Tecemotide: An antigen-specific cancer immunotherapy

Abstract

The identification of tumor-associated antigens (TAA) has made possible the development of antigen-specific cancer immunotherapies such as tecemotide. One of those is mucin 1 (MUC1), a cell membrane glycoprotein expressed on some epithelial tissues such as breast and lung. In cancer, MUC1 becomes overexpressed and aberrantly glycosylated, exposing the immunogenic tandem repeat units in the extracellular domain of MUC1. Designed to target tumor associated MUC1, tecemotide is being evaluated in Phase III clinical trials for treatment of unresectable stage IIIA/IIIB non-small cell lung cancer (NSCLC) as maintenance therapy following chemoradiotherapy. Additional Phase II studies in other indications are ongoing. This review discusses the preclinical and clinical development of tecemotide, ongoing preclinical studies of tecemotide in human MUC1 transgenic mouse models of breast and lung cancer, and the potential application of these models for optimizing the timing of chemoradiotherapy and tecemotide immunotherapy to achieve the best treatment outcome for patients.

Abbreviations

ADT

androgen deprivation therapy

APC

antigen presenting cell

ASI

active specific immunotherapy

BSC

best supportive care

CEA

carcinoembryonic antigen

CI

confidence interval

CONSORT

consolidated standards of reporting trials

CPA

cyclophosphamide

CRT

chemoradiotherapy

CTL

Cytotoxic T-lymphocyte

DMPG

Dimyristoyl phosphatidylglycerol

DPPC

Dipalmitoyl phosphatidylcholine

DTH

delayed-type hypersensitivity

ECOG

Eastern cooperative oncology group

ELISpot

enzyme-linked immunosorbent spot

FACT-L

functional assessment of cancer therapy-lung

Gy

gray

HLA

human lymphocyte antigen

HR

hazard ratio

IFN-γ

interferon gamma

IgG

immunoglobulin G

IL-2

Interleukin 2

INSPIRE

stimuvax trial in Asian NSCLC patients: stimulating immune response

ITT

intent to treat

i.v.

intravenous

KLH

keyhole limpet hemocyanin

LICC

L-BLP25 in colorectal cancer

LR

locoregional

MAP

multiple antigenic peptide

MHC

major histocompatibility complex

MMT

muc1-expressing mammary tumor

MPLA

monophosphoryl lipid A

MUC1

Mucin 1

MUC1.Tg

MUC1 transgenic

NSCLC

non-small cell lung cancer

OH-BBN

N-butyl-N-(4-hydroxybutyl)nitrosamine

OS

overall survival

PBL

peripheral blood lymphocytes

PCR

pathological complete remission

PSA

prostate specific antigen

PyV-mT

polyomavirus middle-T

QOL

quality of life

RCB

residual cancer burden

RECIST

response evaluation criteria in solid tumors

RTX

radiotherapy

START

stimulating targeted antigenic responses to NSCLC

TAA

tumor associated antigen

TGF-β

transforming growth factor β

T_H1

T-helper type I

T_H2

T-helper type II

TNF-α

tumor necrosis factor α

TOI

trial outcome index

VNTR

variable number of tandem repeats

Introduction

The development of antigen-specific cancer immunotherapies has been made possible by the identification of tumor-associated antigens (TAA), of which a vast number have now been discovered.^1,2 One of the best characterized TAAs is mucin 1 (MUC1), which is overexpressed and aberrantly glycosylated in over 90% of adenocarcinomas such as breast and lung cancer.^3,4 While MUC1 is a self-antigen expressed in a number of different epithelial tissues, the normal glycosylation pattern shields the peptide core from immune surveillance. The aberrant underglycosylation of MUC1 in cancer leads to the exposure of the immunogenic tandem repeat regions of the core peptide, which had made MUC1 an attractive target for immunotherapies.^2,5-8 Both cellular and humoral immune responses to MUC1 have been observed in many patients with lung, breast and other adenocarcinomas, but the native immune response is insufficient in controlling tumor growth, which is due to existing tolerance and the development of multiple mechanisms of immune evasion.^4,9-11 Tecemotide, a MUC1-specific cancer immunotherapy, is currently being evaluated in Phase III clinical trials as maintenance therapy following chemoradiotherapy (CRT) for the treatment of unresectable stage IIIA/IIIB non-small cell lung cancer (NSCLC). Recently published clinical trials results suggest that the timing of chemotherapy, radiation, and tecemotide immunotherapy is important with respect to treatment outcome.¹²

Tecemotide, formerly known as L-BLP25 or Stimuvax®, has been developed for the treatment of adenocarcinomas that express MUC1, a member of the membrane-bound O-glycoprotein mucin family. Tecemotide is designed to elicit an antigen-specific cellular immune response against MUC1, which was one of the first TAAs identified by human tumor-specific T-cells.⁸ It is broadly distributed on the apical surface of most simple epithelial tissues, but strong expression of aberrantly glycosylated, tumor-associated MUC1 has been observed in most adenocarcinomas, including lung, breast, stomach, pancreas, colon, prostate, and ovary as well as hematological malignancies such as multiple myeloma. On tumor cells polarity is lost and MUC1 is expressed on the entire cell surface.¹³ The extracellular domain of MUC1 consists of a variable number of tandem repeats (VNTR) of a characteristic 20-amino acid sequence (PDTRPAPGSTAPPAHGVTSA) that is aberrantly glycosylated in tumor tissues.^14-17 The resulting underglycosylation of MUC1 facilitates peptide processing and loading onto human lymphocyte antigen (HLA) molecules, which results in the exposure of a novel and large epitope repertoire bound to HLA molecules that can be recognized by MUC1-specific cytotoxic T-lymphocytes (CTLs).^18,19 It has been shown that immunization of cancer patients with MUC1 peptides results in the generation of both anti-MUC1 antibodies and CTL responses.²⁰ In addition, MUC1 was found to be expressed on activated T cells, exhibiting both immunostimulatory and immunosuppressive functions.²¹ Several MUC1-based immunotherapy approaches such as tecemotide,^12,22-25 TG4010,^26-28 and PANVAC™,^29-31 have been evaluated in clinical trials for a variety of malignancies including NSCLC, metastatic breast cancer, renal cell carcinoma, ovarian cancer and metastatic colorectal carcinoma.

