Accepted Manuscript

Title: Pembrolizumab’s Non-cross Resistance Mechanism of

Action Successfully Overthrown Ipilimumab

Author: <ce:author id="aut0005" biographyid="vt0005"

author-id="S1040842816301986-
1e58935dfa97744dee68b20d7df032b6"> Mohd
Wahid<ce:author id="aut0010"
author-id="S1040842816301986-
b3439d1a5d1f03183e916d65daac696e"> Naseem
Akhter<ce:author id="aut0015"
author-id="S1040842816301986-
6d6750b69f4a8ae83a4a3d9788bbb97a"> Arshad
Jawed<ce:author id="aut0020"
author-id="S1040842816301986-
b613430b4b498400585abdb7b4f9c686"> Sajad A.
Dar<ce:author id="aut0025"
author-id="S1040842816301986-
cab4024af8948a1372d88ae3cfaf2614"> Raju K.
Mandal<ce:author id="aut0030"
author-id="S1040842816301986-
6e6c9fff47feef23c783bd870315b6a4"> Mohtashim
Lohani<ce:author id="aut0035"
author-id="S1040842816301986-
d6bb3ceebe1b5d33ef4f0cdf6ab9705f"> Mohammed Y.
Areeshi<ce:author id="aut0040"
author-id="S1040842816301986-
a9c71063221fa5ce897c86f286d15f2c"> Saif Khan<ce:author
id="aut0045" biographyid="vt0010"
author-id="S1040842816301986-
6950f0921f200135c109bcb84741673e"> Shafiul
Haque

PII: S1040-8428(16)30198-6
DOI: http://dx.doi.org/doi:10.1016/j.critrevonc.2017.01.001
Reference: ONCH 2292

To appear in: Critical Reviews in Oncology/Hematology

Received date: 31-8-2016

Revised date: 1-12-2016
Accepted date: 4-1-2017
Please cite this article as: {http://dx.doi.org/

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Pembrolizumab’s Non-cross Resistance Mechanism of Action
Successfully Overthrown Ipilimumab
Running title: Pembrolizumab’s non-cross resistance MOA successfully overthrown
Ipilimumab

List of authors in order of their authorship

Mohd Wahid1,2, Naseem Akhter3, Arshad Jawed2,4, Sajad A. Dar5, Raju K. Mandal2,6,
Mohtashim Lohani7, Mohammed Y. Areeshi2, Saif Khan7, Shafiul Haque2,8,9*

Authors’ affiliations
1
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central
University), New Delhi-110025, INDIA
2
Research and Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan
University, Jazan-45142, SAUDI ARABIA
3
Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Department of Ophthalmology,
Storm Eye Institute, Medical University of South Carolina, Charleston, SC-29425, USA
4
GE Healthcare, Sector-43, Gurgaon-122002, Haryana, INDIA

Division of Gynecology Oncology, Women’s Health Services, Henry Ford Hospital, Detroit,
5

MI-48202, USA
6
Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raibareli Road, Lucknow-226014,
UP, INDIA
7
Department of Biosciences, Integral University, Lucknow- 226026, UP, INDIA
8
Department of Biosciences, Jamia Millia Islamia (A Central University), New Delhi-110025,
INDIA
9
Centre for Drug Research, Faculty of Pharmacy, Viikki Biocentre-2, University of Helsinki,
Helsinki, FI-00014, FINLAND

*Corresponding author: Dr. Shafiul Haque; Centre for Drug Research (CDR), Faculty of
Pharmacy, Viikki Biocentre-2, University of Helsinki, PO Box 56 (Viikinkaari 5E), FI-00014,
Helsinki, FINLAND; Phone: +358-9-19159182; Fax: +358-9-191-59138
E-mail: shafiul.haque@hotmail.com
Current Address: Research and Scientific Studies Unit, College of Nursing and Allied Health
Sciences, Jazan University, Jazan-45142, SAUDI ARABIA

1
 Highlights

 Pembrolizumab dominates ipilimumab in PFS and OS by 78.49% & 183.2%,

respectively.

 Pembrolizumab’s dominancy is attributed to non-cross resistant mechanism of action.

 Pembrolizumab + BRAF inhibitors have shown synergistic effects in mouse melanoma.

