Adjuvant Granulocyte-macrophage Colony-stimulating Factor Research Articles

Efficacy of a gB + gD-based subunit vaccine and the adjuvant granulocyte-macrophage colony stimulating factor for pseudorabies virus in rabbits.

Pseudorabies (PR), which is caused by the pseudorabies virus (PRV), is a severe infectious disease that causes abortions in adult sows and fatal encephalitis in piglets; the disease can occur in pigs of all ages and other mammals, which can lead to significant economic loss around the worldwide. The new PRV variant invalidated the available commercial attenuated and inactivated vaccines. Consequently, subunit vaccines have been suggested as novel strategies for PR control, while they are usually formulated with adjuvants due to their lower immunogenicity. We aimed to select a safe and efficient adjuvant for subunit vaccines for PR. In our study, glycoprotein B (gB) and glycoprotein D (gD) were expressed based on a baculovirus expression system, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was expressed using an Escherichia coli (E. coli) expression system; subsequently, a gB + gD subunit vaccine adjuvanted by GM-CSF was constructed. A rabbit model infected with a PRV SD-2017 strain was established, the TCID50 and LD50 were measured, and the typical clinical symptoms were observed. After a lethal challenge of 5 LD50 with a PRV SD-2017 strain, the rabbits exhibited typical clinical symptoms, including itching and high temperature, and histopathology revealed severe inflammation in the brain, which is the dominant target organ of PRV. Rabbits immunized with the gB + gD + GM-CSF subunit vaccines produced higher levels of antibodies than those immunized with gB + gD + ISA 201, which was adjuvanted with a frequently used oil adjuvant. The survival rate of rabbits vaccinated with gB + gD + GM-CSF was 100%, which was superior to that of rabbits vaccinated with gB + gD + ISA 201 (80%), inactivated PRV + GM-CSF (60%) and commercial inactivated vaccine (60%) after challenge with PRV SD-2017. These data suggested that the gB + gD + GM-CSF-based subunit vaccine had good protective efficacy against the PRV SD-2017 strain in rabbits and that GM-CSF could be developed as a candidate adjuvant for use in a vaccine regimen to prevent and even eradicate PR.

A first-in-human, phase I, open-label study of a novel cancer vaccine labvax 3(22)-23 and adjuvant GM-CSF in patients with advanced stage adenocarcinomas.

TPS2700 Background: Adenocarcinoma is the most common histologic type of solid tumors and can arise from almost anywhere in the body. Labyrinthin is a novel tumor-specific protein expressed on the cell surface of the majority of adenocarcinomas of various cancer types. Several therapeutic strategies targeting labyrinthin are under development. We hypothesize that vaccination against labyrinthin can elicit strong immune responses against the labyrinthin-positive adenocarcinomas in cancer patients. LabVax 3(22)-23 is a novel anti-tumor vaccine that contains 4 synthetic labyrinthin-based peptides designed to elicit both B-cell and T-cell responses. Sargramostim, a recombinant granulocyte macrophage colony-stimulating factor (GM-CSF), is given as an immunostimulator with LabVax 3(22)-23 to boost the antitumor immune response. Preclinical studies showed that LabVax 3(22)-23 significantly inhibited tumor growth that was augmented by GM-CSF without any significant toxicity in C57/BL6 transgenic mice expressing human PD-1/PD-L1 implanted with the murine colon adenocarcinoma cell line MC-38-huPD-L1. This single institution, first-in-human, phase I trial (UCDCC#296) evaluates LabVax 3(22)-23 and adjuvant sargramostim in patients with labyrinthin-positive metastatic or recurrent adenocarcinoma of any primary tumor site after all standard-of-care therapies. Methods: The primary objective is to assess the toxicity of LabVax 3(22)-23 and adjuvant sargramostim according to NCI CTCAE V5.0. The primary endpoint is dose limiting toxicity (DLT), which is defined as grade ≥ 2 allergic and autoimmune reaction, grade ≥ 3 injection site reaction, any grade 3 toxicities lasting >1 week, or any grade ≥ 4 toxicities. With a sample size of 10 patients, there is 89-97% of chance to observe ≥1 DLT if the true event rate is 20-30%. A secondary endpoint is the preliminary assessment of tumor response rate by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Exploratory objectives measure the effect of LabVax 3(22)-23 on various immune responses (cytokines, anti-labyrinthin antibody production) and the correlation between the level of labyrinthin expression and the efficacy of LabVax 3(22)-23. Eligible patients are required to have labyrinthin expression on their tumor cells by immunohistochemistry and adequate organ function. Patients receive sargramostim subcutaneously and LabVax 3(22)-23 intradermally on weeks 1, 2, 4, 8, and 12 in the absence of disease progression or unacceptable toxicity. After completion of study treatment, patients are followed every 3 months for 1 year. Two patients are enrolled at the time of submission. Clinical trial information: NCT05101356.