The purpose of this review is to discuss the preclinical and clinical development of tecemotide and to highlight ongoing research in human MUC1 transgenic mouse models of breast³² and lung cancer^33,34 that are being utilized with the goal of optimizing the timing of chemotherapy, radiation, and tecemotide immunotherapy to achieve the best treatment outcome for patients.³⁵ As there are presently no effective maintenance therapies for stage III NSCLC following CRT, immunotherapies such as tecemotide offer a potential method of improving overall treatment outcomes.³⁶

Preclinical Development of Tecemotide

BP16

Early preclinical research into the development of MUC1 immunotherapy strategies was conducted using various synthetic peptides from the tandem repeat region of the MUC1 core peptide.³⁷ Mice were immunized with peptides of various lengths conjugated to either dendritic multiple antigenic peptide (MAP-4) or keyhole limpet hemocyanin (KLH) as a carrier molecule and using DETOX, which contains monophosphoryl lipid A (MPLA) and mycobacterial cell wall, as an adjuvant. Each of the peptides tested contained the B and T cell epitope PDTRP in various configurations, along with portions of the 20-amino acid sequence from the MUC1 tandem repeat region. The SP1–16 peptide additionally contained 2 MUC1 tandem repeats plus a universal T-cell epitope from tetanus toxin. Following immunization, antibody, delayed type hypersensitivity (DTH), and antitumor responses were evaluated. To assess DTH and antitumor responses, immunized mice were challenged with 410.4 mouse mammary tumor cells that had been transfected with human MUC1.³⁸ Delayed type hypersensitivity responses were also assessed by peptide challenge in immunized mice. The results showed that peptides conjugated to either KLH (SP1–7-KLH) or MAP-4 (SP1–5 and SP1–6) proved to be the most immunogenic.³⁷ The SP1–6 peptide additionally contained a 19-amino acid sequence (EKKIAKMEKASSVFNVVNS) from the CST-3 peptide of Plasmodium falciparum, which is a universal T-helper epitope.

Mice that had been immunized displayed DTH reactions, indicative of cellular immunity, only in response to challenge with the same or similar peptide, which shows specificity of response. When challenged with mammary tumor cells, immunized mice had DTH responses only when injected with cells that had been transfected with human MUC1. The antitumor effects of these peptides were evaluated in both preventive (immunization) and treatment (active specific immunotherapy or ASI) settings. Whether used prophylactically or as ASI, significant tumor growth inhibition was seen with the MAP-4 conjugated peptides and the KLH-conjugated peptide SP1–7, a 16-mer (GVTSAPDTRPAPGSTA), in particular.³⁷ Active specific immunotherapy with the 3 most promising peptides in this study, SP1–5, a 22-mer (APDTRPAPGSTAPPAHGVTSAP-MAP4), SP1–6, an 8-mer (SAPDTRPA-CST-MAP4) and SP1–7-KLH, was most effective when mice were pretreated with cyclophosphamide (CPA), which is thought to reduce the number and immunosuppressive functionality of T-regulatory cells,^39-43 thereby augmenting the response to immunotherapy.^44,45 Some interesting differences were observed in this study with respect to immune response. Peptides that induced strong antibody and DTH responses were inferior with respect to antitumor effects compared to those that were poor inducers of antibody response. This result is consistent with the hypothesis that for an effective antitumor response, a T_H1 or cellular immune response is superior to a T_H2 or humoral antibody immune response.³⁷

BP24

Based on the results obtained with the SP1–7 peptide (BP16) by Ding et al. in 1993, additional studies were performed using a newer peptide, BP24 (TAPPAHGVTSAPDTRPAPGSTAPP), which was synthesized to include additional T-cell epitopes compared to BP16.⁴⁶ Because there is strong evidence suggesting that multiple pregnancies provide protection against breast cancer, and MUC1 is highly expressed in breast cancer, Agrawal et al. evaluated the proliferation of peripheral blood lymphocytes (PBL) from men and multiparous and nulliparous women in response to exposure to BP24.^47,48 These investigators found that CD4+ T cells from multiparous women, but not nulliparous women or men, proliferated specifically in response to BP24 as well as BP16, the sequence of which is contained within BP24. No significant proliferation was noted in response to a negative control peptide. Proliferative responses were noted in the lymphocytes from several different HLA types in an MHC class II-restricted fashion.⁴⁷ In a follow-up experiment using T lymphocyte cell lines established from the PBLs of multiparious donors, MUC1-specific MHC class I-restricted CTLs were induced by stimulation with autologous antigen-presenting cells (APCs) that had been loaded with BP24 peptide.⁴⁸ This result is consistent with Domenech et al., who showed MHC class I-restricted binding of a 9 amino acid peptide (STAPPAHGV) to HLA-A1, HLA-A2.1, HLA-A3 and HLA-A11,⁴⁹ although BP24 does not fully contain this peptide sequence. This latter experiment was performed under conditions that favored peptide binding to MHC class I molecules, whereas conditions in the earlier study favored MHC class II binding, and no CTL response was observed.⁴⁷

Liposomal BP24

In order to improve cellular or T_H1 type immune responses, the BP24 peptide was prepared in a liposomal formulation, using MPLA as the adjuvant, and then evaluated for immunogenicity and antitumor activity.⁴⁶ Compared to BP16-KLH, the immune response to liposomal BP24 was predominantly T_H1, as assessed by immunizing non-tumor-bearing mice. Mice treated with BP16-KLH showed a much stronger IgG antibody response compared to the BP24-treated mice, suggesting a poorer CTL cellular immune response to the KLH-conjugated peptide. The antitumor activity of liposomal BP24 was evaluated in intravenous, subcutaneous and artificial metastasis tumor models using a human MUC1 transfected mouse mammary carcinoma cell line (410.4). Following intravenous tumor challenge after immunization, mice treated with liposomal BP24 survived significantly longer and formed significantly fewer lung metastases. Mice immunized with liposomal BP24 showed complete protection following subcutaneous tumor challenge with MUC1 transfected cells, while immunization provided no protection against non-transfected cells, indicating specificity of immune response. The artificial metastasis model showed that BP24 immunization again showed complete protection with respect to the formation of lung tumor foci. When used as ASI, BP24-treated mice developed far fewer lung tumor foci compared to control mice or mice treated with empty liposomes containing MPLA. Results of cytokine and antibody analyses in tumor-bearing mice were consistent with a T_H1-polarized immune response.⁴⁶