ABSTRACT

The incidences of melanomas are increasing by leaps and bounds across the globe despite early

detection and intervention. The numbers of patients dying from metastatic melanoma have been

continually increased over the past thirty years. It has been considered as one of the most

therapy-resistant malignancies due to the cross-resistant mechanism developed by the metastatic

cells. With time, many new therapies came and they failed miserably. Ipilimumab, a monoclonal

antibody that works to activate the immune system by targeting CTLA-4 proved to be a boon for

advance melanoma very recently. But it couldn’t stand firmly against the resistant metastatic skin

cancer cells. Now, the new skin cancer drug named pembrolizumab proved as a new miraculous

molecule. It’s a humanized monoclonal antibody that blocks a biological pathway called

programmed cell death-1 (PD-1), which melanoma cells activate to suppress the immune system.

This antibody has surpassed ipilimumab at all the stages of clinical trials because of its non-cross

resistant mechanism to malignant cells. The present review critically analyzes the reasons of

efficacy success of pembrolizumab over ipilizumab shown at various stages of clinical trials.

Keywords: Metastatic skin cancer; Programmed cell death-1; Non-cross resistant, BRAF

inhibitors; Ipilimumab; Pembrolizumab

2
 1. Introduction

The oncology studies have witnessed lots of flip-flops in cancer care. It started with the

introduction of cisplatin in 1970s for the treatment of testicular and ovarian cancers and

continued till date for other molecules to treat various types of cancers or at least delay their

progression. Many a times, tumor cells develop resistance to conventional chemotherapy like

cytotoxic drugs (Grossman and Altieri, 2001) and start harassing the patients again.

The common problem faced by the scientists who boggle their mind and the development of

cancer drugs gets hindered is cross-resistance, which is defined as a tolerance to a medicinal

drug/molecule as a result of exposure to a similarly acting substance. It results due to the

frequent exposure to single anti-neuplastic agents, which in turn make cancerous cells resistant to

some agents of the same class (Riordan and Ling, 1985). Generally, resistant cells keep their

sensitivity to drugs of different classes with alternate mechanisms of cytotoxic action. Hence, the

cells resistant to alkylating agents or antifolates are usually sensitive to unrelated drugs, such as

anthracyclines etc. This resistance is upto some extent may be caused by a process called as

genetic amplification that allows the cancer cells to increase their mortality and invasion

properties due to which many drugs fail (Biedler and Riehm, 1970). The scientists with obstinate

mind again start searching for non-cross resistant drugs and find, if they are lucky. In order to

overcome this resistance, scientists have designed innovative approaches, such as combination

immunotherapy, co-stimulation with cytokines or growth factors etc (Reslan et al., 2009).

Earlier studies showed that BRAF inhibitors shrink melanoma tumors faster than even well-

established treatments like chemotherapy (Chapman et al., 2011; Hauschild et al., 2012).

Unfortunately, the treatment’s success was short lived and the effects subsided in just six months

of the treatment. The cancer cells were able to figure out how to use alternate pathways to grow

3
and divide once again. This eventually leads the melanoma tumors to become drug resistant to

BRAF inhibitors as well. But, the cancer therapy has evolved constantly despite many hurdles

and reached a new and refined epoch called immunotherapy helping tremendously in skin cancer

treatments.

With the advent of innovative technologies and novel discoveries, the skin cancer treatment is

touching incredible heights along with surgery, radiotherapy and chemotherapy (Ascierto and

Marincola, 2015). The checkpoint inhibitors are playing important roles for the treatment of

melanoma patients. These inhibitors act differently by directly acting on the tumor to induce

tumor cell death and further enhance antitumor immune responses to eliminate cancer cells

(Luke and Ott, 2015). The two main checkpoint inhibitors useful for the treatment of advanced

metastatic melanomas act on cytotoxic T-lymphocyte-associated protein 4 (CTLA-4or CTLA4)

also known as CD152 (cluster of differentiation 152) and Programmed cell death 1 (PD-1) or

cluster of differentiation 279 (CD279) protein pathways. The CTLA-4 and PD-1 receptors

rapidly emerged as promising targets for the ferocious advanced melanoma cancer. These

pathways operate at different stages of the immune responses and hence are non-cross resistant

to each other (Fife and Bluestone, 2008).

