Immunotherapy: A New Era

in Cancer Treatment

This is to acknowledge that Dr. Deepak

Nijhawan M.D. PhD has disclosed that he does not have any financial interests or other relationships with commercial concerns related directly or indirectly to this program. Dr.

Nijhawan will not be discussing off-label uses in his presentation. Deepak Nijhawan

Assistant Professor

Division of Oncology and Department of Biochemistry

Dr. Nijhawan supervises a laboratory interested in discovering the mode of action for chemicals that are toxic to cancer cells. These discoveries have the potential to not only unveil new biology, but also lead to potential therapies.

Purpose: A review of the discovery and development of immunotherapeutic drugs for cancer.

Overview: Drugs that manipulate the immune system have recently been shown to have durable clinical responses in patients with melanoma. In this protocol and presentation, we discuss the scientific and clinical milestones that led to that achievement. Each of these milestones was surrounded by controversy and there are lessons to be learned in how the challenges were overcome.

Educational objectives:

o Learn the experimental rationale for cancer

immunosurveillance

o Learn how CTLA-4 was discovered and the rationale

for targeting CTLA-4 in cancer

o Review clinical trials for immunotherapeutic

agents Cancer continues to be a common cause for death, and there is an urgent need for new treatments. Most of our attempts to treat cancer have yielded modest improvement in outcomes measured in weeks to months of prolonged survival.

These modest gains are further compromised by severe, sometimes fatal adverse events.

For more than a century, it has been proposed that the immune system could be harnessed to fight cancer. In 1957,

Burnett proposed that tumors might be recognized as foreign by the immune system and that one strategy to eradicate cancer would be to activate that immune response. In the

50 years since his proposal, immunotherapeutic agents have been developed and are beginning to show durable responses lasting more than three years in some cancer patients.

(Figure 1) In this protocol, I will focus on the controversies surrounding three major milestones in the development of these agents. These milestones are the following: 1) establishing a biological rationale in mouse models that the immune system has a role in cancer surveillance, 2) the discovery of cytotoxic t cell lymphocyte 4 (CTLA-4) as a protein involved in immune checkpoint, and 3) the success of ipilimumab in improving the overall survival of patients with metastatic melanoma in a randomized controlled trial. By achieving these 138 D. Berman et al. / Pharmacology & Therapeutics 148 (2015) 132–153

Together with additional purpose-specific Phase II trials and retro- Cooperative Oncology Group (ECOG) comparing ipilimumab alone at spective subsets analyses, these data have contributed to the knowledge 10 mg/kg or in combination with standard GM-CSF doses. This trial ac- of the effects of ipilimumab in patients with stabilized brain metastases crued 250 patients and did not show a difference between arms in re- (Weber et al., 2011), active brain metastases (Margolin et al., 2012), in sponse rate or in PFS. However, OS favored the combination arm previously untreated patients given the 3 mg/kg dose (K. Margolin (median of 17.5 vs. 12.7 months, HR 0.64, p = 0.014). Even more inter- et al., 2013; Patt et al., 2013), in patients with and without HLA-A2 estingly, the combination arm appeared to be associated with a lower mutations (Wolchok et al., 2010b), with and without BRAF and NRAS incidence of high-grade adverse events (AEs). These unusual character- mutations (Shahabi et al., 2012; Queirolo et al., 2013) and in patients istics could warrant further studies (Hodi et al., 2013a). Other pilot expe- with uveal (Danielli et al., 2012; Luke et al., 2013; Moser et al., 2014; riences with cytokine combinations include peginterferon alfa-2b Piulats Rodriguez et al., 2014) and acral melanoma (Deo, 2014; (Kudchadkar et al., 2014), which is approved for the adjuvant treatment Johnson et al., 2014a; Zimmer et al., 2014). of melanoma, and recombinant interleukin-21 (BMS-982470) (Bhatia Finally, a number of other analyses of ipilimumab effects according et al., 2013), an experimental compound developed by BMS. to baseline characteristics such as age (Chiarion-Sileni et al., 2013), Initial promising results have been reported in a Phase I trial com- baseline lactate dehydrogenase (LDH) levels (Smylie et al., 2009), bining ipilimumab with bevacizumab with durable partial responses good and poor prognostic factors (Schadendorf et al., 2009) and out- (PRs) in 36% of the patients treated (Hodi et al., 2011). comes of prior targeted or immunological therapy (Joseph et al., 2011; Given the dramatic effect of tyrosine kinase inhibitors (TKIs) in Ackerman et al., 2012) have also been reported. patients with melanoma and BRAF and/or MEK mutations, the idea to Long-term follow-up was recently collected and analyzed from all combine such agents with ipilimumab is very appealing. With their completed Phase II and Phase III trials in metastatic disease, as well as different kinetics of antitumor effect (rapid vs. slower immediate from the ongoing follow-up of patients accrued to the EAP (Fig. 3). With tumor shrinkage, limited vs. long-term therapeutic effects for TKIs a large database comprising more than 4800 patients, and a follow-up and ipilimumab, respectively) a Phase I trial of the combination of which currently extends up to 10 years from initiation of ipilimumab, it is ipilimumab and vemurafenib, the first of the BRAF-V600 mutation remarkable that the OS Kaplan–Meier curve appears to show a sustained inhibitors, was jointly initiated by BMS and Roche/Genentech, the two plateau after two to three years from the beginning of therapy, with sponsors. Unfortunately, the concomitant administration of the two about one-fifth of the patients presenting prolonged OS results. 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 commu- 3.1.5. Other experiences with combinations in melanoma nication of the toxicity encountered was generated, including letters to The positive outcome of the Phase III trial of ipilimumab with DTIC the investigators and publications. It is unclear whether a molecular demonstrated that the combination was superior to DTIC alone. However, basis for this interaction exists. Three other TKIs have been tested in due to its design, it did not address the issue of a potential detrimental the clinic concomitantly with ipilimumab: dabrafenib alone and also effect of chemotherapy to immunotherapy. A supportive randomized in a’triplet’ combination with trametinib (Puzanov et al., 2014a) and trial was thus carried out in melanoma patients, and it did not document BMS-098662, a now-abandoned pan-BRAF inhibitor by BMS (Callahan any relevant pharmacokinetic interactions of ipilimumab when combined et al., 2012), and no unacceptable liver toxicity has been reported to with DTIC or with paclitaxel and carboplatin (J. Weber et al., 2013). date. A small randomized trial in patients with melanoma testing the Another new and exciting chapter in the treatment of melanoma con- concomitant administration of paclitaxel, carboplatin and ipilimumab sists of the combination of ipilimumab with other vaccines and/or check- vs. its sequential administration (ipilimumab delayed by one week) point inhibitors. Whereas pilot trials in combination with talimogene seemed to favor in response rate and disease control the sequential laherparepvec (T-VEC), a vaccine that has already reported positive administration (Jamal et al., 2014). results as a single agent in a Phase III trial in melanoma (Puzanov et al., Additional trials have tested combinations of ipilimumab with 2014b)andwithINCB024360,asmallmoleculeinhibitorofindoleamine temozolomide, a drug with similarities to DTIC (Patel et al., 2012), 2,3-dioxygenase (IDO) (Gibney et al., 2014), we will cover the combina- fotemustine, a drug that has received regulatory approval for the treat- tion of checkpoint inhibitors later in this paper. ment of melanoma in certain European countries (Di Giacomo et al., 2012), and low-dose cyclophosphamide, with the intent of affecting 3.1.6. Other tumor types than melanoma regulatory T cells (Pavlick et al., 2014). The effect of cytokines in potentiating cellular immunity has been 3.1.6.1. Prostate cancer. Prostate cancer has provided the setting for a described. Sargramostim (GM-CSF) is one such commercially available three milestones, immunotherapy is poised to become a number of relatively unusual pilot trials with ipilimumab. The initial cytokine. A large randomized Phase II trial was conducted by the Eastern cornerstone in the treatment of cancer. 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-specificantigen(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 FigureFig. 3. Comprehensive 1. Long overallterm survivalclinical results outcomes with ipilimumab in over in metastatic 4,800 melanoma. CI: Confidence interval; OS: Overall survival. induction regimen of ipilimumab 3 mg/kg with or without a single dose metastatic melanoma patients that have received