Liposomal BLP25

To further augment the MUC1 immune response, a serine residue was added to BP24, yielding BP25 (STAPPAHGVTSAPDTRPAPGSTAPP), also known as BLP25.^50,51 This synthetic MUC1 peptide was synthesized to introduce HLA class I epitopes⁴⁹ to complement the HLA class II epitopes that had already been demonstrated for the MUC1 peptide BP24, the sequence of which is completely contained within BP25.⁴⁷ The first experiments with liposomal BP25 (BLP25) examined the generation of MUC1-specific CD4+ and CD8+ T-cell responses in vitro in unprimed lymphocytes from the PBL of human donors.⁵⁰ T cells cultured in the presence of autologous APCs pulsed with liposome-encapsulated BLP25 showed marked proliferation compared to those cultured with APCs that had been pulsed with soluble BLP25. When T-cell proliferation was assessed for antigen specificity, T cells proliferated most strongly in response to liposomal BLP25, with weak responses to liposomal BLP24 and soluble BLP25 and BP24. These lymphocyte responses were found to be T_H1 type based on increased Interferon gamma (IFN-γ) release and minimal IL-4. The lymphocyte subtypes proliferating in response to BLP25 were identified as CD4+ and CD8+ T cells based on flow cytometric analysis and the lack of proliferation in the presence of anti-CD4 and anti-CD8 antibodies. The T cells stimulated by autologous APCs pulsed with BLP25 were able to lyse targets pulsed with HLA class I 9-mer MUC1 peptide epitopes, confirming cytotoxic activity.⁵⁰

As pointed out above, early studies performed by Agrawal et al. on liposomal BLP25 showed that T cells cultured with autologous APCs pulsed with liposomal BLP25 showed marked proliferation compared to T cells cultured with APCs pulsed with soluble BLP25.⁵⁰ This finding is consistent with the results of a study by Guan et al. that showed physical association of the peptide with liposomes, either through encapsulation or surface exposure, is required for a T-cell proliferative response. Peptide alone or peptide combined with empty liposomes failed to elicit an antigen-specific T-cell response in immunized mice.⁵¹ This study also found that the nature of the physical association had a significant effect on the humoral immune response to the BLP25 peptide. Compared to liposomal BLP25, only those mice that were immunized with surface-exposed peptide liposomes developed MUC1-specific antibodies, which suggests that different liposome formulations can produce different immune responses.⁵¹

Tecemotide in Transgenic Tumor-Bearing Mouse Models

Breast cancer

An immune-intact transgenic breast cancer mouse model that expresses human MUC1 as a self-antigen under the control of its own promoter in a pattern consistent with humans⁵² was developed to study MUC1-specific immunotherapy.⁴ To develop this model, MUC1 transgenic (MUC1.Tg) C57BL/6 mice⁵² were crossed with mice transgenic for polyomavirus middle T (PyV-mT) driven by the MMTV promoter.⁵³ The resulting double transgenic MUC1-expressing mammary tumor (MMT) mice spontaneously develop breast cancer that expresses human MUC1. Using this model, Mukherjee et al. evaluated the effects of liposomal BLP25, now known as tecemotide.⁴ In this study, mice that had been immunized with tecemotide developed MUC1 antigen-specific T-cell responses and CTLs that were cytotoxic in vitro against mouse breast cancer cells that had been transfected with human MUC1. The T cells that developed in immunized mice expressed intracellular IFN-γ, consistent with a T_H1 immune response, and were reactive with an MHC class I MUC1 tetramer. Although immunization with tecemotide generated MUC1-specific CTLs, this did not result in a durable antitumor response. A significantly lower tumor burden at 18 weeks was observed in immunized mice compared to controls; however, no significant differences in tumor burden were seen at 24 weeks. Mukherjee et al. found evidence suggesting that tumor evasion mechanisms may have been responsible. Specifically, the expression of transforming growth factor beta (TGF-β), which can create an immunosuppressive tumor microenvironment,⁵⁴ was found to increase as tumors progressed, an effect supported by decreasing numbers of T cells expressing IFN-γ. In addition, the surface expression of MHC class I molecules, required for CTL immune recognition, decreased as tumor burden increased.⁴ Furthermore, immune tolerance may also have contributed to the lack of significant antitumor effects in this study.⁵²

The MMT breast cancer model was later used to show that tecemotide combined with letrozole, an aromatase inhibitor used in the treatment of breast cancer, had additive antitumor effects and significantly increased survival compared to tecemotide alone.³² This was the first preclinical study to demonstrate additive antitumor activity and survival benefits following hormonal therapy combined with immunotherapy. Also assessed in this study were the effects of tamoxifen combined with tecemotide immunotherapy, but this combination resulted in no added antitumor or survival benefits compared to tamoxifen monotherapy, which was not unexpected given previously published results regarding the effects of tamoxifen on immunity.^55-59 Mehta et al. showed that hormonal therapy did not interfere with the immune response to tecemotide and demonstrated a T_H1 immune response as well as the generation of CTLs specific for human MUC1 in their study.³² The antitumor effect of tecemotide was not seen in the study conducted by Mukherjee et al.,⁴ but the timing of tecemotide immunotherapy may have played a role. It is known that cancer specific immunotherapy is most effective with minimal tumor burden.^60-62 In the study by Mukherjee et al., tecemotide immunotherapy began at 7 weeks of age, when tumors were already detectable, and CPA pretreatment was not employed in that study. When Mehta et al. began tecemotide immunotherapy combined with hormonal therapy after tumors had become established, no significant effects of treatment on tumor burden or survival were noted, in agreement with the results of Mukherjee et al.³²