CTLA-4 is a protein present on T-cells that acts as a type of “off switch” to keep the immune

system in check. The inhibition of CTLA-4 may leads to continued activation of a larger number

of T cells, resulting in more effective antitumor responses. It modulates the immune response

early at the time of T-cell activation by antigen presenting cells (APCs). T-cells are activated by

antigen presented on APC in the context of major histocompatibility complex (MHC) (signal 1),

as well as co-stimulatory binding of CD28 to B7 (CD80/86) (signal 2). When T-cell gets

activated, CTLA-4 moves from Golgi apparatus to plasma membrane, where it over comes

4
CD28 in binding to B7 ligands on APCs due to its much higher binding affinity. CTLA-4

binding to B7 ligands inhibits further T-cell activation, limiting the time for T-cell activity. In

this way, the magnitude and duration of initial immune responses is physiologically controlled.

The inhibition of CTLA-4 may leads to continued activation of a large number of T-cells,

resulting in more effective antitumor responses.

The major role of PD-1 is to limit the activity of T-cells in the peripheral tissues occurring at the

time of inflammatory responses because of infections. The PD-1 pathway appears to impact the

T-cell response at the (later) effector stage as well. PD-1 is upregulated on T-cells after persistent

antigen exposure, typically in response to chronic infections or tumors. PDL1 and PD-L2, the

ligands for PD-1, can be expressed by tumor cells, as well as several other hematopoietic and

non-hematopoietic cell types. Expression of PDL1 and PD-L2 are induced by inflammatory

cytokines, predominately interferon-γ (Zou and Chen, 2008). Usually, the PD-1 is more

expressed than CTLA4 and is also induced on other activated non-T lymphocyte subsets,

including B-cells and natural killer (NK) cells and limits their lytic activity. Although, the PD-1

inhibition enhances the activity of effector T-cells in the tissues and in the tumor

microenvironment, it also probably enhances the activity of NK cells in tumors and tissues, and

may also enhances antibody production either indirectly or through direct effects on PD-1 + B-

cells (Terme et al., 2011; Fanoni et al., 2011; Velu et al., 2009).

The BRAF inhibitor named dabrafenib in combination with MEK inhibitor named trametinib

enjoyed a short term success in increasing the progression-free survival, but failed as most of the

patients treated with this combination developed acquired resistance. The immunotherapeutic

molecules that impede CTLA-4 and PD-1 proved themselves better as they were effective

regardless of BRAF status of the patients (Spagnolo et al., 2015).

5
The key players identified after rigorous work done by the scientists that block CTLA-4 and PD-

1 activities are ipilimumab and pembrolizumab, respectively. But pembrolizumab has proved its

mettle more strongly than ipilimumab because of its enhanced potency to cure large number of

patients and keeping important safety points in check.

1.1 Ipilimumab

Ipilimumab is a monoclonal antibody (mAb) that works to activate the immune system by

targeting CTLA-4, a protein receptor which is a negative regulator of T-cell activation. It binds

to CTLA-4 and obstructs the interaction of CTLA-4 with its ligands, B7-1 and B7-2 (Figure 1). It

releases intrinsic and extrinsic inhibitory controls on T-cell proliferation and activation (Walunas

et al., 1994; Peggs et al., 2008; Krummel and Allison, 1995). This process increases the

activation of T-cells and their proliferation leads to anti-tumor immune responses.

Ipilimumab was approved in 2011 for the treatment of unresectable or metastatic melanoma by

U.S. Food and Drug Administration (FDA) and the same was approved by European Medical

Agency in 2011 for the treatment of advanced melanoma because the antibody was capable of

inducing durable objective responses in 10-15% of the patients.

1.2 Pembrolizumab

The interaction of PD-L1 and PD-L2 ligands to the PD-1 receptor found on T cells inhibit their

proliferation and cytokine production. This linkage/upregulation of PD-1 ligands leads to

inhibition of active T-cell immune surveillance of tumors that cause melanoma cancer.

