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Purpose: Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed. Patients and Methods: We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival. Results: Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival. Conclusions: This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients. Significance: The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.

Figure S4. Kaplan–Meier analysis showing PFS of a) CCL13 and b) IL-21. c) Flow cytometry plots showing CCR7 and CD45RA gating among CD3 positive cells in blood samples d) Flow cytometry based comparison between short (n=18) and long (n=6) term survivors with CD3+CCR7+ % (T naive, T stem cell memory and T central memory) analysis using pretreatment blood samples and e) Kaplan–Meier analysis showing Overall survival with median CD3+CCR7+ % (n=24).

Kaplan–Meier analysis showing. PFS (A) and OS (B), respectively using pretreatment plasma CCL21 (pg/mL) measurements based on their median values. C, Correlation between CCL21 (pg/mL) and CD3+CCR7+CD45RA+ (% counts) matched patient samples (n = 23). D, Flow cytometry–based comparison between short- and long-term survivors for CD3+CCR7+CD45RA+ % (T naïve and T stem cell memory) analysis using pretreatment blood samples (n = 24). Statistical test was unpaired two-tailed t tests, assuming equal variance. E, Kaplan–Meier analysis showing OS using median CD3+CCR7+CD45RA+ % values.

IHC magenta showing low mag of CD20 varied staining of TLS-like (borderline-tertiary lymphoid structures) followed by high magnification images of CD20, CD3, CCL21 within serial sections for Pt 2-027 (A), 3-010 (B). Also see Supplementary Fig. S5. Low and high magnification images of CD20+ TLS-like staining for Pt. 2-026 (C) and no TLS sample Pt. 4-017 (D). Kaplan–Meier analysis showing PFS (E) and OS (F), respectively dividing patient population into two groups based on IHC, no-TLS (n = 4) and TLS-like (n = 18). Also see Supplementary Fig. S5. G, Heatmap showing top 10% genes with highest log2 fold change, among all positively and negatively significantly regulated genes comparing no-TLS and TLS-like groups using bulk RNA-seq (log2 Reads Per Kilobase per Million mapped reads [RPKM] normalized values). Arrows represent relevant TLS-based gene signatures. Genes with FDR-adjusted P values H, Individual box plots showing CCL19, CCL21, CCR7, and CD79A changes in expression between the groups. Statistical tests were carried out using DESeq2 and FDR-adjusted P values were denoted with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

A, Kaplan–Meier analysis showing PFS of all patients. B, Kaplan–Meier analysis showing OS of all patients except one. C, Swimmer plot showing time on treatment, time to best response, and duration of response in all patients who received at least one dose of study drug (n = 29) are shown. D, Kaplan–Meier analysis comparing OS between patients with LDH baseline greater or lower than the upper limit of normal (ULN).

A and B, Kaplan–Meier analysis showing PFS and OS of patients with a wildtype BAP1 status and UV-damaged uveal melanoma genome. PFS (C) and OS (D) analyses comparing patients with GNAQ- or GNA11-mutated uveal melanoma. E, Volcano plot showing differentially expressed genes between short term (n = 16) and long term (alive patients, n = 4) from bulk RNA-seq. Genes with FDR-adjusted P values F, Individual box plots showing relevant gene signatures implicating long-term (alive patients) survival. Statistical tests were carried out using DESeq2 and FDR-adjusted P values were denoted with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Baseline characteristics, patient population

A, Heatmap showing TK1 activity value (DuA) from PEMDAC patient plasma samples, grouped within response groups. Each square is a timepoint for each patient and shows TK1 levels from pretreatment to end of study, until otherwise stated. Total plasma samples analyzed for TK1 = 287. B, Longitudinal TK1 activity for individual patients, as shown in A. C and D, Kaplan–Meier analysis showing PFS and OS, respectively for pretreatment TK1 values using a threshold of 150 DuA (median TK1 for all samples = 113). Patients with nonavailability of pretreatment samples were excluded from the analysis. E, IHC of TK1 showing nuclear/cytoplasmic magenta staining in patient biopsies 2-027, 3-012, and 3-010. F, Comparison between pretreatment TK1 values for short- and long-term survivors. G, Correlation between pretreatment TK1 (DuA) and circulating tumor DNA (counts/mL) matched patient samples (n = 21). All statistical tests were unpaired two-tailed t tests, assuming equal variance, with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Supplementary Figure S2. Oncolysis over time The human PaCa3 cell line was infected with 100MOI LOAd(-), LOAd700 or LOAd703 virus, or left uninfected. The cells were plated in a 96-well plate in quadruplicates and analyzed by MTS assay at three time points. The plates were analyzed by spectrophotometry at 490nm as per manufacturer instructions. Uninfected cells remained viable while infected cells were killed over time independently of type of LOAd virus demonstrating that the viruses have oncolytic capacity and that the transgene cassette does not interfere with oncolysis.