ipilimumab. Nearly 20% of patients are alive at least

three years after treatment. 1

A rationale for cancer immunosurveillance.

The discovery of the athymic nude mouse in 1962 provided a critical reagent to determine the role of the immune system in cancer. The athymic nude mouse is a spontaneous mutant that lacks a thymus and as a result lacks mature T lymphocytes. The mouse also lacks hair, which is why it is called nude. These mice are immune deficient evidenced by their inability to reject skin grafts from unrelated mouse strains. Stutman in 1974 administered methylcholanthrene (MCA), a chemical carcinogen that leads to sarcomas in mice, to a nude mouse

(homozygous), nude mouse (heterozygous), or a wild type mouse that readily produces sarcomas after intramuscular injections into mice. 2 The homozygous nude mouse lacked the ability to reject skin graft which confirmed that they were immune-deficient. Nonetheless, all three experimental groups had the same frequency of tumor formation. These observations suggested that mature T lymphocytes and a functional thymus had no impact on tumor formation. The strength of this data silenced much of the enthusiasm for harnessing the immune system to attack cancer.

In the 1990’s there was a resurgence of interest in cancer immunosurveillance new results using genetically engineered mouse models of immune deficiency. By using gene-targeting technology in mice, several groups were able to generate mice lacking genes essential for immune function. These included genes essential for lymphocyte survival such as recombination activating gene (RAG) that results in complete loss of T, B, and natural killer (NK) lymphocytes. In addition, mice were generated that lacked mediators of immune activation such as the interferon gamma receptor or its downstream effector STAT. By eliminating either all lymphocytes or required mediators, these mouse models were more immune-deficient than the athymic nude mouse. Hence, they provided an opportunity to retest

Stutman’s original hypothesis. Surprisingly, Schreiber and colleagues, as well as others found that genetically engineered immune deficient mice lacking RAG, STAT, or IFN gamma receptor all were more likely to form a tumor after an injection of MCA. (Figure 2-3) 3,4 These studies overruled

Stutman’s original claim and resurrected the field of tumor immunology.