Adjuvant GM-CSF Improves Survival in High-risk Stage IIIC Melanoma

Stage III melanoma is associated with an increased risk of recurrence and death. Complete surgical resection remains the best chance for cure. Unfortunately, no adjuvant therapy has demonstrated a consistent improvement in melanoma-specific survival (MSS). We hypothesize that adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF) may improve clinical outcomes. Retrospective cohort study of 317 surgically resected stage III melanoma patients managed with observation or adjuvant GM-CSF at a single institution from 2001 to 2010. Of the 317 stage III patients, 165 (52%) were observed and 152 (48%) were treated with GM-CSF, with a median follow-up of 34 months. Patients treated with GM-CSF tended to be younger (P < 0.0001), had more advanced stage disease (P = 0.002), and were more likely to have had a recurrence before initiation of adjuvant therapy than the observation group (P < 0.0001). Adjuvant GM-CSF seemed to be associated with improved MSS, but this did not reach statistical significance (P = 0.08). Patients with stage IIIC melanoma derived a substantial benefit from adjuvant GM-CSF, with a 52% risk reduction in melanoma-specific death (hazard ratio 0.48; 95% confidence interval, 0.27-0.87; P = 0.02). Despite selecting patients with more advanced stage and a higher incidence of regional relapse, adjuvant GM-CSF was associated with an improved MSS but not disease-free survival in patients with stage IIIC disease. In patients not otherwise eligible for clinical trials, adjuvant GM-CSF treatment is a reasonable option for individuals with resected high-risk melanoma.

Rindopepimut: a promising immunotherapeutic for the treatment of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive glial cell-derived primary tumor. Current standard of care for patients with GBM includes maximal tumor resection plus adjuvant radiotherapy and temozolomide chemotherapy, increasing median overall survival to a mere 15 months from diagnosis. Because these therapies are inherently nonspecific, there is an increased likelihood of off-target and incomplete effects; therefore, targeted modalities are required for enhanced safety and efficacy. Rindopepimut is emerging as a safe and potentially effective drug for the treatment of GBM. Rindopepimut consists of a 14-mer peptide that spans the length of EGF receptor variant III, a mutant variant of EGF receptor found on approximately 30% of primary GBM, conjugated to the carrier protein keyhole limpet hemocyanin. Vaccination with rindopepimut has been shown to specifically eliminate cells expressing EGF receptor variant III. Phase II clinical trials have suggested that vaccination of newly diagnosed GBM patients with rindopepimut plus adjuvant granulocyte-macrophage colony-stimulating factor results in prolonged progression-free and overall survival with minimal toxicity. This review will outline the development of rindopepimut, as well as the current status of this vaccine.

Dendritic cell-activating vaccine adjuvants differ in the ability to elicit antitumor immunity due to an adjuvant-specific induction of immunosuppressive cells.

We questioned whether the vaccine adjuvant combination of TLR-7 ligand agonist, imiquimod, with granulocyte macrophage colony-stimulating factor (GM-CSF) would result in enhanced dendritic cell recruitment and activation with increased antigen-specific immunity as compared with either adjuvant used alone. The adjuvant effects of GM-CSF and imiquimod were studied in ovalbumin (OVA) and MMTVneu transgenic mice using peptide-based vaccines. Type I immunity, serum cytokines, myeloid-derived suppressive cells (MDSC), and regulatory T cells (Treg) levels were examined. Both GM-CSF and imiquimod equally induced local accumulation and activation of dendritic cells. Both adjuvants effectively enhanced OVA-specific T-cell responses. We further evaluated the antitumor efficacy of adjuvant GM-CSF and imiquimod immunizing against murine insulin-like growth factor-binding protein-2 (IGFBP-2), a nonmutated oncoprotein overexpressed in the tumors of MMTVneu transgenic mice. Tumor growth was significantly inhibited in the mice receiving IGFBP-2 peptides with GM-CSF (P = 0.000), but not in imiquimod vaccine-treated groups (P = 0.141). Moreover, the addition of imiquimod to GM-CSF negated the antitumor activity of the vaccine when GM-CSF was used as the sole adjuvant. While GM-CSF stimulated significant levels of antigen-specific T-helper cell (T(H))1, imiquimod induced elevated serum interleukin (IL)-10. Both MDSC and Tregs were increased in the imiquimod-treated but not GM-CSF-treated groups (P = 0.000 and 0.006, respectively). Depleting MDSC and Treg in animals immunized with imiquimod and IGFBP-2 peptides restored antitumor activity to the levels observed with vaccination using GM-CSF as the sole adjuvant. Adjuvants may induce regulatory responses in the context of a self-antigen vaccine. Adjuvant triggered immunosuppression may limit vaccine efficacy and should be evaluated in preclinical models especially when contemplating combination approaches.

