The discovery that cancer cells evade attacks from the immune system by manipulating immune cells at multiple checkpoints has led to the development of the new drug class of immune checkpoint modulators. Three therapeutic antibodies against two different checkpoint targets recently have been FDA approved. Many other drug candidates targeting different checkpoint pathways are in clinical trials.

Affymetrix provides a wide portfolio for the immuno-oncology research space, including ELISA and bead-based multiplex immunoassays for the quantification of these important checkpoint modulators. ProcartaPlex® Human Immuno-oncology Checkpoint Panel enables simultaneous quantification of 14 checkpoint modulators. Singleplex assays are available for each target included in the ProcartPlex Human Immuno-oncology Checkpoint Panel, and combinable with additional analytes of interest.

Immuno-oncology Knowledge wins. Take the 360º view.

Immunoassays for Immuno-oncology Research

Ersteller: Dagmar Dietmann, 29/10/2015Revision 03

PROT13-36 DMR PPX

Filename: Marketing Broschure.xlsx page 1/3

BTLA conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 500000.000 14152.0 14239.5 0.4% 14168 102S2 125000.000 9420.0 9567.0 1.1% 9466 97S3 31250.000 4494.0 4907.5 6.2% 4673 103S4 7812.500 1548.0 1520.0 1.3% 1506 98S5 1953.100 449.0 435.0 2.2% 414 101S6 488.300 127.5 126.0 0.8% 99 100S7 122.100 51.0 52.0 1.4% 24 100

blank 25.5 30.5 12.6% 0mean blank 28.0

GITR conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 100000.00 7548.0 7874.0 3.0% 7646 101S2 25000.00 4920.0 4856.0 0.9% 4823 99S3 6250.00 2111.5 2093.0 0.6% 2037 102S4 1562.50 658.5 657.0 0.2% 592 99S5 390.60 227.5 225.0 0.8% 161 100S6 97.70 118.0 110.0 5.0% 49 103S7 24.40 82.0 88.0 5.0% 20 94

blank 65.0 66.0 1.1% 0mean blank 65.5

HVEM conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 100000.00 13749.0 14247.0 2.5% 13959 101S2 25000.00 7598.0 7915.5 2.9% 7718 98S3 6250.00 2886.0 3123.0 5.6% 2966 102S4 1562.50 838.0 876.0 3.1% 818 99S5 390.60 248.0 229.5 5.5% 200 98S6 97.70 91.0 92.0 0.8% 53 105S7 24.40 50.0 53.5 4.8% 13 94

blank 38.0 40.0 3.6% 0mean blank 39.0

IDO conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 20000.000 24103.5 23990.0 0.3% 23995 98

MFI

Immuno-Oncology Panel (EPX140-15803-901)

MFI

MFI

MFI

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1.000   10.000  100.000  1000.000  10000.000  100000.000  1000000.000  

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1.00   10.00   100.00   1000.00   10000.00  100000.00  

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1.000   10.000   100.000   1000.000   10000.000   100000.000   1000000.000   10000000.000  

MFI  

pg/ml  

Standardrange  of  Analytes  

BTLA   GITR   HVEM   IDO   LAG-­‐3   PD-­‐1   PD-­‐L1  

PD-­‐L2   TIM-­‐3   CD28   CD80   CTLA-­‐4   CD27   CD137

ProcartaPlex® Human Immuno-oncology Checkpoint Panel

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pg/mL

Ersteller: Dagmar Dietmann, 29/10/2015Revision 03

PROT13-36 DMR PPX

Filename: Marketing Broschure.xlsx page 1/3

BTLA conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 500000.000 14152.0 14239.5 0.4% 14168 102S2 125000.000 9420.0 9567.0 1.1% 9466 97S3 31250.000 4494.0 4907.5 6.2% 4673 103S4 7812.500 1548.0 1520.0 1.3% 1506 98S5 1953.100 449.0 435.0 2.2% 414 101S6 488.300 127.5 126.0 0.8% 99 100S7 122.100 51.0 52.0 1.4% 24 100

