Maria Liljefors

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

Abstract A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab2). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab3), were induced by the mAb17-1A therapy. Patients with detectable ab3 after treatment had significantly higher ab2 levels than those not developing ab3. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab2 as well as induction of anti-anti-idiotypic antibodies (ab3), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab3) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab3 (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy.

Augmentation of the immune response with granulocyte-macrophage colony-stimulating factor and other hematopoietic growth factors.

Granulocyte-macrophage colony-stimulating factor is by far the most widely used hematopoietic growth factor to augment immune responses. At present, the best secured effect is as an adjuvant cytokine for vaccination. Granulocyte-macrophage colony-stimulating factor can be delivered as gene-transduced tumor cells, as plasmid DNA, or as the soluble free granulocyte-macrophage colony-stimulating factor protein. Granulocyte-macrophage colony-stimulating factor must be present at the same site as the vaccine component. Granulocyte-macrophage colony-stimulating factor may also augment the effect of therapeutic monoclonal antibodies by enhancing various effector functions such as antibody-dependent cellular cytotoxicity and amplifying an idiotypic network response (i.e., antitumor immunity). It may also be advantageous to combine granulocyte colony-stimulating factor with monoclonal antibodies (neutrophil and monocyte antibody-dependent cellular cytotoxicity) for tumor therapy. However, these growth factors might also induce immune suppression, which may hamper the contemplated effect of the growth factor. It is urgently warranted to better understand these dual effects on the immune system so that we can find optimal uses for the growth factors in various clinical settings.

Natural killer (NK) cell function is a strong prognostic factor in colorectal carcinoma patients treated with the monoclonal antibody 17-1A.

Tumor cells might be susceptible to different effector functions of the immune system. This cytotoxic capacity has been utilized to analyze the prognostic significance of peripheral blood mononuclear cells (PBMC) in patients with metastatic colorectal carcinoma (CRC) treated with the monoclonal antibody (MAb)17‐1A. Such analysis might form the basis for future patient selection and may lead to improvements in therapeutic strategies. Between 1986 and 1998, 73 patients were treated with regimens containing MAb17‐1A. Prior to therapy, the lytic capability of PBMC was assayed against: K562 (4 hr assay), the CRC cell line SW948 (4 hr and 18 hr assays) and antibody‐dependent cellular cytotoxicity (ADCC, 18 hr assay). Since the study was performed over 13 years, the assays were checked for time‐related bias. Reproducibility over time was satisfactory. Patients exhibited a significantly higher cytotoxic capability in all 4 assays compared to healthy control donors. No correlation to clinical outcome was noted for 18 hr ADCC and 18 hr spontaneous cytotoxicity. Pretreatment natural killer (NK) cell cytotoxicity (K562) was significantly related to overall survival (OS), progression‐free survival (PFS), and response rate. OS for patients with high and low NK cell cytotoxicity was 71 vs. 30 weeks, respectively (p = 0.007). NK cell cytotoxicity (K562) was an independent prognostic factor for OS (p = 0.016). Pretreatment NK cell activity is a strong prognostic factor for patients with metastatic CRC receiving MAb17‐1A therapy and is a predictor for OS, PFS and response. These results should be considered when designing antibody‐based therapeutic protocols.

A Phase I safety study of plasmid DNA immunization targeting carcinoembryonic antigen in colorectal cancer patients.

A plasmid DNA vaccine, encoding a truncated form of human CEA fused to a T-helper epitope (CEA66 DNA) was delivered three times intradermally at 2 mg or intramuscularly at 8 mg by Biojector® to patients with colorectal cancer. Prior to the first vaccination, all patients received cyclophosphamide (300 mg/m²) intravenously. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously with each vaccination. All patients completed the vaccine schedule. There were no grade 3 or 4 adverse events (AE). The most frequently reported AE grades 1 and 2 were injection site reactions, fatigue, headache, arthralgia, chest tightness and myalgia. Vaccination with CEA66 DNA in combination with GM-CSF was well tolerated and no signs of autoimmunity have been detected.

Telomerase (GV1001) vaccination together with gemcitabine in advanced pancreatic cancer patients

Telomerase is expressed in 85-90 % of pancreatic adenocarcinomas and might be a target for active cancer immunotherapy. A study was conducted to investigate safety and immunogenicity in non-resectable pancreatic carcinoma patients using a 16-amino acid telomerase peptide (GV1001) for vaccination in combination with GM-CSF and gemcitabine as first line treatment. Three different vaccine treatment schedules were used; [A (n=6), B (n=6) and C (n=5)]. Groups A/B received GV1001, GM-CSF and gemcitabine concurrently. Group C received initially GV1001 and GM-CSF while gemcitabine was added at disease progression. Group D (n=4) was treated with gemcitabine alone. Adverse events (AE) related to vaccination were mild (grades I-II). Grade III AEs were few and transient. An induced GV 1001‑specific immune response was defined as an increase ≥2 above the baseline value in one of the assays (DTH, proliferation, ELISPOT and cytokine secretion assays, respectively). A telomerase‑specific immune response was noted in 4/6 patients in group A, 4/6 patients in group B and 2/5 patients in group C. An induced ras‑specific immune response (antigenic spreading) was seen in 5 of the 17 patients. The cytokine pattern was that of a Th1-like profile. A treatment induced telomerase or ras response was also noted in group D. All responses were weak and transient. A significant decrease in regulatory T-cells over time was noted in patients in groups A and B (p<0.05). Telomerase vaccination (GV1001) in combination with chemotherapy appeared to be safe but the immune responses were weak and transient. Measures have to be taken to optimize immune responses of GV1001 for it to be considered of clinical interest.