Pembrolizumab is a humanized IgG4 immunoglobulin subtype used in cancer immunotherapy

for the treatment of melanoma cancer. It does not activate complement or bind Fc receptors,

6
thereby avoiding cytotoxic effects on T-cells. It interacts with PD-1 receptor and blocks

interaction with its ligands, PD-L1 and PD-L2. The blocking of PD-1 receptor pathway leads to

the blockage of PD-1 mediated negative regulatory T-cell signaling, which leads to potent anti-

tumor response in the patient (Parry et al., 2005) (Figure 2).

Pembrolizumab was approved by the US FDA in September 2014 for the treatment of patients

with unresectable or metastatic melanoma and the disease progression following ipilimumab and,

if BRAF V600 mutation positive, a BRAF inhibitor.

Pembrolizumab surpassed ipilimumab at all the levels of performances comprising primary and

secondary end points. The primary and secondary end points discussed in this review are

progression free survival, overall survival, objective response rate, the duration of response,

safety etc., and are based on already performed studies all over the world.

2. Patients’ Selection

All the enrolled patients were 18 years of age or older having histologically confirmed,

unresectable stage III or IV melanoma and had not received more than one previous systemic

therapy for the advanced stage of the disease (Robert et al., 2015). The known BRAF V600

mutational status was required for the registration of patients with normal lactate dehydrogenase

levels and no significant tumor-related symptoms or evidence of rapidly progressing disease

(Chapman et al., 2011). The Eastern Cooperative Oncology Group (ECOG) performance status

of 0 or 1 (on a 5-point scale) was considered, wherein the higher score indicated greater

disability. The patients who had previously received CTLA-4, PD-1, or PD-L1 inhibitors and

those with ocular melanoma, active brain metastases, or a history of serious autoimmune disease

were not enrolled for the study.

7
 3. Study Design

A total of 834 patients were selected to receive the treatment of advanced melanoma from

September 18, 2013 to March 3, 2014. Out of 834 enrolled patients, 279 patients were randomly

assigned to receive pembrolizumab at a dose level of 10 milligrams per kilogram of body weight

every 2 weeks; 277 patients were randomly assigned to a group to receive 10 milligrams of

pembrolizumab per kilogram of body weight every 3 weeks, whereas 278 patients received four

cycles of ipilimumab at 3 milligrams per kilogram of body weight every 3 weeks.

Randomization was performed according to ECOG performance status (0 versus 1), line of

therapy (first versus second), and PD-L1 expression (positive versus negative) (Robert et al.,

2015).

Pembrolizumab was injected intravenously for a period of 30 min and was continued till disease

progression, the beginning of undesirable side effects, an investigator’s decision or the

withdrawal of patient consent to discontinue treatment, or completed 24 months assigned for the

treatment. The patients who showed complete response and were administered with

pembrolizumab for at least 6 months could discontinue treatment having received at least two

doses beyond the determination of complete response. Ipilimumab was administered

intravenously for a period of 90 min and continued for four cycles or until disease progression,

the beginning of intolerable side effects, an investigator’s decision to discontinue the treatment

or the patient himself has withdrawn consent.

8
3.1 Primary and Secondary End Points

The comparative studies to evaluate the effects of pembrolizumab and ipilimumab on patients

were mainly divided into two classes, viz., (a) First interim analysis, and (b) Second interim

analysis.

3.2 First Interim Analysis

This study was mainly focused on the evaluation of progression-free survival. It was investigated

by Robert et al. (2015), to comparatively assess the main end points of advanced melanoma

cancer by pembrolizumab and ipilimumab. In the first interim analysis for 6-months,

progression-free survival rate observed was 47.3% for the patients who received pembrolizumab

10 mg/kg body weight every 2 weeks. It was 46.4% for the patients who received

pembrolizumab10 mg/kg body weight every 3 weeks, whereas the patients administered with

ipilimumab 3 mg/kg body weight showed 26.5% progression free survival. Median progression-

free survival observed for the treatment methods adopted for pembrolizumab were 5.5 months

and 4.1 months, whereas for ipilimumab, the treatment regimen was 2.8 months. The hazard

ratio for disease progression was 0.58 with p-value less than 0.001 for both, every two weeks as

well as every three weeks pembrolizumab course of therapy vis-à-vis ipilimumab; 95%

confidence interval (CI) was there with values ranging from 0.46 to 0.72 and 0.47 to 0.72,

respectively (Table 1).