blank 25.5 30.5 12.6% 0mean blank 28.0

GITR conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 100000.00 7548.0 7874.0 3.0% 7646 101S2 25000.00 4920.0 4856.0 0.9% 4823 99S3 6250.00 2111.5 2093.0 0.6% 2037 102S4 1562.50 658.5 657.0 0.2% 592 99S5 390.60 227.5 225.0 0.8% 161 100S6 97.70 118.0 110.0 5.0% 49 103S7 24.40 82.0 88.0 5.0% 20 94

blank 65.0 66.0 1.1% 0mean blank 65.5

HVEM conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 100000.00 13749.0 14247.0 2.5% 13959 101S2 25000.00 7598.0 7915.5 2.9% 7718 98S3 6250.00 2886.0 3123.0 5.6% 2966 102S4 1562.50 838.0 876.0 3.1% 818 99S5 390.60 248.0 229.5 5.5% 200 98S6 97.70 91.0 92.0 0.8% 53 105S7 24.40 50.0 53.5 4.8% 13 94

blank 38.0 40.0 3.6% 0mean blank 39.0

IDO conc. pg/ml CV% MEAN blankcorr. % backcalc.S1 20000.000 24103.5 23990.0 0.3% 23995 98

MFI

Immuno-Oncology Panel (EPX140-15803-901)

MFI

MFI

MFI

1  

10  

100  

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100000  

1.000   10.000  100.000  1000.000  10000.000  100000.000  1000000.000  

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1.00   10.00   100.00   1000.00   10000.00  100000.00  

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1.00   10.00   100.00   1000.00  10000.00  100000.00  

10000  

100000  

1  

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100  

1000  

10000  

100000  

1.000   10.000   100.000   1000.000   10000.000   100000.000   1000000.000   10000000.000  

MFI  

pg/ml  

Standardrange  of  Analytes  

BTLA   GITR   HVEM   IDO   LAG-­‐3   PD-­‐1   PD-­‐L1  

PD-­‐L2   TIM-­‐3   CD28   CD80   CTLA-­‐4   CD27   CD137

Standard curves for the ProcartaPlex® Human Immuno-oncology Panel.

1.000 10.000 100.000 1000.000 10000.000 100000.000 1000000.000 10000000.000

Step 2

Dendritic cell

Lym

ph n

ode

Blood vessel

Active T cell

Antigens

Blood vessel

Step 3

Active T cell

Lymph node

Step 4

Step 5

Step 1

Step 6

Tumor microenvironment

Step 7

Tumor cell

Immune response in immuno-oncologyThe cancer immunity cycle1 describes the multiple steps in the immune systems response to a tumor. Several biomarkers, especially checkpoint molecules, play a crucial role in this response. It starts with the engulfment of tumor-derived antigens (TDA) by dendritic cells. Highly specialized dendritic cells mature and present their TDAs to T cells. Primed and activated effector T cells are formed, and tumor infiltration starts. Finally, T cells bind to tumor cells and destroy them. More tumor antigens are released and trigger the immunity cycle further.

Soluble forms of the biomarkers included in the cancer immunity cycles have been described in several publications. 2,3,4,5,6,7,8,9,10

Factors involved in the cancer immunity cycle

Step 3: T cell priming & activation

CD28 PD-L2 CD152/CTLA-4 CD27 CD80 CD137/4-1BB IL-2 IL-12 HVEM GlTR

Step 4: Trafficking T cells to tumors

Fractalkine IP-10 Rantes

Step 5: Infiltration of T cells into tumors

ICAM-1 VEGF Selectins

Step 6: Recognition of cancer cells by T cells

Step 2: Cancer antigen presentation TNF alpha IL-1 IFN alpha IL-10 IL-4 IL-13

Step 1: Release of cancer cell antigens

Step 7: Killing of cancer cells

PD-L1 PD-1 B7.1 IDO BTLA LAG-3 TIM-3 MIC TGF beta

Immuno-oncology checkpoint moleculesCD152/CTLA-4

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibits antigen-specific T cell proliferation and cytokine production, qualifying CTLA-4 as target for cancer immunotherapy. Ipilimumab, an FDA-approved monoclonal antibody against CTLA-4, demonstrated an overall survival (OS) benefit for patients with metastatic melanoma with a response rate of <25%. It has been shown that soluble CTLA-4 levels were statistically higher in patients responding to Ipilimumab treatment (Figure 1).2

PD-1

Programmed cell death protein 1 (PD-1) promotes apoptosis in antigen-specific T cells and reduces apoptosis in regulatory (suppressive) T cells at the same time. Nivolumab and Pembrolizumab are FDA-approved therapeutic antibodies against PD-1 for the treatment of melanoma (response rates: 40%).