Figure 2. Wild type mice (129/Sv) and IFN gamma

receptor null mice were injected with increasing

concentrations of MCA, a chemical carcinogen. The

percent of mice with tumors was higher in the mice

lacking IFN gamma receptor. letters to nature

were obtained in two additional experiments. In total, 30/52 Given the comparable effects on tumour development of eliminat- RAG2-/- mice formed MCA-induced tumours compared to only ing lymphocytes or STAT1-dependent IFNg signalling, either 11/57 wild-type mice (Fig. 1b) (P , 0:0001). The increased tumour individually or together, there is an extensive overlap between the formation observed in RAG2-/- mice was comparable to that two tumour-suppressor systems. observed in 129/SvEv strain, IFNg-insensitive mice that lack To determine whether these tumour-suppressor systems also either the IFNg receptor (IFNGR1) or STAT1, the transcription function to prevent the formation of spontaneous tumours, we factor that plays a dedicated role in mediating signalling by the monitored tumour development in unmanipulated 129/SvEv wild- IFNg and IFNa/b receptors14,15 (12/20 and 17/30, respectively, type mice, RAG2-/- mice and RkSk mice. As neither wild-type nor versus 11/57 wild-type mice; P , 0:001); this data is consistent with RAG2-/- mice showed outward signs of disease during the observa- our previous ®ndings9 (Fig. 1b). Toassess the extent of collaboration tion period, animals were killed and evaluated for neoplasia upon between the lymphocyte- and IFNg/STAT1-dependent tumour- reaching a minimum age of 15 months. At necropsy, 9/11 wild-type suppressor mechanisms, we generated RAG2-/- ´ STAT1-/- mice mice (15±21 months) were free of neoplastic disease, one (16.1 (RkSk mice) and assessed their MCA sensitivity. Mice lacking both months) developed an intestinal adenoma and one (19 months) genes also showed increased susceptibility to MCA-induced carci- displayed a harderian gland cystadenoma, but none had cancer (Fig. nogenesis with 13/18 mice forming tumours compared to 11/57 2a and Supplementary Information). In contrast, 12/12 RAG2-/- wild-type mice (P , 0:0001), but did not display a signi®cantly mice of ages 15±16 months displayed neoplastic lesions in the greater tumour incidence compared to mice that lacked either the intestinal tract and elsewhere (Fig. 2b and Supplementary Informa- RAG2 or STAT1 genes individually (Fig. 1b) (P ˆ 0:127 and 0.140, tion). Half of these mice developed malignant neoplasias (three had respectively). All four groups of mice formed histologically indis- caecal adenocarcinomas, one had an adenocarcinoma at the ileo- tinguishable sarcomas at the injection site. Similar results were caecal junction, one had an adenocarcinoma of the small intestine obtained using male mice, which are known to be more susceptible and one had a lung adenocarcinoma) that grew progressively when to MCA-induced tumorigenesis. Thus, T, NKT and/or B cells are transplanted into naive RAG2-/- mice. Six of eleven RkSk mice essential to suppress development of chemically induced tumours. developed overt mammary-gland carcinomas (one had two mam- mary adenocarcinomas and another carried distinct adenocarcino- mas in the breast and caecum) that were detected well before the mice reached 15 months (Fig. 2c and Supplementary Information). a 100 This result is particularly striking given the extremely low frequency of spontaneous mammary tumours in wild-type mice or RAG2-/- mice of 129 origin (Fig. 2a, b) and the very late appearance (.20 80

–/– 60 RAG2 100 a Wild type 40 80 (15.5–21 months) 60 20 Mice with sarcomas (%) Mice with sarcomas WT 40

0 20 0 80 120 160 Harderian gland Intestine b Days post MCA treatment 0 100 b RAG2–/– 80 (15.0–16.1 months)

80 n=18 60

n=20 40 60 n=52 n=30 Intestine Intestine (Colon) 20 Percentage of mice Percentage 40 Lung 0 c –/– –/– n=57 RAG2 × STAT1 20 80 (11.9–17.5 months) Mice with sarcomas (%) Mice with sarcomas

60 Breast 0 –/– –/– –/– WT RAG2 IFNGR1 STAT1 RkSk 40 Host genotype Colon 20 Lymphocyte-de®cient mice are highly susceptible to MCA-induced tumour FigureFigure 1 3. Mice immune deficient as a result of loss Intestine Mixed development. a, Fifteen female RAG2-/- mice (diamonds) and 15 syngeneic 129/SvEv 0 No tumours Adenoma Adenocarcinoma wild-typeof RAG2 (WT) mice (recombination (squares) were injected activating with a single subcutaneous gene), IFN dose ofgamma 100 mg MCA and observed for tumour development for 160 days. This dose and observation Figure 2 Increased development of spontaneous neoplastic disease in immunode®cient periodreceptor, were determined STAT, to be or the mostboth informative of the based latter on previous two carcinogenesis gene mice. Eleven wild-type 129/SvEv mice (a), 12 RAG2-/- mice (b) and 11 RkSk mice (c) 9 experimentsproducts performed have on thehigher 129/SvEv tumor strain offrequencies mice . Data are representedin response as a to were housed in a room that was free of speci®c pathogens including Helicobacter. Mice percentage of each group of mice bearing tumours at least 9 mm in diameter as a function were killed when they appeared morbid, developed overt masses, or upon reaching an of time.chemicalb, Cumulative carcinogens. tumour formation at 160 days from three independent experiments age of 15±21 months. Organs were examined for gross pathology, and histologic involving female wild-type 129/SvEv mice (n ˆ 57), RAG2-/- mice (n ˆ 52), STAT1-/- sections were analysed microscopically by three independent pathologists. Additional Coincident with the mouse studies, there was mounting mice (n ˆ 30), IFNGR1-/- mice (n ˆ 20) and RAG2-/- ´ STAT1-/- (RkSk) mice (n ˆ 18) details of tumour formation and histologic sections from representative mice can be evidenceafter injection of ofcancer 100 mg MCA.immunosurveillance in humans. Patients viewed as Supplementary Information. whose immune system is compromised have an increased 1108 © 2001 Macmillan Magazines Ltd NATURE | VOL 410 | 26 APRIL 2001 | www.nature.com incidence of cancer. Most of these cancers have a viral etiology and include squamous cell cancer related to human papilloma virus (HPV) and epstein bar virus (EBV).