3.3 Second Interim Analysis

The second interim analysis focused mainly on overall survival rate estimation of the patients

enrolled in the study. The investigation was carried out basically for 12 months for which all the

9
patients were followed-up. The number of deaths occurred during the follow-up period were

289.The overall survival rates observed were 74.1% for the patients administered with

pembrolizumab every two week (hazard ratio for death as compared with the ipilimumab group,

0.63; 95% CI, 0.47 to 0.83; P<0.0005) and 68.4% for the patients who received pembrolizumab

after every three weeks (hazard ratio for death as compared with the ipilimumab group, 0.69;

95% CI, 0.52 to 0.90; P = 0.0036). The overall survival percentage for the patients who received

ipilimumab was 58.2 (Table 1).

Pembrolizumab evidently dominated ipilimumab in both, the first and the second interim

analyses for progression-free and overall survival of the patients, which are considered as the

primary end points of advanced melanoma. Furthermore, it continued to lead over ipilimumab

for objective responses as well. The observed facts stand out in the study conducted by Robert et

al., (2015). The patients in the pembrolizumab treatment group showed 33.7% objective response

rates for every two-weeks dosage regimen and 32.9% for every three-weeks dosage

administration, whereas those in the ipilimumab treatment group demonstrated 11.9%. The

statistically observed values of the clinical trials for two pembrolizumab dosing regimens

exhibited no apparent differences in the objective response rates. The iota of differences present

is based on line of therapy and the expression of ligands for the 'programmed death 1' protein

(termed 'PD-L1’).

4. Safety

The safety of the patients undergoing treatment during clinical trials or after the launch of the

drug in a market is of utmost importance. The prime concerns of safety for the drugs are

treatment-related adverse events, which are classified in grades of various severities. The

10
treatment-related adverse reaction of pembrolizumab and ipilimumab were also compared,

wherein the grades of 3 to 5 severity were lower in the pembrolizumab groups (13.3% and

10.1%) than in the ipilimumab group (19.9%). The ipilimumab group reported only one

treatment-related death of the patient. The patient had a history of type-2 diabetes and died from

cardiac arrest secondary to metabolic imbalances associated with ipilimumab-induced diarrhea.

5. Opinion

Pembrolizumab overthrown ipilimumab at all the stages of the treatment whatever may be the

parameter of clinical trials like primary or secondary end-points. Pembrolizumab has

significantly improved the progression-free survival as well as overall survival with very less

number of high grade-toxic incidences in treated patients. Quantitatively, during the first interim

analysis of 6-months, pembrolizumab exceeds in approximately 78.49% of the patients for

progression free survival. The parameter for overall survival measured for 12 months period also

exceeded in almost 27% of the patients. Furthermore, it continued to dominate for the objective

responses and safety features as well. The objective response rate tracked in the treatment cases

increased to about 183.2%. Additionally, the treatment-related adverse reactions of

pembrolizumab vis-à-vis ipilimumab were lower (i.e. 13.3% vs. 19.9%). The increased values of

the mentioned beneficial parameters for the treatment of unresectable or metastatic melanoma

patients prove the superiority of pembrolizumab.

The major question arises why pembrolizumab is dominating ipilimumab at all the level of

performances albeit both are immune check-point inhibitors. Different possibilities exist and

have been suggested by the scientists across the globe. An international group of scientists

conducted a clinical trial to check the effectiveness of pembrolizumab over ipilimumab. They

11
infused the patients with pembrolizumab who had progressed on ipilimumab and were tested

positive for BRAF mutations, and were also treated with a BRAF inhibitor (dabrafenib; Tafinlar,

Glaxo SmithKline) and vemurafenib (Zelboraf, Roche) or a MEK inhibitor (trametinib;

Mekinist, Glaxo SmithKline), or both. The overall responses to pembrolizumab in these cases of

patients with refractory melanoma were lower than those previously reported for pembrolizumab

in melanoma patients who were treatment-naïve or had not previously received ipilimumab. But

the results were found to be in line with those reported for nivolumab in ipilimumab-treated

melanoma patients. This suggests that PD inhibition is a distinct therapeutic approach from that

of CTLA-4 blockade (the mechanism of action of ipilimumab), even though both are

immunotherapies and immune checkpoint inhibitors (Pembrolizumab works in melanoma after

ipilimumab) http://www.medscape.com/viewarticle/828738#vp_1 (Accessed on 13.02.2016)].