PD-1 ligands PD-L1 and PD-L2

It has been shown that PD-L1 and PD-L2 are expressed on various tumor cells. Some studies indicate that high levels of soluble PD-1 ligands in tumor cells correlate with lower survival and response rates.7,11

CD27

The TNF receptor superfamily member CD27 is required for generation and long-term maintenance of T cell immunity. The interaction between CD27 and its ligand, CD70, is implicated in regulating cellular immune responses to cancer.9

CD80

CD80 is present on activated B cells and monocytes and functions as costimulatory signal which is needed for T cell activation and survival. CD80 has two ligands, CD28 and CTLA-4. Soluble CD80 restores T cell activation and overcomes PDL-1 mediated immune suppression.8

HVEM-BTLA

Herpes virus entry mediator (HVEM) is also known as TNF receptor superfamily member 14. HVEM is expressed on certain tumor cell types (e.g., melanoma) and on tumor-associated endothelial cells. Interestingly, HVEM is the ligand of B- and T-lymphocyte attenuator (BTLA). It is exceptional that a TNF family member interacts with an immunoglobulin supergene family member. Evidence suggests that the HVEM-BTLA pathway is an inhibitory checkpoint for dendritic cell homeostasis in lymphoid tissue.

LAG-3

Lymphocyte activation gene 3 protein (LAG-3) is another immune checkpoint receptor, which is being investigated in various ongoing trials. It has been shown that soluble human LAG-3 amplifies tumor-specific immunity.7

Figure 1: Overall 5-year survival of patients treated

with Ipilimumab.

Overall survival (5 years) of patients treated with Ipilimumab comparing

those with greater than 200 pg/mL serum of soluble CTLA-4 (sCTLA-4)

to those with less than or equal to 200 pg/mL. sCTLA-4 levels were

quantified using human sCD152/CTLA-4 Platinum ELISA from Affymetrix.

Leung et al. CTLA4 levels and ipilimumab efficacy

Table 2 | Demographics of 11 melanoma patients not treated with

ipilimumab.

Demographic sCTLA4 ≤200

pg/mL (n %)

sCTLA4 >200

pg/mL (n %)

p-Value

Sex 0.34

Male 3 (100) 6 (75)

Female 0 (0) 2 (25)

Age 0.40

Average 41 52

Primary 0.17

Trunk 2 (66.7) 3 (37.5)

Extremity 0 (0) 5 (62.5)

Head and neck 1 (33.3) 0 (0)

M status 0.38

M1a 5 (33.3) 4 (50)

M1b 0 (0) 2 (25)

M1c 2 (66.7) 2 (25)

FIGURE 1 | Serum sCTLA4 levels correlate with clinical benefit toipilimumab treatment. sCTLA4 levels were measured by ELISA andindividual values plotted according to clinical response. Values areexpressed as the mean value of triplicate wells. The sensitivity of the ELISAwas 100 pg/mL. Two-tailed Mann–Whitney test was used to evaluate thesignificance of the differences between patients who received (n = 9) or didnot receive (n = 5) benefit from ipilimumab treatment. The cutoff sCTLA4level for prediction of clinical benefit was determined using a threshold of200 pg/mL based on ROC curve analysis.

for patients with sCTLA4 ≤200 pg/mL and sCTLA4 >200 pg/mL,respectively (Figure 2). Multivariable analysis showed that nocovariate other than elevated sCTLA4 level was associated withprolonged 5-year OS (Table 3).

Table 3 | Univariable Cox Proportional hazard regression analysis of

5-year overall survival.

Variable p-Value Hazard ratio 95% CI

Elisa <200 vs. >200 0.04 5.29 1.06–26.4

Male vs. female 0.97 0.97 0.26–3.66

Age at diagnosis 0.52 0.97 0.90–1.06

Breslow depth 0.67 1.02 0.94–1.11

Ulcerated vs. non-ulcerated 0.51a NA NA

M1abc 0.32

M1a vs. M1c 0.20 3.19 0.55–18.59

M1b vs. M1c 0.64 0.60 0.07–5.35

aBy log-rank test (Hazard ratio approached zero so log-rank test was done instead).