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma.Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses.Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells.Conclusions: LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. Clin Cancer Res; 23(19); 5846–57. ©2017 AACR.

Ovarian cancer is commonly treated with debulking surgery, followed by a combination of a taxane and a platinum-based chemotherapy. However, >70% of patients relapse within 3 years with tumors resistant to platinum-based agents. 5-year survival is ranging from 30-40%. Thus, delaying recurrence of disease is a high unmet need in ovarian cancer. Combining the chemotherapy regime with immunostimulatory gene therapy utilizing oncolytic viruses as gene vehicles is an attractive treatment option targeting alternative pathways to induce tumor cell death, control tumor growth and prevent development of metastasis. LOAd703 is an oncolytic adenovirus derived from the LOAd platform of replication-restricted Ad5/35 adenoviruses. LOAd703 encodes for the human immunostimulatory genes trimerized, membrane-bound CD40L (TMZ-CD40L) and 4-1BBL, expressed under a CMV promotor which enables gene expression in both tumor cells and tumor stroma while replication is restricted to tumor cells. Herein, we investigated the effect of combining LOAd703 with paclitaxel and cisplatin in vitro and in vivo. Four human ovarian cancer cell lines (CAOV3, COV362, OVCAR3 and PEA2) were treated with LOAd703 alone, in combination with paclitaxel and/or cisplatin or were left untreated. LOAd703 efficiently killed the ovarian cancer cell lines by oncolysis, as evaluated by an MTS cell viability assay. This effect was further improved when LOAd703 was given in combination with paclitaxel and/or cisplatin. Flow cytometry revealed that the ability of LOAd703 to induce expression of the immunostimulatory transgenes TMZ-CD40L and 4-1BBL, was not negatively affected by the combination with paclitaxel and/or cisplatin. The combination treatment was further tested in an in vivo model where BALB/c nu/nu mice were xenografted with 5 million OVCAR3 cells. The mice were subsequently treated with LOAd703 (1 × 10e9 FFU/mouse), both alone as well as in combination with paclitaxel (6mg/kg) and cisplatin (2.5mg/mg). The treatments were given over the course of 4 weeks where LOAd703 was administered intratumorally twice per week while chemotherapy was given intraperitoneally once per week. The results showed that the combination of LOAd703 and the chemotherapeutics controlled tumor growth significantly better than either LOAd703 alone or the standard treatment with paclitaxel and cisplatin. In conclusion, LOAd703 efficiently killed ovarian cancer cell lines in vitro, and could potentiate the effect of standard-of-care chemotherapy. This effect was confirmed in vivo, in which the combination therapy had a better tumor control than the other treatment groups. A clinical trial is ongoing to confirm the effect of adding LOAd703 to different treatment regimes in ovarian cancer (NCT03225989). Citation Format: Sedigheh Naseri, Jonas Härdin, Emma Eriksson, Tanja Lövgren, Gustav Ullenhag, Angelica Loskog. Immunostimulatory gene therapy with oncolytic viruses potentiates the effect of paclitaxel and cisplatin in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 686.