Further to this, the non-cross resistant mechanism of action has been suggested by many other

scientists as well (Weber et al., 2011). Purified peripheral blood CD4+ and CD8+ T- cells were

isolated from the patients suffering from melanoma cancer both pre- and post-treatment of

ipilimumab and gene expression changes were observed in CD4+ T-cell compartment when the

patients were administered with ipilimumab whereas biopsies done on patients treated with anti–

PD-1 antibody showed predominately a CD8+ infiltrate. It was also explained that biochemical

pathways catalyzed by CTLA-4 and PD-1/PD-L1 were biochemically and molecularly distinct.

Additionally, it was also apprehended that possibly CTLA-4 preferentially augments CD4+ T-

cells and PD-1 acts predominately on CD8+ T-cells, which might be the probable reason for non-

cross-resistance observed in the study (Weber et al., 2011; Hamid et al., 2013).

Earlier, the checkpoint inhibitors that target programmed cell death-1 receptor (PD-1) and its

ligand (PD-L1) have demonstrated synergistic effects with BRAF inhibitors in a mouse model of

12
melanoma. The combinations may act as an alternative option for the BRAF monotherapy

resistant patients. These experiments may also hold true for BRAF-pembrolizumab combination

though no such results have been published yet. The clinical trials combining BRAF and

pembrolizumab strategies are ongoing and in coming time, the new melanoma model may

provide therapeutic insights, including optimal timing and sequence of therapy (Cooper et al.,

2014). Hence, the suggested new synergistic combination of BRAF inhibitors with

pembrolizumab has tremendous potential to provide greater heights to the skin cancer treatments.

Conflict of Interest

The authors have declared no conflicts of interests.

Acknowledgements

Authors are grateful to Jazan University, Saudi Arabia, and Jamia Millia Islamia, New Delhi,

India for providing the necessary facilities for this research study.

13
References

Ascierto, P.A., Marincola, F.M., 2015. 2015: The Year of Anti-PD-1/PD-L1s Against Melanoma

and Beyond. EBiomedicine 2, 92-93.

Biedler, J.L., Riehm, H., 1970. Cellular resistance to actinomycin D in Chinese hamster cells in

vitro: Cross-resistance, radioautographic, and cytogenetic studies. Cancer Res 30, 1174-

1184.

Chapman, P.B., Hauschild, A., Robert, C., Haanen, J.B., Ascierto, P., Larkin, J., Dummer, R.,

Garbe, C., Testori, A., Maio, M., Hogg, D., Lorigan, P., Lebbe, C., Jouary, T.,

Schadendorf, D., Ribas, A., O'Day, S.J., Sosman, J.A., Kirkwood, J.M., Eggermont, A.M.,

Dreno, B., Nolop, K., Li, J., Nelson, B., Hou, J., Lee, R.J., Flaherty, K.T., McArthur, G.A.;

BRIM-3 Study Group. 2011. Improved survival with vemurafenib in melanoma with

BRAF V600E mutation. New England Journal of Medicine 364(26), 2507-2516.

Cooper, Z.A., Juneja, V.R., Sage, P.T., Frederick, D.T., Piris, A., Mitra, D., Lo, J.A., Hodi, F.S.,

Freeman, G.J., Bosenberg, M.W., McMahon, M., Flaherty, K.T., Fisher, D.E., Sharpe,

A.H., Wargo, J.A., 2014. Response to BRAF inhibition in melanoma is enhanced when

combined with immune checkpoint blockade. Cancer Immunol Res (7), 643-654.

Fanoni, D., Tavecchio, S., Recalcati, S., Balice, Y., Venegoni, L., Fiorani, R., Crosti, C., Berti,

E., 2011. New monoclonal antibodies against B‑cell antigens: possible new strategies for

diagnosis of primary cutaneous B‑cell lymphomas. Immunol Lett 134, 157-160.

Fife, B.T., Bluestone, J.A., 2008. Control of peripheral T-cell tolerance and autoimmunity via the

CTLA-4 and PD-1 pathways. Immunol Rev 224, 166-182.