Lung cancer

As tecemotide has primarily been evaluated clinically in lung cancer, a human MUC1.Tg, immune intact, lung tumor-bearing mouse model³⁴ has been developed to evaluate the effects of tecemotide in a preclinical or “postclinical” setting to refine the protocols of clinical trials, e.g. the Phase III START trial of tecemotide as maintenance therapy following CRT.¹² Using the chemical carcinogen urethane to induce the development of lung tumors, this mouse model was developed in human MUC1.Tg C57BL/6 mice and used to assess the efficacy of tecemotide.³⁴ After 10 weekly doses of 0.75 mg/g urethane, mice were administered either one or 2 cycles of tecemotide, each consisting of 8 weekly 10-μg doses. Three days prior to the first dose of tecemotide in each cycle, each mouse received a single 100-mg/kg dose of CPA as pretreatment. The results showed that pretreatment with CPA potentiated the tecemotide-induced T_H1 cytokine responses, and proinflammatory cytokines were increased with distinctive kinetics. Two cycles of tecemotide immunotherapy administered during tumor progression resulted in a significant reduction in the number of lung tumor foci, whereas single cycle treatment was ineffective.³⁴ Clinically, a primary cycle of tecemotide immunotherapy consisting of 8 weekly doses is given, followed by maintenance doses every 6 weeks until disease progression. In light of the findings in the preclinical lung cancer mouse model, the continued dosing of tecemotide appears to be necessary.

This urethane-induced human MUC1.Tg lung cancer mouse model has also been used in preliminary studies examining the effects of cisplatin chemotherapy, radiotherapy, and the combination of cisplatin and tecemotide immunotherapy. These studies were designed to lay the groundwork for future studies that will address the optimal timing of CRT and tecemotide immunotherapy. As detailed below, results of the Phase III START trial revealed some important differences in overall survival in patients who received concurrent CRT followed by tecemotide immunotherapy compared to those who received sequential CRT.¹² Preclinical studies utilizing the lung cancer mouse model developed by Wurz et al.³⁴ showed that the combination of 2 cycles of tecemotide immunotherapy concurrent with 4 cycles of cisplatin chemotherapy resulted in a significant and additive reduction in the number of lung tumor foci, with elevated IFN-γ levels and MUC1-specific immune responses. Moreover, neither cisplatin nor radiation interfered with the immune response to tecemotide. Although the translational relevance of this observed modest reduction in lung tumor foci is unclear, the results of these studies were important in that tecemotide did not interfere with the activity of cisplatin, a key first-line chemotherapeutic used in the treatment of NSCLC, and that the immune response to tecemotide was sustained during concurrent radiation treatment.³³ Kao et al. recently discussed the use of this lung tumor model in addressing the differences between concurrent and sequential CRT followed by tecemotide.³⁵

Bladder cancer

A human MUC1.Tg mouse model of invasive transitional cell bladder carcinoma was developed in immune intact C57BL/6 mice for the purpose of immunotherapy development.⁶³ In this model, bladder cancer is induced using the bladder carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN), and the bladder tumors that develop are positive for the expression of human MUC1. Although not specifically evaluated for antitumor effects in this model, tecemotide treatment resulted in a T_H1 polarized serum cytokine profile and a MUC1-specific T cell response as assessed by enzyme-linked immunosorbent spot (ELISpot) immunoassay.⁶³ As bladder cancer, particularly transitional cell carcinoma, is another epithelial cell cancer that expresses MUC1 at a high level,^64-66 this disease, where high rates of recurrence are seen despite the best treatments available, is thus a logical candidate for MUC1-targeted immunotherapies such as tecemotide.

Clinical Development of Tecemotide

The clinical development of tecemotide began in the late 1990s, and the results of the first Phase I study were published in 2001. As of July 2013, a total of 14 clinical trials of tecemotide against a variety of cancers, including NSCLC, prostate cancer, colorectal carcinoma, and breast carcinoma had been initiated or completed. Results of the published studies are discussed below, beginning with an early clinical trial of BP16-KLH in breast cancer patients. Research on BP16-KLH led to the development of liposome encapsulated MUC1 peptides, culminating in liposomal BLP25, now known as tecemotide. Completed clinical trials are summarized in Table 1, whereas Table 2 summarizes all ongoing clinical trials of tecemotide.

Table 1.

Completed Clinical Trials of Tecemotide

Study Identifier Indication	Description	Patients Enrolled	Reference
EMR 63325–002 Stage IIIB or IV NSCLC	Phase l randomized open-label safety and dose comparison study.	17	24
EMR 63325–003 Stage IIIB or IV NSCLC	Phase II open-label safety and immunogenicity study.	8	70
EMR 63325–004 Stage IIIB or IV NSCLC	Phase II open-label, dose-escalation study to determine safety and immunogenicity of tecemotide in combination with liposomal IL-2.	18	70
NCT00157209 EMR 63325–005 Stage IIIB or IV NSCLC	Phase IIB open-label, randomized study to test safety and efficacy of tecemtotide plus BSC compared to BSC alone.	171	22, 23
NCT00157196 EMR 63325–006 Unresectable stage III NSCLC	Phase II open-label study to assess safety of tecemotide made with a new formulation of the immunoadjuvant MPL®.	22	71
NCT00409188 EMR 63325–001 (START) Unresectable stage III NSCLC	A multicenter randomized double-blind placebo-controlled Phase III trial of tecemotide versus placebo in patients with unresectable stage III NSCLC.	1513	12
EMR 63325–007 Prostate cancer	Phase II open-label trial to test safety and efficacy of tecemotide in patients with rising PSA values following radical prostatectomy.	16	72
NCT01094548 EMR 63325–008 Multiple myeloma	Phase II open-label, dose-escalation study to determine safety and efficacy of L-BLP25 in multiple myeloma, either chemotherapy naïve, slowly progressive and asymptomatic or with stage II/III in stable response/plateau following anti-tumor therapy.	34	In preparation

Table 2.