In the LOKON002 phase I/II clinical trial (NCT03225989), therapy with LOAd703 (delolimogene mupadenorepvec) is investigated in combination with gemcitabine-based chemotherapy in patients with advanced cancer. LOAd703 is an immunostimulatory gene therapy utilizing an oncolytic adenovirus that is engineered to express two transgenes (trimerized membrane-bound CD40L (TMZ-CD40L) and 4-1BBL), which are key players in the induction of an anti-tumor immune response. Herein, we present the preliminary immunological evaluation of the first 27 patients enrolled in the trial. The primary tumor was pancreatic cancer (n=16), colorectal cancer (n=5), ovarian cancer (n=3) or biliary cancer (n=3). Patients received a maximum of eight intratumoral LOAd703 treatments, given biweekly, and chemotherapy was administered at the same time according to standard protocol. Matched tumor biopsies from the injected tumor lesion were taken at baseline and after six LOAd703 treatments, and analyzed for gene expression with nCounter® PanCancer Immune Profiling Panel from NanoString (n=12; 7 patients analyzed so far). Blood samples were collected for isolation of serum samples and peripheral blood mononuclear cells, and these were analyzed with multiplex assays (Meso Scale Diagnostics) (n=15) and flow cytometry (n=27), respectively. Preliminary results indicate that after treatment, most tumors displayed a strong inflammatory profile with an upregulation of gene profiles, which have been shown to predict responses to immune checkpoint inhibitor therapy (T cell inflamed profile (Ayers et al.) & T effector and IFNγ associated profile (Fehrenbacher et al.)). We also noted an upregulation of genes connected to dendritic cell activation and antigen presentation (MHC class I and II, CD80, CD86, TAP1/2), as well as adenoviral-response genes. Moreover, we observed systemic treatment effects after three LOAd703 treatments, as shown by an increase in serum levels of pro-inflammatory cytokines and chemokines (IFNγ, IL-15, CXCL10, CCL2, IL-8, IL-6), as well as an increased percentage of CD8+ effector memory T cells (CD45RA-CCR7-) and PD-1+ CD8+ T cells in the blood. The fractions of natural killer (NK) cells (CD14-CD3-CD56+CD16+) and M2-like macrophages (CD11b+CD163+) were reduced in the blood at that time point. In conclusion, LOAd703 therapy in combination with chemotherapy generated an inflamed tumor microenvironment in tumors that are normally seen as immunologically “cold”. Hence, LOAd703 may be able to prime tumors for immune checkpoint inhibitors or other immunotherapies, such as adoptive T or NK cell transfer. Citation Format: Jessica Wenthe, Emma Eriksson, Sedigheh Naseri, Linda Sandin, Amanda Hahn, Sandra Irenaeus, Anders Sundin, Justyna Leja-Jarblad, Gustav Ullenhag, Tanja Lövgren, Angelica Loskog. Immunostimulatory gene therapy targeting CD40/4-1BB in combination with chemotherapy induces an inflammatory gene profile in tumors from patients with advanced disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6663.

In the past decade, immunotherapy with checkpoint inhibitors has revolutionized the field of oncology. Checkpoint inhibitors have been approved for several types of cancer and thousands of patients in Sweden now receive oncological immunotherapy annually. Immune-related side effects are common and can occur in almost any organ. These side effects are different from those that occur with traditional oncological treatments. The side effects are usually mild, but can be serious and even lethal. In a short time, health care providers have had to readjust to be able to handle these side effects. Early and correct diagnosis of immune-related side effects, proper management and a multidisciplinary approach is crucial. Here, we give an overview of the presentation, diagnosis and treatment of immune-related side effects, with emphasis on endocrine, rheumatologic and skin toxicity.

In the past decade, immunotherapy with checkpoint inhibitors has revolutionized the field of oncology. Checkpoint inhibitors have been approved for several types of cancer and thousands of patients in Sweden now receive oncological immunotherapy annually. Immune-related side effects are common and can occur in almost any organ. These side effects are different from those that occur with traditional oncological treatments. The side effects are usually mild, but can be serious and even lethal. In a short time, health care providers have had to readjust to be able to handle these side effects. Early and correct diagnosis of immune-related side effects, proper management and a multidisciplinary approach is crucial. Here, we give an overview of the presentation, diagnosis and treatment of immune-related side effects, with emphasis of those occurring in gastrointestinal organs, lungs and kidneys.