Clinical effects of monoclonal antibody 17-1A combined with granulocyte/macrophage-colony-stimulating factor and interleukin-2 for treatment of patients with advanced colorectal carcinoma

Abstract Granulocyte/macrophage-colony-stimulating factor (GM-CSF) has previously been indicated to enhance the therapeutic effect of the anti-colorectal carcinoma mAb17-1A as well as to augment in vivo immune effector functions. In vitro interleukin-2 (IL-2) augmented GM-CSF-induced antibody-dependent cellular cytotoxicity, a mechanism considered to be of significance for the therapeutic effect of mAb. A treatment regimen was elaborated that combined mAb17-1A (400 mg at day 3 of a 10-day treatment cycle) with the simultaneous administration of GM-CSF (250 μmg/m2 once daily) and IL-2 (2.4 × 106 U/m2 twice daily) for 10 days. The treatment cycle was repeated once a month. Twenty patients with advanced colorectal carcinoma were included in the study. One patient obtained a partial remission and 2 patients stable disease for 7 and 4 months respectively. The median survival time from the start of mAb therapy was 8 months. Owing to allergic reactions, the planned mAb17-1A dose had to be reduced by repeated infusions. At the fourth treatment cycle only 25% received the planned mAb dose. In 3 patients the GM-CSF and IL-2 dose was reduced because of side-effects. The subjective tolerability of the treatment was considered good or acceptable in more than 80% of the patients. The increment in white blood cell subsets induced by the cytokines decreased by increasing number of courses. This particular regimen did not augment the therapeutic effect of mAb17-1A anticipated from in vitro data but rather hampered the clinical effect of the antibody. The reason for this is not clear but a possibility might be the induction of immune suppression in vivo resulting from an impaired human anti-(mouse Ab) and anti-idiotypic antibody response as well as antibody-dependent cellular cytotoxicity, on the basis of a comparison of mAb17-1A/GM-CSF/IL-2- and mAb17-1A/GM-CSF-treated patients.

MAb17-1A and cytokines for the treatment of patients with colorectal carcinoma.

CO17-1A/GA73-3/EpCam/KSA is a cellular adhesion molecule expressed on the majority of tumor cells in most patients with colorectal carcinoma. One of the first mouse monoclonal antibodies (MAbs) for therapeutic use was produced against this particular tumor associated antigen (MAb17-1A). MAb17-1A has served as a model for the development of antibody therapy. It exerts therapeutic effects through antibody dependent cellular cytotoxicity (ADCC), induction of an idiotypic network cascade and maybe also by complement activation. Addition of cytokines that augment these functions, mainly granulocyte macrophage-cerebrospinal fluid (GM-CSF), seemed to improve the clinical efficacy as well as chemotherapeutic agents (5-Fu). In advanced disease the clinical effect is, however, modest while the most beneficial clinical situation seems to be the adjuvant setting. Twenty years have passed since the EpCam antigen was identified as a target structure for immunotherapy, but still we do not know how to optimally use this target. The antigen might, however, be a rewarding structure to utilize for therapy. Preclinical and clinical trials are ongoing aimed at improving passive and active immunotherapy using CO17-1A/EpCam as a target antigen in colorectal carcinoma.

A Phase I Dose-Escalation Study of Lenalidomide in Combination with Gemcitabine in Patients with Advanced Pancreatic Cancer

Purpose Lenalidomide have both immunomodulatory and anti-angiogenic properties which could confer anti-cancer effects. The aim of this study was to assess the feasibility of combining lenalidomide with the standard treatment gemcitabine in pancreatic cancer patients with advanced disease. Patients and Methods Eligible patients had locally advanced or metastatic adenocarcinoma of the pancreas. Patients received lenalidomide days 1–21 orally and gemcitabine 1000 mg/m2 intravenously (days 1, 8 and 15), each 28 day cycle. Three cohorts of lenalidomide were examined (Cohort I = 15 mg, Cohort II = 20 mg and Cohort III = 25 mg daily). The maximum tolerated dose (MTD) of lenalidomide given in combination with gemcitabine was defined as the highest dose level at which no more than one out of four (25%) subjects experiences a dose-limiting toxicity (DLT). Patients should also be able to receive daily low molecular weight heparin (LMWH) (e.g. dalteparin 5000 IU s.c. daily) as a prophylactic anticoagulant for venous thromboembolic events (VTEs). Twelve patients (n = 4, n = 3 and n = 5 in cohort I, II and III, respectively) were enrolled in this study. Results Median duration of treatment was 11 weeks (range 1–66), and median number of treatment cycles were three (range 1–14). The only DLT was a cardiac failure grade 3 in cohort III. Frequent treatment-related adverse events (AEs) (all grades) included neutropenia, leucopenia and fatigue (83% each, but there was no febrile neutropenia); thrombocytopenia (75%); dermatological toxicity (75%); diarrhea and nausea (42% each); and neuropathy (42%). Discussion This phase I study demonstrates the feasibility of the combination of lenalidomide and gemcitabine as first-line treatment in patients with advanced pancreatic cancer. The tolerability profile demonstrated in the dose escalation schedule of lenalidomide suggests the dosing of lenalidomide to be 25 mg daily on days 1–21 with standard dosing of gemcitabine and merits further evaluation in a phase II trial. Trial Registration ClinicalTrials.gov NCT01547260

