it. Indeed PD-L1 expression can be induced by cytokine exposure, CTLA-4 expression on the TIL can be increased by nivolumab treatment, and PD-1 expression is induced in T cells after CTLA-4 blockade (Curran et al., 2010; M. Selby et al., 2013). In patients previously exposed to ipilimumab, the sequential treatment with nivolumab produced 4/8 responses in PD-L1 positive and 1/13 responses in PD-L1 negative melanoma patients (28.8 antibody staining) (Callahan et al., 2013). Thus, the observation of tumor responses even in the PD-L1 negative population was confirmed. More importantly, the concomitant administration of ipilimumab and nivolumab produced

1645 1646

5. Conclusions

1664

The progress in understanding the immune system in general and the immune checkpoint modulation in particular is allowing a new chapter to be written in the history of drug development. It is clear that a new modality of cancer treatment is being introduced which would integrate with or possibly substitute traditional forms of therapy. The availability of multiple agents with different mechanisms of immunologic response activation poses the challenge but also offers the opportunity for further innovation. What was presented only a few years ago as the promise of a novel approach is now delivering results that, if confirmed, might exceed those predictions (Fig. 5). The continuous connectivity between drug developers and academic investigators, bench scientists and clinical oncologists is the formula that can accelerate this process.

1665

Conflict of interest statement

1680

D

P

6/13 responses in PD-L1 positive and 9/22 responses in PD-L1 negative patients: the latter observation suggests that the combined regimen efficacy might not depend solely upon ligand expression (Callahan et al., 2013). In this trial, the combination regimen produced responses in patients, irrespective of their baseline absolute lymphocyte count, and increases of HLA-DR+ CD4+ and CD8+ T-cells and of Ki67+ CD4+ and CD8+ T cells were also produced. However, low levels of myeloidderived suppressor cells correlated with better response. Notwithstanding the differences in assays, it appears that ligand overexpression correlates with better responses when blocking the PD-1/PD-L1 pathway. However, given the plasticity of the immune system and the observation of antitumor effects even in the PD-L1 negative population, it will be important to correlate ligand expression with the hoped-for long-term effects of treatment. Thus, it is expected that validation of the ligand expression as a predictive biomarker will be possible only upon the availability of long-term results from Phase III trials. Fortunately the vast majority of such trials have been designed prospectively with this need in mind.

E

T

1612 1613

4.3.3. Predictive factors (PD-1/PD-L1 pathway) The assessment of potential pharmacodynamic and predictive biomarkers at the tumor and microenvironmental level is even more important in the case of nivolumab, due to the expression of the PD-L1 ligand on tumors and TILs, and of PD-L2 on macrophages and dendritic cells. Differences in the type of assays utilized to assess ligand expression must be considered when reviewing the current information. In the Phase I trial by Topalian (Topalian et al., 2012) a murine antitumor PD-L1 monoclonal antibody, 5H1, was utilized on formalinfixed, paraffin-embedded new or archived specimens. Positivity was defined by a 5% expression threshold. With this technique, 9/25 responses were seen in PD-L1 positive and 0/17 in PD-L1 negative patients. A different approach, which utilized an automated assay and a different monoclonal antibody, 28-8, was also tested, identifying 7/16 responses in PD-L1 positive and 3/18 responses in PD-L1 negative patients. In this series, which considered ≥ 5% cell membrane staining as a positive result, PD-L1 positivity appeared also to correlate with longer PFS and OS (Grosso et al., 2013). Other studies with other monoclonals affecting the PD-1/PD-L1 pathway have adopted other assays, cut-off and evaluation criteria, including PD-L1 expression on both tumor cells and TILs (Powderly et al., 2013; Daud et al., 2014).

C

1610 1611

E

1608 1609

R

1606 1607

R

1604 1605

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might be associated with shorter OS in both prostate cancer and melanoma (Fong et al., 2014). The existence of immunologic predictive biomarkers has been also interrogated with serial tumor biopsies conducted in a Phase II trial of ipilimumab in melanoma (Hamid et al., 2011). Whereas there was no correlation between the baseline presence of TIL and clinical benefit (defined as CR, PR or SD), an increase of TILs after treatment was associated with clinical benefit. FOXP3 and IDO expression at baseline immunostaining of leukocytes, however, did correlate with positive clinical benefit for ipilimumab treatment.