Multivariable analysis incorporating all the above variables showed that all the

other variables except (ELISA) were not significant. Thus, the univariable model

with ELISA is the final model.

0.00

0.20

0.40

0.60

0.80

1.00

0 12 24 36 48 60

Months from Ipilimumab

Ove

rall s

urv

iva

l

CTLA-4 >200

CTLA-4 <200

p= 0.02

FIGURE 2 | Overall survival after ipilimumab treatment is greater inpatients with serum CTLA4 levels above 200 pg/mL foripilimumab-treated patients. Overall survival curves (5 years) of patientstreated with ipilimumab comparing those with greater than 200 pg/mLserum sCTLA4 to those with less than or equal to 200 pg/mL.

To determine whether sCTLA4 levels correlate with survivalin general, rather than only in those patients who are treatedwith anti-CTLA4, we also tested stage IV melanoma patients whohad not received ipilimumab (n = 11). Using the same cutoff of200 pg/mL, the two survival curves were not statistically different,with 33.4 and 29.6 months median OS for patients with sCTLA4≤200 pg/mL and sCTLA4 >200 pg/mL, respectively (p = 0.60)(Figure 3).

DISCUSSIONHere, we report serum sCTLA4 levels of ipilimumab-treatedpatients with advanced melanoma from samples collected prior toipilimumab therapy. Our preliminary findings show that elevatedserum levels of sCTLA4 are associated with clinical benefit to ipil-imumab in this initial cohort. Patients with elevated sCTLA4 alsoshowed a significant survival benefit over those with low sCTLA4

www.frontiersin.org May 2014 | Volume 4 | Article 110 | 3

1.00

0.80

0.60

0.40

0.20

0.000 12 24 36 48 60

Months from Ipilimumab

Ove

rall

surv

ival

CTLA-4 >200

CTLA-4 <200

p= 0.02

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References

ProcartaPlex® Immunoassays

1. Chen D. S., et al. Oncology meets immunology: the cancer-immunity cycle. Immunity 39(1):1–10 (2013). 2. Leung A. M., et al. Clinical benefit from ipilimumab therapy in melanoma patients may be associated with serum CTLA4 levels. Frontiers in Oncology 4:1–5 (2014). 3. Ostrand-Rosenbert S., et al. Novel strategies for inhibiting PD-1 pathway-mediated immune suppression while simultaneously delivering activating signals to tumor-reactive T cells. Cancer Immunology, Immunotherapy 64:1287–1293 (2015). 4. Heo S. K., et al. The presence of high level soluble herpes virus entry mediator in sera of gastric cancer patients. Experimental and Molecular Medicine 44(2):149–158 (2012). 5. Taylor L., et al. Identification of a soluble OX40 isoform: development of a specific and quantitative immunoassay. Journal of Immunological Methods 255(1–2):67–72 (2001). 6. Casati C., et al. Soluble Human LAG-3 Molecule Amplifies the In vitro Generation of Type 1 Tumor-Specific Immunity. Cancer Research 66(8):4450–4460 (2006). 7. Wang L., et al. Serum levels of soluble programmed death ligand 1 predict treatment response and progression free survival in multiple myeloma. Oncotarget 1(6):41228–41236 (2015). 8. Haile S. T., et al. Soluble CD80 Restores T Cell Activation and Overcomes Tumor Cell Programmed Death Ligand 1–Mediated Immune Suppression. Journal of Immunology 191(5):2829–2836 (2013). 9. Huang J., et al. Soluble CD27-pool in humans may contribute to T cell activation and tumor immunity. Journal of Immunology 190(12):6250–6258 (2013). 10. Tieu R., et al. TIM-3, a Possible Target for Immunotherapy in Cancer and Chronic Viral Infections. Austin Virology and Retro Virology 1(2): 1–12 (2014). 11. Okazaki T., et al. PD-1 and PD-1 ligands: from discovery to clinical application. International Immunology 19(7):813–824 (2007).