DNA immunization targeting carcinoembryonic antigen in colorectal cancer patients

Active specific immunotherapy targeting carcinoembryonic antigen (CEA) may induce antigen-specific humoral and cellular responses in cancer patients. Plasmid DNA, encoding tumor antigens, represents a novel approach of delivering conformational antigens. Here we report immune data of an explorative study using CEA66-DNA (non-glycosylated cytoplasmic CEA) and tetwtCEA-DNA (wild type glycosylated and secreted CEA) for immunization in combination with cyclophosphamide and GM-CSF in the adjuvant setting of radically operated colorectal cancer (CRC). 10 patients received intradermal (id) or intramuscular (im) CEA66-DNA delivered by needle-free Biojector at weeks 0, 2, 6 (part 1). 10 patients received tetwtCEADNA id by needle injection and electroporation at weeks 0 and 12 (part 2). In part 3, 6 patients were primed with CEA66-DNA and boosted with tetwtCEA-DNA. A significant increase of CD4+ effector memory, CD8+ effector and CD8+ effector memory T cells was seen in part 1. An immune response against CEA atleast one time point was noted in 15/20 (75%) patients in parts 1 and 2 together. The frequency of patients mounting a CEA-specific cellular immune responses was significantly higher in part 1 (100%) than in part 2 (50%) (p=0.03). In part 3, 5/6 (83%) patients showed a CEA-specific immune response after a prime-boost protocol. The higher CEA-specific T cell responses seen in part 1, may indicate reduced immunological tolerance induced by the non-glycosylated intracellularly produced CEA66-DNA immunogen. Humoral responses determined by ELISA were low. Further studies are warranted to optimize vaccination schedules to induce both cellular and humoral anti-CEA responses of clinical significance. Clinical trials.gov.identification number (parts 2/3) is NCT01064375. Abbrevations CEA: Carcinoembryonic Antigen; CRC: Colorectal Carcinoma; DFS: Disease free survival; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunospot; EP: Electroporation; GM-CSF: Granulocyte-macrophage colony-stimulating factor; HRP: Horseradish peroxidase; id: intradermal; IFN-γ: interferon γ; IL-4: Interleukin-4; IL-10: Interleukin-10; im: Intramuscular; OS: Overall survival; PBMC: Peripheral blood mononuclear cells; PBS: Phosphate buffered saline; PHA: Phytohemagglutinin; PPD: Purified protein derivative of tuberculin; sc: Subcutaneous; SI: Stimulation index; SIIR: Sustained induced immune response; STIIR: Single time point induced immune response; TAA: Tumor associated antigens; TCV: Therapeutic cancer vaccines; TNF-α: Tumor necrosis factor-α Introduction Colorectal carcinoma (CRC) is a major cause of cancer-related mortality. Despite introduction of new drugs, a large proportion of patients remain incurable. Adjuvant chemotherapy is standard for the treatment of stage III colon cancer and increases the 5-year survival rate to more than 70% and is routinely also used in stage II colon cancer patients with high-risk of relapse. Several chemotherapeutic agents approved for metastatic CRC have however failed to improve the prognosis for patients with stages II and III CRC [1]. New therapeutic approaches are needed and immunotherapy may offer a novel targeted therapeutic option [1]. The goal of therapeutic cancer vaccines (TCV) is to induce a robust long-lasting immune response with limited toxicity [2]. Most tumor cells express tumor-associated antigens (TAA), which might act as targets for the immune system [3]. A commonly expressed TAA in gastrointestinal cancer is the carcinoembryonic antigen (CEA) which has been explored in immunotherapy trials [4-11]. Vaccination targeting CEA in humans was shown to induce antigen-specific humoral, CD4+ helper as well as CD8+ cytotoxic T-cell (CTL) responses [8,11-14]. Immunisation with proteins comprising multiple CD4+ and CD8+ T cell epitopes, have failed to induce a robust CD8+ response which may partly be explained by defect peptide processing of exogenous proteins not entering the MHC class I antigen presentation pathway. DNA contains immunostimulatory CpG sequences that stimulate cytosolic DNA-sensing pathways, interferon regulatory factors and Fas-FasL Correspondence to: Håkan Mellstedt, MD, PhD, Prof, Department of Oncology and Pathology (Radiumhemmet), Cancer Centre Karolinska, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden, Tel: +46851774641; E-mail: hakan.mellstedt@karolinska.se