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Fig. 5. The promise and the present of metastatic melanoma therapy with immune. checkpoint inhibitors.

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All the authors of this review are employed by the Bristol-Myers 1681 Squibb Company. 1682 Acknowledgments

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A., Koyama, S., Carretero, J., Altabef, A., Tchaicha, J. H., Christensen, C. L., et al. (2013). Activation of the PD-1 pathway contributes to immune escape in EGFRdriven lung tumors. Cancer Discov 3, 1355–1363. Altomonte, M., Queirolo, P., Testori, A., Ascierto, P., Danielli, R., Di Giacomo, A. M., et al. (2009). The Italian experience on the feasibility and safety of ipilimumab therapy in pretreated metastatic melanoma patients. Eur J Cancer 7(Suppl. 2), 585 (Abstract 9328). Amin, A., DePril, V., Hamid, O., Wolchock, J., Maio, M., Neyns, B., et al. (2009). Evaluation of the effect of systemic corticosteroids for the treatment of immune-related adverse events (irAEs) on the development or maintenance of ipilimumab clinical activity. J Clin Oncol 27(Suppl. 15) (Abstract 9037). Amin, A., Plimack, E. R., Infante, J. R., Ernstoff, M. S., Rini, B. I., McDermott, D. F., et al. (2014). Nivolumab (anti-PD-1; BMS-936558, ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC). J Clin Oncol 32(Suppl. 5) (Abstract 5010). Ansell, S. M., Geyer, S. M., Hurvitz, S., Fernando, D., Habermann, T. M., Inwards, D. J., et al. (2006). Phase I/II study of ipilimumab (MDX-010), an anti-CTLA-4 monoclonal antibody, in patients with follicular non-Hodgkin lymphoma. Blood 108 (Abstract 2729). Ansell, S. M., Northfelt, D. W., Flinn, I., Burris, H. A., Dinner, S. N., Villalobos, V. M., et al. (2014). Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies. J Clin Oncol 32(Suppl. 15) (Abstract 3024). Antonia, S. J., Brahmer, J. R., Gettinger, S. N., Man Chow, L. Q., Juergens, R. A., Shepherd, F. A., et al. (2014). Nivolumab (anti-PD-1; BMS-936558, ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC). 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M., Yang, J. C., et al. (2005). Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4. J Clin Oncol 23, 6043–6053. Awada, A., Rolfo, C. D., Rottey, S., Ysebrant de Lendonck, L., Schroyens, W. A., Offner, F., et al. (2014). A phase I, first-in-human study of ARGX-110, a monoclonal antibody targeting CD70, a receptor involved in immune escape and tumor growth in patients with solid and hematologic malignancies. J Clin Oncol 32(Suppl. 15) (Abstract 3023). Bashey, A., Medina, B., Corringham, S., Pasek, M., Carrier, E., Streicher, H., et al. (2005). Phase I study of a neutralizing monoclonal anti-CTLA4 antibody (MDX-010) in patients with relapse of malignancy after allogeneic hematopoietic stem cell transplantation. Blood 106 (Abstract 2017). Bauman, J. E., Gooding, W. E., Clump, D. A., Kim, S., Karlovits, B. J., Heron, D. E., et al. (2014). Phase I trial of cetuximab, intensity modulated radiotherapy (IMRT), and the anti-CTLA-4 monoclonal antibody (mAb) ipilimumab in previously untreated, locally advanced head and neck squamous cell carcinoma (PULA HNSCC). J Clin Oncol 32(Suppl. 5) (Abstract TPS6104).

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are too many to be individually named, but it is on their behalf that this review has been written. We are truly indebted to the academic basic scientists and clinical investigators who have collaborated over the years in this remarkable journey, sharing ideas and experiences. Finally, we are most grateful to all the cancer patients, irrespective of the outcome of their treatment, for having the courage to undertake the clinical investigations which, thanks to them, can benefit a large number of patients. Professional editorial assistance was provided by Rebecca Turner, Ward Pedersen, and Karin McGlynn at StemScientific and was funded by Bristol-Myers Squibb.