The Response to Repeated Vaccination with PR1 and WT1 Peptides Admixed in Montanide Adjuvant Is Transient and Fails to Induce Sustained Anti-Leukemia Responses in Patients with Myeloid Malignancies.

Abstract 4096Poster Board III-1031We previously demonstrated the immunogenicity of a combined vaccine approach employing two leukemia-associated antigenic peptides, PR1 and WT1 (Rezvani Blood 2008). Eight patients with myeloid malignancies received one subcutaneous 0.3 mg and 0.5 mg dose each of PR1 and WT1 vaccines in Montanide adjuvant, with 100 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF). CD8+ T-cell responses against PR1 or WT1 were detected in all patients as early as 1 week post-vaccination. However, responses were only sustained for 3-4 weeks. The emergence of PR1 or WT1-specific CD8+ T-cells was associated with a significant but transient reduction in minimal residual disease (MRD) as assessed by WT1 expression, suggesting a vaccine-induced anti-leukemia response. Conversely, loss of response was associated with reappearance of WT1 transcripts. We hypothesized that maintenance of sustained or at least repetitive responses may require frequent boost injections. We therefore initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies. Five patients with acute myeloid leukemia (AML) and 2 patients with myelodysplastic syndrome (MDS) were recruited to receive 6 injections at 2 week intervals of PR1 and WT1 in Montanide adjuvant, with GM-CSF as previously described. Six of 7 patients completed 6 courses of vaccination and follow-up as per protocol, to monitor toxicity and immunological responses. Responses to PR1 or WT1 vaccine were detected in all patients after only 1 dose of vaccine. However, additional boosting did not further increase the frequency of PR1 or WT1-specific CD8+ T-cell response. In 4/6 patients the vaccine-induced T-cell response was lost after the fourth dose and in all patients after the sixth dose of vaccine. To determine the functional avidity of the vaccine-induced CD8+ T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of PR1 and WT1 peptides (0.1 and 10 μM) was measured by IC-IFN-γ staining. Vaccination led to preferential expansion of low avidity PR1 and WT1 specific CD8+ T-cell responses. Three patients (patients 4, 6 and 7) returned 3 months following the 6th dose of PR1 and WT1 peptide injections to receive a booster vaccine. Prior to vaccination we could not detect the presence of PR1 and WT1 specific CD8+ T-cells by direct ex-vivo tetramer and IC-IFN-γ assay or with 1-week cultured IFN-γ ELISPOT assay, suggesting that vaccination with PR1 and WT1 peptides in Montanide adjuvant does not induce memory CD8+ T-cell responses. This observation is in keeping with recent work in a murine model where the injection of minimal MHC class I binding peptides derived from self-antigens mixed with IFA adjuvant resulted in a transient effector CD8+ T cell response with subsequent deletion of these T cells and failure to induce CD8+ T cell memory (Bijker J Immunol 2007). This observation can be partly explained by the slow release of vaccine peptides from the IFA depot without systemic danger signals, leading to presentation of antigen in non-inflammatory lymph nodes by non-professional antigen presenting cells (APCs). An alternative explanation for the transient vaccine-induced immune response may be the lack of CD4+ T cell help. In summary these data support the immunogenicity of PR1 and WT1 peptide vaccines. However new approaches will be needed to induce long-term memory responses against leukemia antigens. To avoid tolerance induction we plan to eliminate Montanide adjuvant and use GM-CSF alone. Supported by observations that the in vivo survival of CD8+ T-effector cells against viral antigens are improved by CD4+ helper cells, we are currently attempting to induce long-lasting CD8+ T-cell responses to antigen by inducing CD8+ and CD4+ T-cell responses against class I and II epitopes of WT1 and PR1. Disclosures:No relevant conflicts of interest to declare.