ProcartaPlex® Immuno-Oncology Checkpoint Panel

EPX140-15803-901

BTLA PD-L2

GlTR TIM-3

HVEM CD28

IDO CD80

LAG-3 CD137/4-1BB

PD-1 CD27

PD-L1 CD152/CTLA

ProcartaPlex® Simplex Kits Part Number

IL-2 EPX01A-10221-901

Fractalkine EPX01A-12121-901

CD152/CTLA4  EPX01A-10276-901

CD27  EPX01A-10286-901

CD80  EPX01A-10291-901

GlTR  EPX01A-12210-901

ICAM-1 EPX01A-10201-901

IL-10 EPX01A-10215-901

IFN alpha EPX01A-10216-901

IL-4 EPX01A-10225-901

IL-12p70 EPX01A-10238-901

CD154 (CD40 Ligand) EPX01A-10239-901

IL-1 alpha EPX01A-10243-901

VEGF-A EPX01A-10277-901

IP-10 EPX01A-10284-901

Rantes EPX01A-10287-901

CD137/4-1BB EPX01A-10289-901

CD28 EPX01A-10290-901

TWEAK EPX01A-12006-901

VEGF-D EPX01A-12076-901

IL-12p40 EPX01A-12090-901

TSLP EPX01A-12164-901

LAG-3 EPX01A-12211-901

PD-L1 EPX01A-12212-901

IDO EPX01A-12213-901

PD-1 EPX01A-12214-901

PD-L2 EPX01A-12215-901

BTLA EPX01A-12217-901

HVEM EPX01A-12218-901

TIM-3 EPX01A-12219-901

ELISA Format Part NumberBTLA Platinum ELISA BMS2217CD137/4-1BB Platinum ELISA BMS289CD152/CTLA4 Platinum ELISA BMS276CD27 Instant ELISA® BMS286INSTCD28 Platinum ELISA BMS290CD80 Instant ELISA® BMS291INSTGlTR Platinum ELISA BMS2210HVEM Platinum ELISA BMS2218ICAM-1 Platinum ELISA BMS201

Platinum ELISA BMS241Instant ELISA® BMS201INST

IDO Platinum ELISA BMS2213IFN alpha Ready-SET-Go!® ELISA 88-7389

Instant ELISA® BMS216INSTPlatinum ELISA BMS216

IL-1 alpha Platinum ELISA BMS243/2IL-10 Ready-SET-Go!® ELISA 88-7106

High Sensitivity ELISA BMS215HSInstant ELISA® BMS215INSTPlatinum ELISA BMS215/2

IL-12p70 High Sensitivity ELISA BMS238HSIL-13 Ready-SET-Go!® ELISA 88-7439

Instant ELISA® BMS231INSTPlatinum ELISA BMS231/3

IL-1 beta Ready-SET-Go!® ELISA 88-7261 High Sensitivity ELISA BMS224HSInstant ELISA® BMS224INSTPlatinum ELISA BMS224/2

IL-2 Ready-SET-Go!® ELISA 88-7025High Sensitivity ELISA BMS221HSInstant ELISA® BMS221INSTPlatinum ELISA BMS221/2

IL-4 Ready-SET-Go!® ELISA 88-7046 High Sensitivity ELISA BMS225HSInstant ELISA® BMS225INSTPlatinum ELISA BMS225/2

IP-10 Instant ELISA® BMS284INSTPlatinum ELISA BMS6018

LAG-3 Platinum ELISA BMS2211PD-1 Platinum ELISA BMS2214PD-L1 Platinum ELISA BMS2212PD-L2 Platinum ELISA BMS2215Rantes Instant ELISA® BMS287/2INSTTIM-3 Platinum ELISA BMS2219VEGF-A Platinum ELISA BMS277/2OX40 (CD134) Platinum ELISA BMS296CD154 (CD40 Ligand) Platinum ELISA BMS239

Instant ELISA® BMS239INSTCD258 (LIGHT) Ready-SET-Go!® ELISA 88-7258 CD276 (B7-H3) Ready-SET-Go!® ELISA 88-50370 CD40 Platinum ELISA BMS265TSLP Ready-SET-Go!® ELISA 88-7497TWEAK Instant ELISA® BMS2006INST