However, the risk of other solid tumors not know to have a viral etiology are also higher. For instance in a epidemiologic analysis of cancer outcomes in patients who are received a cadaveric renal transplant and are medically immunosuppressed, the risk of melanoma is 4-5 fold higher. 5

A more direct link between the immune system and cancers comes from studies analyzing and quantifying the number of lymphocytes in a tumor, so called tumor infiltrating lymphocytes (TIL). (Figure 4) The number of lymphocytes invading the tumor correlated with improved survival outcomes in both melanoma and colorectal carcinoma. 6 7 TIL in Melanoma/Clemente et al. 1305

% overall suwival ’“F-7

80 p= ,0003 60 1 i: :I 40

u, m )C 20

kBrisk * Non-brisk *Absent 0 0 12 24 36 48 60 72 84 96 108 120 months

FIGURE 2. Distribution of 285 Stage I and II (AJCC) melanoma patients FIGURE 1. Presence of tumor infiltrating lymphocytes and overall sur- according to brisk, nonbrisk, and absent tumor infiltrating lymphocytes vival in 285 Stage I and II (AJCC) cutaneous melanoma patients. Figure 4. The number of lymphocytes infiltratingand a thickness.

melanoma lesion was quantified and designated “brisk”,

thickness“non- inbrisk”, millimeters; or absent. and regression. Patients The with results a “brisk” de- rive from independent histopathologic review by two dif- ferentresponse pathologists had (C.G.C.better and survival M.C.M.) outcomes. on separate occa-] sions. Diagnostic differences between the two patholo- gists’ assessments were resolved by consensus. The histologic review was performed without knowledge of outcome. Vertical growth phase was determined by the histo- logic parameters described by Clark et aL3z4 The cuta- neous melanomas in horizontal growth phase, ten in situ and four with evidence of papillary dermis invasion, were not further analyzed. The mitotic rate was evaluated ac- cording to the method reported by Schmoeckel and Braun-Falco,’ and the patients were subdivided into three groups (0 mitoses/mm2,between 0.1 and 6 mitoses/mm2 and, more than 6 mitoses/mm’ with a microscopic field FIGURE 3. Ten-year survival of 285 Stage I and II (AJCC) cutaneous diameter of 0.63 mm, X40). melanoma patients according to brisk, nonbrisk, and absent tumor infil- Tumor infiltrating lymphocytes were classified, ac- trating lymphocytes and thickness. cording to Elder et a1.’ and Clark et al.,” into three catego- ries: brisk, nonbrisk, and absent. According to the criteria reported by Elder et al. and Clark et al. and used in our review, tumor infiltrating lymphocytes in cutaneous mel- but did not infiltrate the melanoma (totally absent). In anoma are defined as follows: 1) “brisk” (see Fig. 4), if cutaneous melanomas with evidence of multiple foci of the lymphocytes were present throughout the substance vertical growth phase, the lymphocytic infiltrate was con- of the vertical growth phase (diffuse) or were present and sidered to be brisk if all the neoplastic foci presented infiltrating across the entire base of the vertical growth lymphocytic infiltration and to be absent (focally absent) phase (peripheral); 2) “nonbrisk” (see Fig. 5), if the lym- if only one focus was free of lymphocytes (Fig. 6). In the phocytes were in one or more foci of the vertical growth case of evidence of regression, the same rules were ap- phase, either dispersed throughout (“diffuse”) or situated plied. focally in the periphery, so-called peripheral lymphocytes Thickness was evaluated according to the method of (for ease of understanding and communication we have Breslod from the upper portion to the depth of the tumor suggested the terms diffuse and peripheral to facilitate at its thickest part. The strata of thickness were those description of patterns that have the same prognostic reported by van der Esch et al.’ significance.); or 3) “absent” (see Fig. 6),if there were no Histologic regression is characterized by absence of lymphocytes or if the lymphocyte were actually present melanoma cells in a focal region of horizontal growth

The discovery of CTLA-4 as an immune checkpoint regulator.

In order to effectively manipulate the immune system in order to target cancer required a detailed understanding of the proteins involved in the immune response. The activation of a specific T cell clone as an initial response to a foreign agent depends on the binding of the major histocompatibility complex protein in complex with a peptide to the T cell receptor (TCR). Activation of the immune system requires a co-stimulatory signal in addition to the MHC-TCR interaction. The MHC-TCR interaction is, however, not sufficient to activate T cells. T cell activation requires a second signal through the binding of

B7-1 or B7-2 (hereafter referred to as B7) to CD28, which is constitutively expressed on T lymphocytes. (Figure 5) 8

This second signal acts as a necessary co-stimulator to activate a particular T cell clone.

Figure 5. The two signal model of T cell activation.

CD28 binding to B7-1/2 is a necessary co-stimulatory

signal to activate T cells.

CTLA-4 was discovered as a mRNA that was upregulated in activated T cell lymphocytes 9, and the first clue to its function came from its similarities to CD28. CTLA-4 is 75% identical at the protein sequence to CD28, and like CD28, binds to B7 proteins with high affinity. 10 Allison and his colleagues developed an in vitro system of T cell activation in which they triggered interleukin 2 release by crosslinking antibodies to CD28 and TCR. Antibodies that activate CTLA-4 inhibit IL-2 release by T cells. 11 These observations suggested that CTLA-4 binds to B7 as an Published August 1, 1995