Ongoing Clinical Trials of Tecemotide

Study Identifier Indication	Status	Description	Patients Planned	Reference
NCT00960115 EMR 63325–009 Unresectable stage III NSCLC	Closed to enrollment	Combined Phase I/II study in Japanese subjects with stage III unresectable NSCLC following primary chemotherapy.	205	69
NCT00828009 EMR 63325–600 (ECOG 6508) Unresectable stage III NSCLC	Open to enrollment*	Phase II study of tecemotide and bevacizumab in unresectable stage IIIA and IIIB NSCLC after definitive CRT.	55	79
NCT01015443 EMR 63325–012 (INSPIRE) Unresectable stage III NSCLC	Open to enrollment*	Phase III clinical trial of tecemotide in Asian subjects with stage III, unresectable, NSCLC who have demonstrated either stable disease or objective response following primary CRT.	420	25,36
NCT02049151 EMR 63325–021 (START2) Unresectable stage III NSCLC	Open to enrollment*	A multicenter randomized double-blind placebo-controlled Phase III trial of tecemotide vs. placebo in patients with completed concurrent CRT for unresectable stage III NSCLC.	1002	73
NCT01423760 EMR 63325–011 Tumor type as per feeder trial	Enrollment on invitation	Open label trial to collect long-term data on subjects who have received tecemotide in previous clinical trials.	262	—
NCT01507103 EMR 63325–013 (SPRINT) Rectal cancer	Closed to enrollment*	Multi-center, randomized, open-label, mechanistic trial of the biological effects of tecemotide in rectal cancer subjects undergoing neoadjuvant CRT.	124	76
NCT01496131 EMR 63325–015 Prostate cancer	Open to enrollment*	A randomized Phase II study of tecemotide in combination with standard ADT and RTX for newly diagnosed, high-risk prostate cancer.	48	78
NCT01462513 EMR 63325–602 (LICC) Colorectal cancer	Open to enrollment*	Multicenter, multinational, randomized (2:1), double-blind, placebo-controlled Phase II trial in colorectal carcinoma after curative resection of hepatic metastases.	159	74,75
EMR 63325–603 (ABCSG 34) Breast cancer	Open to enrollment*	Multicenter, randomized (1:1), open label, 2-arm trial in the pre-operative treatment of women with primary breast cancer.	400	77

*as of June 2014.

Early clinical development

Based on the preclinical results seen with BP1–7-KLH (also known as BP16-KLH),³⁷ which is an ASI with MUC1 used as a vaccine construct, a Phase I study was conducted in 16 metastatic breast cancer patients treated with the BP16-KLH vaccine plus the adjuvant DETOX-B. This vaccine, which was the precursor to tecemotide (BLP25) now in Phase III clinical trials, employed a synthetic 16-amino acid peptide derived from MUC1 conjugated to KLH as the immunogenic carrier protein. Patients received a total of 4 10-μg doses of BP16-KLH by subcutaneous injection. One week prior to the first and third doses, each patient received a single, low dose of CPA (300 mg/m²). The use of CPA is thought to enhance antitumor immunity through increasing DTH humoral and cellular immune responses and inhibiting T-suppressor function.⁶⁷ Only 3 of the 16 patients developed weak anti-MUC1 IgG responses, in contrast to strong IgG antibody responses against KLH in all of the patients. Using PBLs isolated from 11 patients, 7 developed HLA class I (HLA-A2, HLA-A1, and HLA-A11)-restricted CTL effectors capable of killing MUC1-expressing human breast cancer cell line targets after only a single in vitro stimulation with synthetic BP16 MUC1 peptide. No safety concerns were noted in this study.⁶⁸

During the course of early clinical studies with BP16-KLH, development of liposomal vaccine formulations based on a longer MUC1 peptide (BLP25), which contains epitopes with the capacity to bind to several HLA class I molecules including HLA-A11, HLA-A3, HLA-A2.1 and HLA-A1, was begun in order to provide stronger protection against tumor cell challenge and to increase cellular immune responses.⁵⁰ The development of BLP25 was built on liposomal BP24, which contains epitopes that bind to HLA class II molecules. The final vaccine product, tecemotide, is comprised of BLP25 lipopeptide encapsulated with MPLA and 3 different lipids: cholesterol, dimyristoyl phosphatidylglycerol (DMPG) and dipalmitoyl phosphatidylcholine (DPPC) in multilamellar liposomes. Tecemotide is designed to facilitate uptake by APCs such as dendritic cells so that the peptide is processed and presented via class I and class II HLA molecules, thereby eliciting a CTL-mediated MUC1-specific cellular immune response.

Phase I

The safety and immunogenicity of 2 dose levels of tecemotide (20 and 200 μg) were evaluated in 17 patients with unresectable stage IIIB or IV NSCLC in an uncontrolled Phase I clinical trial.²⁴ In this trial, the 12 patients who completed the treatment protocol received 4 doses of either 20 μg or 200 μg tecemotide administered in weeks 0, 2, 5, and 9, followed by maintenance vaccinations at 12-week intervals. Three days prior to initiating ASI with tecemotide, each patient received a single dose of CPA 300 mg/m² (maximum dose 600 mg). Although no antitumor responses were observed, median survival time was 5.4 months in patients receiving 20 μg compared to 14.6 months in patients treated with 200 μg tecemotide, a difference that was not statistically significant due to the small number of patients. As expected, tecemotide treatment induced primarily a cellular immune response. Among the 12 evaluable patients, 5 developed CTL responses against MUC1-positive tumor cell lines, and no significant humoral immune responses were noted in any of the patients. Another 5 patients out of the 12 evaluable were found to have preexisting MUC1-specific CTLs prior to tecemotide immunotherapy and were not included in the group of patients considered positive for CTL activity following tecemotide immunization. This trial demonstrated that tecemotide can be safely administered and is well tolerated in stage IIIB and stage IV NSCLC patients.²⁴

A second, non-randomized, open-label Phase I study, conducted as part of a Phase I/II study, assessed the safety of tecemotide in Japanese patients following primary CRT for stage III NSCLC.⁶⁹ Tecemotide at a dose of 1000 μg was administered by subcutaneous injection weekly for 8 weeks, with 1000-μg maintenance doses administered every 6 weeks thereafter until disease progression. Three days prior to the first dose of tecemotide, each patient received a single 300-mg/m² dose of CPA intravenously. Adverse events considered related to tecemotide treatment were arthralgia, myalgia, and nausea. No serious adverse events were observed.⁶⁹ This trial showed that tecemotide is well tolerated in Japanese NSCLC patients, with a safety profile consistent with previous clinical trials. The immunogenicity of tecemotide was not studied in this trial.