Grossman, D., Altieri, D.C., 2001. Drug resistance in melanoma: mechanisms, apoptosis, and

new potential therapeutic targets. Cancer Metastasis Rev 20(1-2), 3-11.

14
Hamid, O., Robert, C., Daud, A., Hodi, F.S., Hwu, W., Kefford, R., Wolchok, J.D., Hersey, P.,

Joseph, R.W., Weber, J.S., Dronca, R., Gangadhar, T.C., Patnaik, A., Zarour, H., Joshua,

A.M., Gergich, K., Elassaiss-Schaap, J., Algazi, A., Mateus, C., Boasberg, P., Tumeh,

P.C., Chmielowski, B., Ebbinghaus, S.W., Li, X.N., Kang, S.P., Ribas, A., 2013. Safety

and Tumor Responses with Lambrolizumab (Anti–PD-1) in Melanoma. The New England

Journal of Medicine 369(2), 134-144.

Hauschild, A., Grob, J.J., Demidov, L.V., Jouary, T., Gutzmer, R., Millward, M., Rutkowski, P.,

Blank, C.U., Miller, W.H Jr., Kaempgen, E., Martín-Algarra, S., Karaszewska, B., Mauch,

C., Chiarion-Sileni, V., Martin, A.M., Swann, S., Haney, P., Mirakhur, B., Guckert, M.E.,

Goodman, V., Chapman, P.B., 2012. Dabrafenib in BRAF-mutated metastatic melanoma: a

multicentre, open-label, phase 3 randomised controlled trial. Lancet 380(9839), 358-365.

Krummel, M.F., Allison, J.P., 1995. CD28 and CTLA-4 have opposing effects on the response of

T cells to stimulation. J Exp Med 182, 2459-2465.

Luke, J.J., Ott, P.A., 2015. PD-1 pathway inhibitors: The next generation of immunotherapy for

advanced melanoma. Oncotarget 6(6), 3479-3492.

Parry, R.V., Chemnitz, J.M., Frauwirth, K.A., Lanfranco, A.R., Braunstein, I., Kobayashi, S.V.,

Linsley, P.S., Thompson, C.B., Riley, J.L., 2005. CTLA-4 and PD-1 receptors inhibit T-

cell activation by distinct mechanisms. Mol Cell Biol 25, 9543-9553.

Peggs, K.S., Quezada, S.A., Allison, J.P., 2008. Cell intrinsic mechanisms of T-cell inhibition

and application to cancer therapy. Immunol Rev 224, 141-65. doi: 10.1111/j.1600-

065X.2008.00649.x.

Pembrolizumab works in Melanoma after Ipilimumab.

http://www.medscape.com/viewarticle/828738#vp_1 (Accessed February13, 2016).

15
Reslan, L., Dalle, S., Dumontet, C., 2009. Understanding and circumventing resistance to

anticancer monoclonal antibodies. MAbs 1(3), 222-229.

Riordan, J.R., Ling, V., 1985. Genetic and biochemical characterization of multidrug resistance.

Pharmacol Ther 28, 51-75.

Robert, C., Schachter, J., Long, G.V., Arance, A., Grob, J.J., Mortier, L., Daud, A., Carlino,

M.S., McNeil, C., Lotem, M., Larkin, J., Lorigan, P., Neyns, B., Blank, C.U., Hamid, O.,

Mateus, C., Shapira-Frommer, R., Kosh, M., Zhou, H., Ibrahim, N., Ebbinghaus, S., Ribas,

A.; KEYNOTE-006 investigators., 2015. Pembrolizumab versus Ipilimumab in Advanced

Melanoma. The New England Journal of Medicine 372(26), 2521-2532.

Spagnolo, F., Ghiorzo, P., Orgiano, L., Pastorino, L., Picasso, V., Tornari, E., Ottaviano, V.,

Queirolo, P., 2015. BRAF-mutant melanoma: treatment approaches, resistance

mechanisms, and diagnostic strategies. OncoTargets Ther. 8, 157–168.

Terme, M., Ullrich, E., Aymeric, L., Meinhardt, K., Desbois, M., Delahaye, N., Viaud, S.,

Ryffel, B., Yagita, H., Kaplanski, G., Prévost-Blondel, A., Kato, M., Schultze, J.L.,

Tartour, E., Kroemer, G., Chaput, N., Zitvogel, L., 2011. IL‑18 induces PD‑1‑dependent

immunosuppression in cancer. Cancer Res 71, 5393-5399.