in Fig. 2 suggested that soluble, bivalent anti-CTLA-4 anti- ment presented in Fig. 2 most likely result from perturba- body was effective in blocking B7-mediated signals, but was tion of B7/CTLA-4 interactions. inefficient in providing signals. We next examined the effects CD28 and CTLA-4 Deliver Quantitatively Opposing Sig- of using anti-hamster Ig to cross-link CD3, CD28, and nals. The preceding data indicate that CTLA-4 cross-linking CTLA-4 singly or together. As shown in Fig. 4, no prolifer- in the presence of CD28 signaling can inhibit IL-2 secretion ation was obtained when CD3, CD28, or CTLA-4 were cross- and proliferation. We next sought to determine whether sig- linked individually. As expected, cross-linking of CD3 to- naling above the threshold for CTLA-4 inhibition is inde- gether with CD28 resulted in potent costimulation, while pendent of the magnitude of CD28 costimulation, or whether cross-linking of CD3 and CTLA-4 had no effect. Co-cross- the threshold increases as CD28 signaling increases. To ad- linking of CTLA-4 together with CD3 and CD28 consis- dress this issue, T cells were stimulated by incubation with tently resulted in a 5- to 10-fold reduction in proliferation. polystyrene beads coated with a constant amount of anti- This inhibition was largely reversed by the addition of IL-2 CD3 and varying amounts of anti-CD28 and anti-CTLA-4. to the cultures, suggesting that the effect is not caused by As shown in Fig. 5, costimulation with increasing amounts toxicity. Finally, cross-linking of CTLA-4 with CD3 and of anti-CD28 in the absence of anti-CTLA-4 resulted in a CD28 also resulted in a profound decrease in IL-2 produc- gradual increase in proliferation, reaching at the highest dose tion in the cultures (Fig. 4 B). These results demonstrate that a 1,500-fold increase over that obtained with anti-CD3 alone. CTLA-4 can deliver signals that inhibit T cell responses to The addition of increasing amounts of anti-CTLA-4 reduced TCR ligation, and that the effects observed in the experi- that proliferation in a stepwise manner at all doses of anti- CD28. These results suggest that T cells integrate signals

from CD28 with signals from CTLA-4, and the balance Downloaded from mAb X-linked of these signals regulates the magnitude of the response to C~rlt TCR ligation. aCD3,Ctrl1 aCD2S,Ctrl1 Discussion aCTLA-4,Ctrl1 The results presented here clearly demonstrate that CTLA-4 aCD3,aCD28.Ctrl-[~!!!~:.~:.~:.! !~:.i:.i:.~:.i iii~il ![[~:.i:. i:.i;.i:.~i~:. i:.i:.i:.i :.iii!i:. i:.i:. ~ I- - I

does not serve as a functional alternative to CD28 in providing jem.rupress.org ac~D3aca~A-4aCTL:~~ costimulatory signals to T cells. This finding is in agreement with earlier studies showing that CTLA-4 did not replace CD28 function in CD28 mutant mice (37). The finding that anti-CTLA-4 increases proliferation of T cells activated by antagonistaCD3,aCD28,aCTLA-4JL2 to '-~CD28 i!i!ii!iiii::!!ii~i!:i!i!i!::~i~i!::~i~i!::!::!~ leading]-I to a dampening of T cell I I I I i anti-CD3 and anti-CD28 is in agreement with the results 0 50000 100000 150000 20~C00 250000

of Linsley et al. (24). However, the fact that a similar result on July 1, 2015 activation. (Figure 6) cpm is obtained when blocking antibodies to B7 are included sug-

MAb )[-linked gests that this apparent cooperativity of CTLA-4 is in fact a result of removal of preexisting inhibitory B7-CTLA-4 in- aCD28~"CU'[-~ aCTLA-4-',Ctrl-

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Figure 4. Cross-linkedanti-CTLA-4 can diminish both proliferation and lymphokineFigure production 6. Activating by purified LN antibodies T cells. 10s BALB/c of LNCD3 T (TCR complex) cells were cultured with the indicated hamster antibodies together with controland hamster CD28 antibodies. lead Anti-hamsterto release Ig antibody of IL2, was addeda marker at 20 ofFigure T cell 5. Proliferation in responseto a constant CD3 signal is affected /~g/ml to cross-link. Where indicated, anti-CD3 was added at 5 ~tg/ml, by the relativeconcentrations of CD28 and CTLA-4 signals. 107 5 ~tM anti-CD28activation. was added at 4 ~g/ml,This anti-CTLA-4is inhibited was added by atactivation 20 #g/ microspheres of CTLA were- coated with 1 ~tg/ml of anti-CD3, the indicated con- ml, and controlwas addedto normalizeantibody concentration at 30 p.g/ml. centrations of anti-CD28 and anti-CTLA-4, and control hamster Ig con- (a) Calls4. were cultured for 72 h, pulsed with 1 ~Ci [3H]thymidine,and stituting a total antibody concentration of 5 pg/ml. 10s coated spheres harvestedafter an additional 16 h. (b) Supernatants wereremoved and ana- were incubated in 96-well cultures with 10s purifiedLN T calls for 72 h, lyzed forThe II.-2 upregulation production at 48 h ofusing CTLA an ELISA-4, detectiontherefore, system. is consideredpulsed with 1 ~tCi a [3H]thymidine, and harvested after an additional 16 h.

“checkpoint” on the degree462 ofOpposing the Effectsimmune of CD28response. and CTLA-4 on T Cell Response

Consistent with this hypothesis, Mak and colleagues generated mice lacking CTLA-4 and showed that they develop a massive expansion of their lymphocytes and manifest symptoms of auto-immune disease. 12

Allison and colleagues proposed that antagonism of

CTLA-4 might stimulate the immune system to attack an existing cancer. In a landmark experiment, they discovered that tumors derived from mouse cancer cell lines regressed after treatment with neutralizing antibodies to CTLA-4.

Moreover, when these mice were rechallenged with the same cancer cells, they were refractory to tumor formation.