Phase II

Following completion of the Phase I safety trial, 2 early Phase II studies were conducted in previously treated stage IIIB and stage IV NSCLC patients to establish the tecemotide dose and treatment schedule.⁷⁰ In the first study, performed in 8 patients, a higher dose and more frequent immunization schedule (1000 μg weekly x 8 weeks) compared to the Phase I study were employed, in addition to maintenance immunizations every 6 weeks. Compared to baseline measurements, 6 of the 8 patients showed significant, MUC1-specific T-cell proliferative responses following tecemotide immunization. A MUC1-specific cytokine response was also detected in these 6 patients, as shown by intracellular tumor necrosis factor alpha (TNF-α). Median survival in this trial was 9.6 months.⁷⁰ In the other early Phase II trial, a total of 18 patients were administered the 1000-μg dose of tecemotide in combination with 2 different doses of liposomal IL-2, an immune modulator. A MUC1-specific T-cell proliferative response was seen in 2 of the 10 patients given the lower dose of IL-2, one of whom had TNF-α-expressing CD4+ T cells. Of the 8 patients who received the higher dose of IL-2 in combination with tecemotide, 6 showed MUC1-specific T-cell proliferative responses, and 4 of these 6 demonstrated TNF-α-expressing CD4+ T cells. In this latter early Phase II study, median survival was 8.7 months for those patients who received the lower dose of IL-2 and 17.8 months for patients who were given the higher dose of IL-2 in combination with tecemotide.⁷⁰

Based on the promising results of the 2 early Phase II studies, a multicenter, open-label, randomized Phase IIB trial was conducted in a total of 171 patients with stage IIIB or stage IV NSCLC.²³ Patients were randomized to receive either best supportive care (BSC) (83 patients) or tecemotide plus BSC (88 patients). As in previous clinical studies, patients randomized to the tecemotide arm received a single 300-mg/m² dose of CPA prior to the first dose of tecemotide. Tecemotide was administered by subcutaneous injection weekly for 8 weeks at a dose of 1000 μg spread over 4 anatomic sites, and then every 6 weeks thereafter as maintenance therapy. Overall median survival time was 4.4 months longer with tecemotide plus BSC (17.4 months) compared with BSC alone (13.0 months).²³ The greatest difference in median survival was seen in the subset of patients with stage IIIB locoregional (LR) disease, where median survival was 13.3 months with BSC alone and 30.6 months with tecemotide plus BSC.^22,23 Although this difference did not reach the level of statistical significance, it was definitely worthy of further study. Quality of life, as assessed by the Functional Assessment of Cancer Therapy-Lung (FACT-L) questionnaire and the Trial Outcome Index (TOI), was maintained longer in patients who received tecemotide compared to those who received only BSC.²³ Of the 88 patients who received tecemotide, T-cell immune responses were assessed in 78. MUC1-specific proliferative T-cell responses as a result of tecemotide immunotherapy were observed in 16 patients, only 2 of whom had stage IIIB LR disease. Among the tecemotide-treated patients with a positive T-cell proliferative response, median survival was 27.6 months compared to 16.7 months in those patients with a negative proliferative response.²³ In confirmation of the initial analysis, an updated survival analysis showed that the overall 3-year survival rate was 31% with tecemotide plus BSC compared to 17% with BSC alone. In patients with stage IIIB LR disease, 3-year survival was 49% with tecemotide and 27% with BSC alone, but this difference was not quite statistically significant.²² No significant safety issues were encountered in this study.²³

Another open-label Phase II trial in 22 patients with unresectable stage IIIA/IIIB NSCLC evaluated the safety of a new formulation of tecemotide.⁷¹ Patient survival was the secondary endpoint of this study. Tecemotide liposomes contain MPLA as the adjuvant, and while this was not changed in the new formulation, changes in the manufacturing process resulted in changes to the MPLA chain composition. The design of this study was as described for the Phase IIB study. Results of this trial showed that the new tecemotide formulation was well tolerated, had a safety profile similar to the original formulation,⁷¹ and the 2-year survival rate was comparable to that reported by Butts et al. for patients with LR stage IIIB NSCLC.²² The new tecemotide formulation was thus considered safe for use in Phase III clinical development.

A Phase II pilot study of tecemotide in prostate cancer was performed in 16 patients who had experienced prostate specific antigen (PSA) failure following radical prostatectomy.⁷² All patients received CPA pretreatment followed by weekly tecemotide (1000 μg) for 8 weeks, followed by tecemotide maintenance dosing every 6 weeks for up to one year. A total of 15 patients completed the primary treatment period and 10 completed the maintenance period. After 8 weeks treatment, 8 patients experienced stable or decreased PSA; however, by the end of the maintenance period, only one patient had stable PSA, with all others progressing. Interestingly, 6 of the 15 patients who completed the primary treatment experienced a more than 50% increase in their PSA doubling time compared to their baseline measurements. The most common adverse events were nausea, fatigue and injection site reactions. These results suggest that tecemotide may be useful in prolonging PSA doubling time, potentially delaying the start of hormone deprivation therapy in this patient population.⁷²