Low tumor immunogenicity and a cold tumor microenvironment are two main characteristics occurring in different cancer types responsible for immunotherapy resistance. Immunostimulatory gene therapy using oncolytic viruses (OVs) has been developed as a new promising cancer therapy for its capacity to induce an anti-tumor immune response and sensitize the non-responding patients to checkpoint blockade therapy. Lokon adenoviruses (LOAds) are serotype 5/35 oncolytic adenoviruses that in addition to their oncolytic capacity encode for immunostimulatory molecules. Here we have evaluated the anti-tumor effects of trimerized membrane-bound (TMZ)-CD40L-armed LOAd700 and TMZ-CD40L/4-1BBL-armed LOAd703 in a variety of solid tumors to understand the underlying tumor modulating mechanisms. A panel of eight different human solid cancer cell lines (T24, PEA2, Mel526, H727, Skov3, MiaPaca2, H29 and A549) was infected with LOAd(-), a virus without transgenes, LOAd700 or LOAd703 and assessed for their response in different assays. MTS assay was used to measure the cell viability, flow cytometry for evaluating the cell surface markers and luminescent assay for measuring apoptosis (caspase 3/7 activity). LOAd viruses efficiently killed all tested cell lines. However, the oncolytic capacity of CD40L-armed LOAd700 and LOAd703 was significantly higher than LOAd(-) in two cell lines, T24 and PEA2, which were found to express the CD40 receptor. Death receptor TRAIL-R1 was significantly upregulated in T24 infected with LOAd703 and PEA2 infected with either LOAd700 or LOAd703. TRAIL-R2 was upregulated post LOAd infection in 7/8 cell lines where the upregulation was significant in T24, PEA2 and H29. Interestingly, the immunogenic cell death marker calreticulin, a damage-associated molecular pattern (DAMP) known as an “eat-me” signal to dendritic cells, was upregulated post LOAd infection in all cell lines. The increase in calreticulin level was statistically significant in 4/8 cell lines (T24, PEA2, Mel526 and H29) post LOAd700 and/or LOAd703 infection. Furthermore, LOAd700 and LOAd703 increased the effector caspase 3/7 activity in 5/8 cell lines (T24, PEA2, Mel526, Skov3 and MiaPaCa2) indicating an augmented apoptosis level. In conclusion, CD40L-armed LOAd viruses were, in addition to their high oncolytic capacity, demonstrated to induce apoptosis. Upregulation of cell surface calreticulin as well as the death receptors TRAIL-R1 and -R2 indicated generation of a more immunogenic phenotype of cancer cells. Thus, our findings suggest that CD40L-armed LOAd viruses could be inducers of apoptosis and immunogenic cell death in a variety of cancer types with an augmented effect in cancer cells expressing the CD40 receptor. Citation Format: Sedigheh Naseri, Mariela Mejia Cordova, Jessica Wenthe, Ann-Charlotte Hellström, Tanja Lövgren, Angelica Loskog, Emma Eriksson, Gustav Ullenhag. Induction of tumor cell apoptosis & immunogenic cell death by CD40L-armed oncolytic adenoviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3501.

Background ATOR-1015 is a human CTLA-4 x OX40 targeting IgG1 bispecific antibody developed as a first in class tumor-localizing CTLA-4 antibody for improved efficacy and reduced toxicity. Methods The study (NCT03782467) is a first-in-human dose escalation study followed by an expansion part. In the dose escalation patients with refractory solid malignancies are enrolled and the expansion part will enroll patients with cutaneous or mucosal malignant melanoma. Patients receive ATOR-1015 intravenously Q2W as a single agent until confirmed progressive disease, unacceptable toxicity or withdrawal of consent. Intra-patient dose escalation is allowed. The primary objective is to assess the safety and tolerability of ATOR-1015. Secondary objectives include pharmacokinetics, immunogenicity, pharmacodynamics, and clinical efficacy as assessed by iRECIST. Pharmacodynamic analyses include serum cytokines, immunophenotyping of peripheral blood mononuclear cells. Tumor biopsies before and after ATOR-1015 will be analyzed. Results As of June 26, 2020, 23 patients have been exposed to ATOR-1015. The median age of the patients is 54 years (range 40–72). Patients have received a median of 5 prior lines of therapy (range 1–16). Most common cancer type is colorectal cancer.Dose levels from 0.043 mg to 600 mg have been evaluated and declared safe. Dose escalation is ongoing, and currently two patients have been enrolled at 750 mg dose level. The median time on study was 8.4 weeks (range 0.1–34.3). Five patients are on study and 18 patients have discontinued. Reasons for discontinuation included clinical deterioration (n=10), disease progression (n=5), death due to disease progression (n=2), and investigator’s decision (n=1). Twelve of the 23 patients experienced a drug-related adverse event (AE). Two patients experienced a grade 3 drug-related AE, for all other patients AEs were grade 1 or 2. Infusion-related reactions (IRR) were reported in nine patients. Predominant symptoms of the IRR were chills, rash and pain. Potentially immune-related AEs grade 1 were reported in three patients: one patient had rash, one vitiligo, and one exanthema and eczema. No dose-limiting toxicities have occurred. Best response is stable disease. Pharmacokinetic data show dose proportional kinetics up to 600 mg. Preliminary biomarker analysis shows pharmacodynamic activity of ATOR-1015. Conclusions ATOR-1015 has been safe and well-tolerated up to 600 mg. Currently 750 mg is under evaluation. Best response is stable disease. Following the dose escalation phase, an expansion cohort for patients with advanced malignant melanoma will be initiated. Ethics Approval The study is approved by the Ethic Boards in Sweden and Denmark.