(Figure 7) 13

300 themselves.Others, such as engineeringtu- protein G-purifiedfrom hybridoma supernatants and 9 _ _ G Sa1 N only quantified by ultravioletspectrophotometry. E 250- To Control morcells to expressMHC classII molecules 19. E. R. Kearney et al., J. Immunol. 155, 1033 (1995). e ~~~~Anti-CTLA-4 (26, 28, 29) or to producegranulocyte-mac- 20. M. F. Krummel,T. J Sullivan, J. P. Allison, Int. Im- 200- rophagecolony-stimulating factor (27, 30, munol., in press. 21. J. G. Gribben et al., Proc. Natl Acad. Sci. U.S.A. 92, 0150- 31) or pulsing dendritic cells with tumor 811 (1995). E antigen ex vivo (32, 33), seek to enhance 22. P. Waterhouse et al., Science 270, 985 (1995). 100- antigen presentation,antigen transfer,or 23. S. E. Townsend, F. W. Su, J. M.Atherton, J. P. Allison, both. Thus,CTLA-4 blockade, by removing Cancer Res. 54, 6477 (1994). 51 BLiml0 colon carci- 50- noma cells were transfected with a plasmid construct potentially competing inhibitory signals, containing the gene for murine B7-1 and cloned by 0, limitingdilution. Fresh cultures of tumor cells were 0 20 40 50 70 maybe a particularlyuseful adjunct to other 10 30 60 established from early passage frozen stocks and ttt Days aftertumor injection therapeuticapproaches involving the co- maintained in culturefor no more than 30 days before stimulatorypathway. use. Tumor cells were harvested by trypsinization FigureFig. 4.7. Treatment Cancer cell lineswith derivedanti-CTLA-4 from a mouse reduces were the from tissue culture plates, washed three times in se- growth of the murinefibrosarcoma Sal N. Groups injected into syngeneic animals who were either REFERENCESAND NOTES rum-freemedium, and suspended at 4 x 107 cells per untreated,of five mice treated were with injected a control subcutaneously antibody, or treated in the milliliter.Expression of B7-1 molecules on transfected cells was verified by flow cytometry before injection. withflank hamster with aantibodies suspension to CTLA of- 4.1 x Arrows106 Salindicated N fibrosar- 1. D. L. Mueller, M. K. Jenkins, R. H. Schwartz, Ann. coma cells. Treated groups were injected intra- Rev. ImMUnol.7, 445 (1989). V51 BLim10 and wild-type 51 BLim10 tumor cells do treatment time points. Mice treated with CTLA-4 not express detectable amounts of B7-1, B7-2, or peritoneallywith 100 Vg of anti-CTLA-4 or irrele- 2. P. S. Linsleyand J. A. Ledbetter, ibid. 11, 191 (1993). showed evidence of tumor regression. CTLA-4as determined by flow cytometric analyses. vant hamster control antibody at days 0, 3, and 6 3. C. H. June, J. A. Bluestone, L. M. Nadler, C. B. Thompson, ImmLInol.Today 15, 321 (1994). 24. J. A. Gross, E. Callas, J. P. Allison,J. Immunol. 149, as indicated by the arrows. All control animals 380 (1992). These findings were consistent with an immune attack 4. J. P. Allison, Curr. Opin. Immunol. 6, 414 (1994). were killedby day 30. Two of five animals treated 5. L. Chen, S. Ashe, W. A. Brady, I. Hellstrom, K. E. 25. D. R. Leach, M. F. Krummel,J. P. Allison, data not on thewith tumor anti-CTLA-4 stimulated by CTLAremained -4 inactivation. tumor-free These at day 55. Hellstrom et al., Cell 71, 1093 (1992). shown. 26. S. Baskar, L. Glimcher, N. Nabavi, R. T. Jones, S. 6. S. E. Townsend and J. P. Allison, Science 259, 368 Ostrand-Rosenberg, J. Exp. Med. 181, 619 (1995). (1993). 27. A. Y. C. Huang et al., Science 264, 961 (1994). 7. S. Baskar et al., Proc. Nat!. Acad. Sci. U.S.A. 90, 28. S. Ostrand-Rosenberg, A. Thakur, V. Clements, J. able, rapidly growing tumors within 7 days, 5687 (1993). Immunol. 144, 4068 (1990). P. A. A. D. Curr. whereas only two mice treated with anti- 8. J. Allison, Hurwitz, R. Leach, Opin. 29. D. R. Leach and G. N. Callahan, ibid. 154, 738 Immunol. 7, 682 (1995). CTLA-4 had tumors by day 30, and one (1995). 9. M. K. Jenkins, Immunity1, 443 (1994). additional mouse developed a tumor around 30. G. Dranoff et al., Proc. Natl. Acad. Sci. U.S.A. 90, 10. J. A. Bluestone, ibid. 2, 555 (1995). 3539 (1993). day 40 after injection. The remaining mice 11. P. S. Linsley,J. Exp. Med. 182, 289 (1995). 31. H. I. Levitsky,A. Lazenby, R. J. Hayashi, D. M. Par- were still tumor-free 70 days after injection. 12. J. P. Allison and M. F. Krummel,Science 270, 932 doll, J. Exp. Med. 179,1215 (1994). In another experiment, control mice inject- (1995). 32. V. Flamand et al., Eur. J. Immunol. 24, 605 (1994). 13. J. F. Brunet et al., Nature 328, 267 (1987). 33. S. Grabbe, S. Beissert, T. Schwarz, R. D. Granstein, ed with 4 X 105 SaiN tumor cells also 14. K. Harperet al., J. Immunol. 147, 1037 (1991). Immunol. Today 16,117 (1995). developed rapidlygrowing tumors, whereas 7 15. P. S. Linsley etal., J. Exp. Med. 174, 561 (1991). 34. We thank S. Ostrand-Rosenberg and R. Warrenfor of 10 mice treated with anti-CTLA-4 were 16. P. S. Linsley et al., Immunity1, 793 (1994). providing tumor lines. Supported by NIH grants 17. T. L. Walunas et al., ibid., p. 405. CA57986, CA09179, and CA40041. tumor-free by day 25 after injection (25). 18. M. F. Krummeland J. P. Allison, J. Exp. Med. 182, Our results indicate that removing in- 459 (1995). Antibodies used in these studies were 17 November 1995; accepted 16 January 1996 hibitory signals in the costimulatory path- way can enhance antitumor immunity. Al- though it has been shown that anti- Light-inducedDegradation of TIMELESSand CTLA-4 interferes with signals that nor- mally down-regulate T cell responses in Entrainmentof the Drosophila CircadianClock vivo (17, 18), the exact mechanisms of antitumor immunity elicited by CTLA-4 Michael P. Myers, KarenWager-Smith, blockade are not clear. In the case of B7- AdrianRothenfluh-Hilfiker, Michael W. Young* negative tumors, antigens are most likely transferred to and presented by host APCs Two genes, period (per) and timeless (tim), are required for production of circadian (27), where CTLA-4 blockade might effect rhythms in Drosophila. The proteins encoded by these genes (PER and TIM)physically T cell responses in two nonexclusive ways. interact, and the timing of their association and nuclear localization is believed to promote First, removal of inhibitory signals may low- cycles of per and tim transcription through an autoregulatory feedback loop. Here it is er the overall threshold of T cell activation shown that TIM protein may also couple this molecular pacemaker to the environment, and allow normally unreactive T cells to because TIM is rapidly degraded after exposure to light. TIM accumulated rhythmically become activated. Alternatively, CTLA-4 in nuclei of eyes and in pacemaker cells of the brain. The phase of these rhythms was blockade might sustain proliferation of ac- differentially advanced or delayed by light pulses delivered at different times of day, tivated T cells by removing inhibitory sig- corresponding with phase shifts induced in the behavioral rhythms. nals that would normally terminate the re- sponse, thus allowing for greater expansion of tumor-specific T cells. Regardless of the mechanism, it is clear Circadian rhythms, found in most eu- be entrained by environmentalcycles of that CTLA-4 blockade enhances antitumor karyotes and some prokaryotes(1), are light or temperature.Circadian rhythms responses. Most importantly, we have ob- -24-hour rhythmsgoverned by an internal producedin constant darknesscan also be served these effects against unmanipulated, clock that functionsautonomously but can reset by pulses of light. Such light pulses wild-type tumors. Current methods of en- will shift the phaseof the clock in different hancing antitumor immunity generally re- Howard Hughes Medical Institute, National Science directions(advance or delay) and to a vary- Foundation Science and Technology Center for Biologi- quire the engineering of tumor cells (8). cal Timing, and the Laboratory of Genetics, The Rock- ing extent in a mannerthat dependson the Some of these methods, such as the induc- efeller University, 1230 York Avenue, New York, NY time of light exposure(2). tion of B7 expression, rely on enhancing the 10021, USA. In the fruit fly Drosophilamelarnogaster, costimulatory activity of the tumor cells *To whom correspondence should be addressed. two genes, period(3) and timeless(4), are