Phase III

The first Phase III clinical trial of tecemotide, designated START (stimulating targeted antigenic responses to NSCLC), was a randomized, placebo-controlled, double-blind trial conducted in 1,513 patients from 33 different countries.¹² The START trial, the results of which were recently published, assessed the safety, efficacy and tolerability of tecemotide in patients with unresectable, locally advanced stage IIIA/IIIB NSCLC who had not progressed after initial concurrent or sequential CRT. Patients were randomized 2:1 to treatment with tecemotide or placebo. Patients assigned to the tecemotide arm received CPA pretreatment (300 mg/m² to a maximum of 600 mg) followed by 806 μg tecemotide weekly for 8 weeks and then every 6 weeks thereafter until disease progression or withdrawal. A total of 829 tecemotide-treated and 410 placebo-treated patients were included in the efficacy analysis. In March 2010, clinical trials of tecemotide, including the START trial, were put on hold for enrollment and treatment after a case of encephalitis occurred in a Phase II trial of tecemotide for multiple myeloma. Subsequent investigations of this patient, an overall safety analysis of the use of tecemotide in NSCLC, and introduction of safety measures by protocol amendment led to the clinical hold being lifted in June 2010. As a result, the sample size of the START trial was adjusted and 274 excluded patients were replaced, so that 1,200 patients were needed to observe the anticipated number of events in the modified intention-to-treat (ITT) analyses for efficacy. The modified ITT population did not introduce bias to the statistical analysis. The US. FDA under Special Protocol Assistance and several European regulatory authorities approved the amendment and modification of the ITT population before the analysis. For a detailed consolidated standards of reporting trials (CONSORT) diagram and description of the population, as well as for details on the clinical hold impact, please refer to the respective recent publication.¹² The primary endpoint of overall survival prolongation in patients who received CPA and tecemotide was not significantly different compared to those who received saline and placebo (HR 0.88 [95% CI 0.76–1.03]; p = 0.126). However, a favorable effect of tecemotide was observed in a prospectively planned analysis of the predefined subgroup of patients receiving initial concurrent CRT (HR 0.78 [95% CI 0.64–0.95]; p = 0.016), with a 10.2-month improvement in median overall survival (from 20.6 months to 30.8 months), whereas no survival benefit of tecemotide was seen in the sequential CRT subgroup. The improved OS outcome in patients receiving initial concurrent CRT appeared to be consistent across respective patient subsets.¹²

In the START trial, the safety analysis set consisted of 1,024 patients who received tecemotide, 372 (36%) of whom received it for more than 52 weeks. These analyses confirmed the favorable safety and tolerability profile of tecemotide. There were no clinically concerning differences between tecemotide and placebo for any adverse event reported. Adverse events of special interest such as injection-site reactions or flu-like symptoms were reported to be infrequent and rarely greater than grade 2. Potential immune-related diseases or events were seen in less than 3% of patients and with similar frequency in the 2 groups.¹²

While the reason for the difference in survival outcome between concurrent and sequential CRT is unknown, it suggests that the timing of chemotherapy, radiotherapy, and tecemotide immunotherapy is important. The studies recently reported by Kao et al.³³ have laid the groundwork for future studies that will address the optimal timing of CRT and tecemotide immunotherapy as discussed by Kao et al.³⁵ and DeGregorio et al.³⁶ In order to verify the results of the START trial showing a significant increase in overall survival in patients receiving concurrent CRT followed by tecemotide, a new Phase III trial (START2) has been initiated to study tecemotide maintenance therapy after concurrent CRT.³⁶

Ongoing clinical trials

Phase III

The recently initiated pivotal phase III study START2⁷³ intends to confirm the results observed in the predefined subset of 806 patients treated with concurrent CRT who experienced improved overall survival (OS; adjusted HR 0.78, 95% CI 0.64–0.95; p = 0.016) in the START trial.¹² The START2 study is a global, randomized, double-blind, placebo-controlled Phase III trial investigating tecemotide in patients with unresectable stage III NSCLC who did not progress after completing first-line concurrent CRT 4–12 weeks before randomization. Concurrent CRT is defined as ≥2 cycles of platinum-based chemotherapy that overlaps with radiotherapy (total tumor dose ≥60 Gy, single fraction dose ≥1 .8 Gy); any other therapy for NSCLC constitutes an exclusion criterion. Patients will be stratified by response to CRT (stable disease or objective response) and region (North America and Australia; Western Europe; Rest of World), and randomized on a 1:1 basis to tecemotide (806 μg lipopeptide) or placebo. A single dose of CPA (300 mg/m²) or saline will be given i.v. 3 d prior to the first dose of tecemotide or placebo, respectively. Eight weekly subcutaneous injections will be administered initially, followed by maintenance injections every 6 weeks thereafter until disease progression or discontinuation. The primary objective is OS. Secondary objectives are time to symptom progression (Lung Cancer Symptom Scale), progression-free survival, time to progression, and safety. Approximately 1,002 patients will be enrolled. Sample size was calculated for a hazard ratio of 0.77 corresponding to an increase in median OS from 20 to 26 months in the placebo/tecemotide arm, respectively, a power of 90%, and one-sided significance level of 2.5%. The START2 trial will be conducted in more than 20 countries worldwide, excluding Asia. The study started recruiting in 2014.⁷³

While the START and START2 trials have focused on patients with a Caucasian background, parallel studies had been initiated to investigate tecemotide in patients with Asian genetic background. There are currently 2 ongoing clinical trials of tecemotide in Asian patients with unresectable stage III NSCLC, one Phase II study in Japan⁶⁹ and one Phase III study in China, Hong Kong, South Korea, Singapore, and Taiwan.²⁵ In both studies, the same treatment regimens used in the START trial are being employed. The tecemotide dose reported for earlier clinical trials was 1000 μg, but patients were found to have actually received about 930 μg or 806 μg based on an updated density determination of tecemotide prior to lyophilization and the respective applied syringe types for reconstitution. It was decided that the dose actually applied in the Phase III study START (806 μg) would be carried forward into the subsequent Phase III trial START2. However, the 930-μg dose was maintained unchanged for the Asian trial INSPIRE.²⁵