Velu, V., Titanji, K., Zhu, B., Husain, S., Pladevega, A., Lai, L., Vanderford, T.H., Chennareddi,

L., Silvestri, G., Freeman, G.J., Ahmed, R., Amara, R.R., 2009. Enhancing SIV-specific

immunity in vivo by PD‑1 blockade. Nature 458, 206-210.

Walunas, T.L., Lenschow, D.J., Bakker, C.Y., Linsley, P.S., Freeman, G.J., Green, J.M.,

Thompson, C.B., Bluestone, J.A., 1994. CTLA-4 can function as a negative regulator of T

cell activation. Immunity 1(5), 405-413.

16
Weber, J.S., Yu, B., Hall, M., Morelli, D., Yu, D., Zhang, Y., Zhao, X., Sarnaik, A., Wang, W.,

2011. Pharmacodynamic and predictive markers of ipilimumab on melanoma patients’ T-

cells. Program and abstracts from the 2011 Annual Meeting of the American Society of

Clinical Oncology. Chicago, Illinois, Abstract 2503.

Zou, W., Chen, L., 2008. Inhibitory B7-family molecules in the tumour microenvironment. Nat

Rev Immunol 8, 467-477.

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Biographies

Shafiul Haque completed his doctoral degree in biotechnology and accomplished fabulous

blend of managerial knowledge by completing MBA, PG Diploma in Pharma Regulatory

Affairs, and Certificate course on Intellectual Property Rights. He worked for Bio-Rad

laboratories as an Application Specialist (genomics & proteomics) for EMEA region & West

India. He completed his Post-doctoral research from the University of Helsinki, Finland. Later,

he worked as a Research Consultant for King Saud University and currently he is leading the

Research & Scientific Studies Unit of the College of Nursing & Allied Health Sciences, Jazan

University, Saudi Arabia. His areas of interest are cancer/ tuberculosis genetics, bioprocess,

bioinformatics, medical education, and infectious diseases.

Mohd Wahid completed his PhD in biochemistry from Jamia Millia Islamia, New Delhi, India.

He attained outstanding blend of techno-legal knowledge by completing PG Diplomas in

Pharmaceutical Regulatory Affairs and in Intellectual Property Laws. He was a manager at R&D

center of Dr. Reddy’s Laboratories Ltd. Hyderabad (India) and supervised different projects

dealing with therapeutic proteins/antibodies development, and also participated in their clinical

trials. Also, he worked as an Intellectual Property Manager in Glenmark Research Centre,

Mumbai (India) for the development of New Biological Entities (NBEs). Currently, Dr. Wahid is

working as an Asst. Professor of biochemistry at the College of Nursing & Allied Health

Sciences, Jazan University, Saudi Arabia. He expertise included infectious diseases,

immunology, and cancer biology.

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Figure Legends

Figure 1. Ipilimumab mechanism of action showing interaction with CTLA-4 receptor.

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Figure 2. Pembrolizumab interaction with PD-1 allowing T-cell to function by releasing a brake
on the immune system.

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Table 1: Comparative clinical effectiveness of pembrolizumab and ipilimumab (interim analysis)

Pembrolizumab Ipilimumab % change

No. of patients 277 279 278
Dosage 10 mg/kg every two 10 mg/kg every three 3 mg/kg every three
weeks weeks week
First interim analysis, 6 months
Progression free 47.3% 46.4% 26.5% Max. 78.49%
survival (% of patients)
Median: Months 5.5 4.1 2.8 Max. 96.4%
(95% CI) (3.4-6.9) (2.9-6.9) (2.8-2.9)
Hazard ratio 0.58 0.58 -- --
vs. Ipilimumab (0.46-0.72) (0.47-0.72)
P<0.001 P<0.001
Interim Analysis, 12 months
Overall survival 74.1 68.4 58.2 Max. 27
(% of patients)
Hazard ratio vs. 0.63 0.69 -- --
Ipilimumab 0.47 to 0.83 0.52 to 0.90
(95% CI) P<0.0005 P = 0.0036
Abbreviations: CI, confidence interval

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