1736 SCIENCE * VOL. 271 * 22 MARCH 1996

This content downloaded from 128.32.19.34 on Wed, 17 Jul 2013 12:44:45 PM All use subject to JSTOR Terms and Conditions studies inspired the clinical development of analogous humanized antibodies – Tremelimumab (Pfizer) and Ipilimumab

(Bristol Myers Squib – BMS) for the treatment of patients with malignant melanoma.

The clinical development of Ipilimumab and the failure of

Tremelimumab.

CTLA-4 knockout mouse as mentioned earlier have a profound expansion of lymphocytes leading to early lethality, which led to apprehension about the development of CTLA-4 antagonists for the treatment of patients.

Nonetheless, two different companies, Pfizer and Bristol

Myers Squibs, advanced tremelimumab and ipilumumab, respectively, to the clinic.

Tremelimumab is a IgG2 humanized antibody, which is less likely to activate complement in an antibody dependent cytotoxicity. In early clinical development, investigators used plasma IL-2 levels following injection of tremelimumab as an in vivo pharmacodynamics marker for efficacy. 14 Using this marker, they established that the pharmacokinetics efficacy of tremelimumab could last 90 days after a single dose at 15 mg/kg. Phase I/II testing of tremelimumab compared 10 mg/kg dosed monthly versus 15 mg/kg dosed every 90 days. Both doses had a 10% objective response rate, however, the 15 mg/kg dose had an improved toxicity profile and was chosen as the dosage in phase III testing. Of note in phase II testing, the median time to a response was 21 weeks and there were numerous durable responses lasting over 2 years. Based on these results, tremelimumab was tested in a randomized control Phase III trial against standard of care chemotherapy at a dose of 15 mg/kg with the primary endpoint of overall survival. Although there were ~10% objective and durable response rates in the tremelimumab arm, the change in overall survival was not significant (p=0.12). 15

In contrast, a similar phase III trial using ipilimumab, a IgG1 antibody targeting CTLA-4, did result in improved overall survival and received fast track approval by the FDA. Given that these agents were both antibodies to the same target, CTLA-4, it is worth considering why one trial succeeded and the other failed.

In early phase I/II testing with ipilimumab, investigators noted that patients were exhibiting delayed responses, sometimes even after treatment started. (Figure

8) 16 For example after 12 weeks of treatment, 63/186 patients had stable disease and of these 45 patients showed Cancer Immunity (17 January 2008) Vol. 8, p. 1

Figurea steady 4 decline in their overall tumor through week 24. At week 24 he received his first dose of ipilimumab maintenance therapy (10 mg/kg) and continues to receive maintenance therapy q12wk. The axillary lymph node initially detected at the week 12 scan was confirmed to contain metastatic melanoma by needle biopsy. However, imaging showed it regressed beginning at week 39, with the most recent dimensions being 0.9 cm x 2.3 cm, decreased from a peak size of 1.6 cm x 2.7 cm. He has now received 2 doses of maintenance therapy at weeks 28 and 39 after initial ipilimumab treatment and continues to have SD at week 48. This pattern of response, with some lesions shrinking or stable in the presence of a single new lesion, is relatively rare with conventional therapies. It is in this type of patient that an appreciation of the biologic mechanism of action of ipilimumab is vital for decision making.