The multinational Phase III clinical trial INSPIRE (tecemotide trial in Asian NSCLC patients: stimulating immune response) is designed to assess the efficacy of tecemotide plus BSC, compared to placebo plus BSC, on overall survival time in patients of East Asian ethnicity with locally advanced, unresectable stage III NSCLC who had documented stable disease or an objective response following completion of primary CRT based on Response Evaluation Criteria in Solid Tumors (RECIST) criteria.²⁵ Secondary objectives include time to symptom progression, time to progression, progression-free survival time, time to treatment failure and safety. Patients will be randomized 2:1 to treatment with tecemotide plus BSC or placebo plus BSC. The results of the START trial have led to the modification of the INSPIRE trial to exclude sequential therapies and concentrate on concurrent therapy options.³⁶ The projected enrollment is 420 patients.²⁵

Phase II

Several Phase II studies are ongoing in colorectal cancer, rectal cancer, prostate cancer, breast cancer, and lung cancer. In a randomized, double-blind, placebo-controlled Phase II trial (L-BLP25 In Colorectal Cancer, or LICC), a total of 159 patients with stage IV colorectal adenocarcinoma following curative resection of hepatic metastases will be randomized 2:1 to receive treatment with tecemotide or placebo. Tecemotide is being administered at a dose of 930 μg weekly for 8 weeks, followed by maintenance dosing every 6 weeks thereafter for the first 2 y and every 12 weeks in the third year until recurrence. The primary endpoint is recurrence-free survival, and the secondary endpoints are overall survival, safety, and tolerability.^74,75

In rectal cancer, the multi-center, randomized, 3-arm, open-label, Phase II mechanistic trial, designated SPRINT (NCT01507103), is designed to investigate the immune response to tecemotide treatment in rectal cancer patients undergoing neoadjuvant CRT.⁷⁶ The immune response to tecemotide (primary endpoint) will be evaluated based on the local response in the tumor and the MUC1- and carcinoembryonic antigen (CEA)-specific response tested in blood. In this 3-arm study (1:1:1 randomization) 24 subjects per arm receive either tecemotide (806 μg weekly for 8 weeks) concomitant with standard CRT with or without a prior single dose of CPA (300 mg/m²), or standard CRT alone. This study has completed recruitment and results are expected in 2015.⁷⁶

In breast cancer, the randomized, 2-arm, controlled, open-label Phase-II study (Austrian Breast & Colorectal Cancer Study Group-34) is investigating tecemotide in the preoperative treatment of women with primary breast cancer.⁷⁷ A total of 400 patients will receive either standard chemotherapy or standard hormonal therapy, with or without tecemotide. The primary objective is assessment of histopathological response to preoperative standard of care treatment with or without tecemotide immunotherapy when measured by Residual Cancer Burden (RCB0/I versus RCBII/III) at the time of surgery. Secondary objectives include assessment of pathological complete remission (pCR; an absence of invasive cancer cells in surgical specimen) at the time of surgery, immunological parameters, quality of life and safety.⁷⁷

Triggered by encouraging Phase I data in prostate cancer, a randomized, controlled Phase II study of tecemotide in combination with standard androgen deprivation therapy (ADT) and RTX for newly diagnosed, high-risk prostate cancer patients was initiated at the US. National Cancer Institute (NCT01496131).⁷⁸ A total of 48 patients will receive RTX/ADT with or without combined tecemotide treatment (1:1 randomization). Primary outcome will be to assess immune parameters (change from baseline in the ELISpot level of MUC1-specific T cells at 2 and 6 months after radiation). Secondary outcomes include time to progression, further immune-related parameters and safety.⁷⁸

In lung cancer, the ongoing Phase II Japanese study mentioned earlier had included 168 patients with unresectable stage III NSCLC following primary CRT who were randomized 2:1 to treatment with either BSC alone or BSC plus tecemotide weekly for 8 weeks followed by maintenance dosing every 6 weeks thereafter until disease progression. More than 90% of patients had received concurrent CRT pretreatment. The primary endpoint of this study is overall survival. This event-driven study is in the follow-up phase and results are expected in 2015.⁶⁹

Finally, the ongoing open-label, Eastern Cooperative Oncology Group (ECOG) Phase II study (NCT00828009)⁷⁹ is investigating the combination of tecemotide and bevacizumab after CRT in a total of 55 patients with newly diagnosed stage IIIA or stage IIIB unresectable NSCLC. The primary endpoint of this interventional study is to determine the safety of this combination treatment. Secondary endpoints are overall survival and progression-free survival in patients treated with this combination regimen.⁷⁹

Conclusions

Tecemotide is a liposome-based, antigen-specific cancer immunotherapy designed to elicit a cellular immune response against MUC1, a TAA that is overexpressed and aberrantly glycosylated in epithelial cancers such as lung, breast and prostate cancer. Tecemotide has been in clinical development for over 13 years, and clinical trials have shown very promising results in regard to prolongation of overall survival and improvement in quality of life in patients with locally advanced, unresectable stage IIIA/IIIB NSCLC. While results of the first and largest Phase III clinical trial were disappointing in that overall survival was not significantly increased with tecemotide following primary CRT compared to placebo, a notable survival benefit was observed in the predefined subset of patients treated with concurrent CRT followed by tecemotide compared to those patients treated with sequential CRT. Safety analyses of tecemotide studies have revealed an overall favorable safety and tolerability profile. In the START study, as yet the largest controlled study of tecemotide, there were no clinically relevant differences between tecemotide and placebo observed with respect to safety and tolerability.¹² A new Phase III trial (START2) will address concurrent CRT regimens followed by tecemotide maintenance therapy, while an ongoing Phase III trial in Asian patients has been modified to focus on concurrent CRT options. A number of Phase II clinical trials are ongoing and may provide additional mechanistic insights as well as potential activity signals in indications other than lung cancer. Studies utilizing an immune-intact, MUC1 transgenic lung cancer mouse model are currently underway that seek to address the mechanisms underlying the differences between concurrent and sequential CRT so that the optimal timing of chemotherapy, radiotherapy and tecemotide immunotherapy may be defined to achieve the most beneficial treatment outcome for patients.

Disclosure of Potential Conflicts of Interest

GTW and CJK declare no conflicts. MW is an employee of Merck KGaA, and MWD is principal investigator of a research grant received from Merck KGaA.