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A., Hamid, O., Weber, J. S., Pavlick, A. C., Hodi, F. S., Amin, A., et al. (2013). Ipilimumab retreatment following induction therapy: the expanded access program (EAP) experience. J Clin Oncol 31(Suppl. 15) (Abstract 9041). Margolin, K., Wong, S. L., Penrod, J. R., Song, J., Chang, I., Hebden, T., et al. (2013). Effectiveness and safety of first-line ipilimumab (IPI) 3 mg/kg therapy for advanced melanoma (AM): evidence from a U.S. multisite retrospective chart review. Eur J Cancer 49(Suppl. 2) (Abstract 3742). Masters, G., Barreto, L., Girit, E., & Jure-Kunkel, M. (2009). Antitumor activity of cytotoxic T-lymphocyte antigen-4 blockade alone or combined with paclitaxel, etoposide, or gemcitabine in murine models. J Immunother 32, 994. McDermott, D., Haanen, J., Chen, T. -T., Lorigan, P., & O'Day, S. (2013). Efficacy and safety of ipilimumab in metastatic melanoma patients surviving more than 2 years following treatment in a phase III trial (MDX010-20). Ann Oncol 24, 2694–2698. 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Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med 3, 682–685. Merchant, M. S., Baird, K., Wexler, L. H., Rodriguez-Galindo, C., & Mackall, C. (2012). Clin Oncol 30(Suppl. 15) (Abstract 9545). Moser, J., Pulido, J., Dronca, R. S., Markovic, S., & Mansfield, A. S. (2014). The Mayo Clinic experience with the use of kinase inhibitors (KIs), ipilimumab, and bevacizumab in the treatment of metastatic ocular melanoma. J Clin Oncol 32(Suppl. 15). Motzer, R. J., Rini, B. I., McDermott, D. F., Redman, B. G., Kuzel, T., Harrison, M. R., et al. (2014). Nivolumab for metastatic renal cell carcinoma (mRCC): results of a randomized, dose-ranging phase II trial. J Clin Oncol 32(Suppl. 5) (Abstract 5009). Neyns, B., Weber, J. S., Lebbé, C., Maio, M., Harmankaya, K., Hamid, O., et al. (2013). Ipilimumab (Ipi) retreatment at 10 mg/kg in patients with metastatic melanoma previously treated in phase II trials. 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pH-dependent antigen-binding antibodies as a novel therapeutic modality.

[Development of HPLC analysis methods for therapeutic monoclonal antibodies].

Use of monoclonal antibodies in vivo as a therapeutic strategy for alloimmune or autoimmune reactivity: the Besançon experience.

The geriatric institution as a therapeutic modality.

Monoclonal antibodies: pharmacokinetics as a basis for new dosage regimens?

Immunomodulatory Monoclonal Antibodies in Combined Immunotherapy Trials for Cutaneous Melanoma.

Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications.

Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies.

Glypican-3 antibodies: a new therapeutic target for liver cancer.

Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.

Development of human monoclonal antibodies to diphtheria toxin: A solution for the increasing lack of equine DAT for therapeutic use?

Anti-idiotype monoclonal antibodies as vaccines for human cancer.

Therapeutic ultrasound as a treatment modality for chronic rhinosinusitis.

Daratumumab, Elotuzumab, and the Development of Therapeutic Monoclonal Antibodies in Multiple Myeloma.

Hydrogen-deuterium exchange mass spectrometry as an emerging analytical tool for stabilization and formulation development of therapeutic monoclonal antibodies.

New frontiers in oncology: biosimilar monoclonal antibodies for the treatment of breast cancer.

Monoclonal antibodies as reagents.

Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: development and optimization of a washing step.

Integrating novel therapeutic monoclonal antibodies into the management of head and neck cancer.

Monitoring therapeutic monoclonal antibodies in brain tumor.

Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM).

A new pediatric protocol for rapid desensitization to monoclonal antibodies.

Combination epigenetic and immunotherapy overcomes resistance to monoclonal antibodies in hematologic malignancies: A new therapeutic approach.

SA-4-1BBL as a novel adjuvant for the development of therapeutic cancer vaccines.