Case 5 A male patient aged 63 years from study 4 presented with a

more than 15-year history of skin, soft tissue, and lymph node metastases. His history is complicated by prolonged Figure 8. The case of a 50 yo man with melanoma lymphopenia due to long-term temozolomide use, which metastatic to the liver who received ipilimumab resulted in symptomatic Pneumocystis carinii pneumonia and 4 monotherapy. After treatment, he exhibited subsequent years of lymphopenia. He experienced PD when previously treated with vaccines, IL-2, temozolomide, and radiographic progression (top right) even though other biochemotherapy. After approximately 12 weeks of 3 mg/kg markers of disease (LDH) were reduced. These ipilimumab (q3wk, plus gp100 peptides) treatment, he

“lesions” responded over the course of several months. experienced a near complete response (CR) based on imaging and he also had an immune reconstitution syndrome consisting One year later, he had no evidence of disease. of a sarcoid-like reaction in his airways which lasted for 2 to 3 burden. weeks and caused mild shortness of breath. These symptoms resolved without parenteral corticosteroids. This patient Even more surprising, 10 of 57 patients with continues to be in near-CR, and, along with cases 1 and 2, progressive disease at 12 weeks eventually showed a partial demonstrates a correlation between lymphoid reconstitution response. Based on these observations, these and clinical response to ipilimumab.

Discussion The cases in this report illustrate the variable kinetics of response that commonly appear with ipilimumab therapy. In the authors' experience, responses to ipilimumab have been observed prior to (case 1), at (cases 4 and 5), and after (cases 2 and 3) 12 weeks of induction dosing with ipilimumab. The complexity of the immune response and the impact of individual patient status on the immune system creates Response to ipilimumab therapy of patient case 2. (A) Radiographic progres- challenges for the prediction of the time course of response. sion of disease at week 12, followed by a clinical response and near complete reso- However, given the time required to produce a sufficient anti- lution of disease following another course of ipilimumab induction therapy. (B) tumor immune response, it is likely that the induction-dosing Lymphocyte recovery consistent with immune reconstitution accompanied by a schedule (12 weeks) may need to be completed before clinical decline in LDH and alkaline phosphatase, reflective of decreasing tumor burden. benefit is observed. This is in marked contrast to chemotherapy where response is usually detectable soon after treatment. This Case 4 is probably because ipilimumab, unlike chemotherapy, does not have a direct effect on tumor cells, but exerts its effects through A male patient aged 60 years from study 1 was diagnosed with educating the immune system. Subsequently, the time taken to melanoma in late 2005 when he was found to have a large generate an anti-tumor immune response can result in a longer burden of disease in axillary lymph nodes and possible liver time to response. Indeed, the case studies described herein metastasis on computed tomography (CT) scans. He progressed provide proof of this concept. It is reasonable to hypothesize that on temozolomide and had a palliative lymph node dissection the immune system is activated during ipilimumab induction performed. He was then treated with temozolomide, cisplatin, treatment between weeks 1 and 12. After week 12, the first time and vinblastine and exhibited PD of his liver and abdominal point for measuring ipilimumab efficacy and tumor assessment, lymph nodes. At week 12, after the full induction schedule of 4 some patients enter a maintenance phase and receive doses of ipilimumab, radiologic measurements revealed that his ipilimumab q12wk. Periodic re-inhibition of CTLA-4 through liver lesions had regressed and his other lesions were stable or ipilimumab maintenance dosing should theoretically re-activate smaller. However, a new 2 cm axillary lymph node lesion was the anti-tumor immunologic activity established through the also noted on the same scan. The only toxicity was a mildly induction phase, ensuring continued activity, and help the pruritic rash. The patient experienced slow improvement/SD immune system to recognize and respond to any new tumor

4 of 7 www.cancerimmunity.org investigators established new criteria for measuring the response following immune therapy that is based on total tumor burden. In this case, evidence of a new lesion but stable or partial responses in existing lesions would be considered stable disease based on a change in total tumor burden. These experiences influenced the protocol for the phase III ipilimumab trials. For example, patients were given four cycles of therapy every 3 weeks unless they had a decline in performance status, high grade toxicity, or clear progressive disease. Unlike in the tremelimumab, immune related toxicities were aggressively managed with high dose steroids and/or anti-TNF agents, and if the toxicity resolved, therapy was restarted. Furthermore, patients who showed delayed responses were offered a reinduction regimen in which they received four additional cycles of therapy. Ultimately, at least in part because of these elements in the protocol, ~60% of patients completed

Ipilimumab treatment were as ~13% completed tremelimumab treatment. 17 It is my opinion that this is the primary reason why trememlimumab failed. Other possibilities noted by the authors are that no crossover was allowed from the chemotherapy to tremelimumab arm and that several patients in the chemotherapy arm received ipilimumab leading to improvements in survival in that arm. Immunotherapeutic agents have begun to cure patients with malignant melanoma. The development of new immunomodulatory agents that antagonize either PD-1 or its ligand PD-L1 have shown even better clinical outcomes either alone or in combination with ipilimumab.

Furthermore, the types of cancers that are responding to these agents have expanded beyond melanoma to also include lung, bladder, and colorectal cancers. Now, there is little doubt that immunotherapy will become a mainstay in cancer treatment and there is optimism that some patients will achieve durable remissions. In my opinion, there were three key milestones in this achievement which we have discussed here: the basis for cancer immunosurveillance in mice, the discovery of CTLA-4 and the proof of principle experiments of anti-CTLA-4 anti-tumor efficacy in mice, and finally the clinical trial of ipilimumab in melanoma.

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