Great advances in the systemic treatment in malignant melanoma patients Up until a few years ago there was no systemic treatment with the capacity to prolong overall survival for patients with advanced malignant melanoma. Several new treatments have been established in clinical practice in recent years and they belong to two main groups; checkpoint inhibitors and inhibitors of an intracellular pathway (MAP kinase). The latter drugs are only active in melanomas with an activating BRAF mutation, which is present in half of the patients. Side effect management is important and in particular for the checkpoint inhibitors, which activate the immune system and frequently provoke immune related reactions. The introduction of these new treatments has substantially improved prognosis and some patients might even be cured. Based on ongoing research, more therapeutic breakthroughs can be anticipated in the years to come.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

In order to obtain a sustained cytotoxic T lymphocyte (CTL) response against cancer cells it is preferable to have CTLs directed against multiple peptide epitopes from numerous tumor-associated antigens.We used a Flow Cytometry-based interferon (IFN)-gamma secretion assay with peptide-pulsed C1R-A2 as antigen-presenting cells to analyze whether CD8+ T cells directed against any of 24 HLA-A*0201-binding peptides from 15 prostate-associated proteins can be found in the peripheral blood of patients with localized prostate cancer. We also investigated whether multiple prostate antigen-specific CD8+ T cells can be generated simultaneously, from a naïve T cell repertoire. In that case, dendritic cells (DCs) from peripheral blood of healthy donors were divided in six portions and separately pulsed with six peptides. The peptide-pulsed DCs were then pooled and used to stimulate autologous T cells. The T cells were re-stimulated with peptide-pulsed monocytes.We found prostate antigen-restricted CD8+ T cells in the peripheral blood in 48 out of 184 (26.1%) analyzed samples from 25 cancer patients. This is significantly higher than 17 out of 214 analyzed samples (7.9%) from 10 healthy age-matched male individuals (P = 0.0249). In the cases when antigen-specific T cells could not be detected, we were able to generate IFN-gamma-producing CD8+ T cells specific for up to three prostate antigens simultaneously from a naïve T cell repertoire.CD8+ T cells directed against prostate antigen peptides can be found in, or generated from, peripheral blood. This indicates that such T cells could be expanded ex vivo for adoptive transfer to prostate cancer patients.

Malignant melanoma is a disease capable of rapid progression and rapidly developing metastases. Angiogenesis is a key event signalling tumour progression and elevated levels of angiogenic markers may indicate metastatic disease. No previously published work has, so far, examined plasma vascular endothelial growth factor (VEGF) and its receptor, VEGFR-1, in melanoma. This study investigated circulating levels of the angiogenic factors, VEGF-A and -D, their receptors 1-3 and hepatocyte growth factor (HGF)/scatter factor, in patients shortly after primary surgery for localized malignant melanoma. Elevated circulating levels of VEGF and its receptors, and of HGF, were found postoperatively, possibly derived from the reactive stroma adjacent to the tumours. Using univariate analysis, a correlation between levels of VEGFR-1 and relapse was found, but a correlation between the investigated angiogenic factors and survival could not be established. The results of the present study indicate that production of these angiogenic factors may be due to sources other than malignant melanoma cells.

Monoclonal antibodies (MAbs) have different modes of action and toxicity profile compared to chemotherapeutics, which makes it interesting to combine these drugs. Addition of cytokines to MAb therapy may also augment immune effector functions utilized by MAb. In an effort to improve the therapeutic effect of a MAb-based regimen in colorectal carcinoma (CRC) patients, the effects of a combination of alpha-interferon (alpha-IFN), 5-fluorouracil (5-FU), granulocyte-macrophage colony-stimulating factor (GM-CSF) and mouse MAb17-1A was evaluated in 27 patients with metastatic disease. alpha-IFN was given s.c. once daily for 5 consecutive days and at days 4 and 5, 5-FU was administered as a daily i.v. bolus injection. After 2 days rest, GM-CSF was given s.c. once daily, days 8-14 and on day 10, MAb17-1A was given i.v. The treatment cycle was repeated every 4th week. One patient achieved a partial remission and 13 patients showed a minor response or stable disease3 months, inducing an overall response rate of 54%. Responding patients survived significantly longer than non-responding patients (p=0.021). Median overall survival time for all patients was 75 weeks and progression-free survival time 15 weeks. Adverse events related to alpha-IFN, GM-CSF and 5-FU were as expected. The frequency of patients with an immediate-type allergic reaction (ITAR) against MAb17-1A at the 1st, 2nd, 3rd and 4th treatment cycles was 11%, 52%, 62% and 64% respectively. The planned MAb17-1A dose had to be reduced by repeated infusions. No patient received full dose of MAb17-1A from the 3rd cycle and onward. Compared to historical control patients treated with MAb17-1A alone, the present combination regimen seemed to improve the response rate (54% vs 15%) as well as progression-free survival (15 vs 7 weeks; p0.05).