Neutralizing activity and cellular immune responses induced in mice after immunization with apoptotic HIV-1/murine leukemia virus infected cells

Dendritic cells present microbial antigens to T cells after uptake of apoptotic vesicles from infected cells. We previously reported that immunizations with apoptotic HIV-1/murine leukemia virus (MuLV) infected cells lead to induction of both cellular and humoral immune responses as well as resistance to mucosal challenge with live HIV-1/MuLV infected cells. Here we extended those studies and investigated whether apoptotic cells from HIV-1/MuLV infected cells stimulate the production of HIV-1 neutralizing activity. We compared different routes of administration and were able to induce p24- and Nef-specific cellular proliferation after intraperitoneal (i.p.), intranasal (i.n.), subcutaneous (s.c.) and intramuscular (i.m.) immunizations. Serum IgG and IgA antibodies directed against gp160, p24, or Nef were also produced regardless of immunization route used. However, the induction of mucosa-associated IgAs from faeces or vaginal secretions were detected only after either i.p. or i.n. immunizations. We were able to measure neutralizing activity in sera of mice after i.p. and i.n. immunization. Neutralizing reactivity was also detected after s.c. and i.m. immunizations in the presence of the cytokine adjuvant granulocyte macrophage-colony stimulating factor (GM-CSF). Conclusively we show induction of cellular and humoral immune responses including neutralizing activity after immunization with apoptotic HIV-1/MuLV infected cells in mice. The results from this study support further evaluations using apoptotic cells as antigen delivery system for vaccination against HIV-1 in other animal models.

Phenotypic and Functional Analysis of Dendritic Cells and Clinical Outcome in Patients With High-Risk Melanoma Treated With Adjuvant Granulocyte Macrophage Colony-Stimulating Factor

Granulocyte macrophage colony-stimulating factor (GM-CSF) can induce differentiation of dendritic cells (DCs) in preclinical models. We hypothesized that GM-CSF-stimulated DC differentiation may result in clinical benefit in patients with high-risk melanoma. We conducted a prospective trial in patients with high-risk (stage III B/C, IV), resected melanoma, with GM-CSF 125 microg/m(2)/d administered for 14 days every 28 days. Patients underwent clinical restaging every four cycles, with DC analysis performed at baseline and at 2, 4, 8, and 12 weeks. Of 42 patients enrolled, 39 were assessable for clinical outcome and DC analysis. Median overall survival was 65 months (95% CI, 43 to 67 months) and recurrence-free survival was 5.6 months (95% CI, 3 to 11 months). GM-CSF treatment caused an increase in mature DCs, first identified after 2 weeks of treatment, normalizing by 4 weeks. Patients with decreased DCs at baseline had significant increases in DC number and function compared with those with "normal" parameters at baseline. No change was observed in the number of myeloid-derived suppressor cells (MDSCs). Early recurrence (< 90 days) correlated with a decreased effect of GM-CSF on host DCs, compared with late or no (evidence of) recurrence. GM-CSF treatment was associated with a transient increase in mature DCs, but not MDSCs. Greater increase of DCs was associated with remission or delayed recurrence. The prolonged overall survival observed warrants further exploration.

Granulocyte macrophage colony stimulating factor as adjuvant therapy for resected stage III/IV melanoma: Retrospective review of a single institutional experience

19002 Background: Preliminary data suggesting positive clinical outcome in patients treated with adjuvant granulocyte macrophage colony stimulating factor (GM-CSF) following surgical resection of stage III/IV melanoma already exist (Spitler et al). A compassionate use protocol for the treatment of such patients with the “Spitler” regimen (GM-CSF administered at 250 mcg/day s.c. for 14 days of a 28 day cycle) has been ongoing at Mayo Clinic. Herein we present a retrospective review. Methods: Between 1998 and 2006, data was collected from 30 patients rendered disease free by surgery who received adjuvant GM-CSF. GM-CSF was administered per the “Spitler” regimen, although one patient received a reduced dose of 125mcg/day. The Kaplan-Meier approach was used to estimate relapse free survival. Results: Thirty patients (14 Female) with a median age of 53 and excellent performance status were evaluated. Most patients had resected stage III melanoma (Stage II/III/IV: 1/20/9). Median followup was 33 months (3–96). Twenty one patients were treated with GM-CSF for at least 12 months. Overall, relapse-free survival at 1 year was estimated to be 69% (95% confidence interval (CI) of 54% to 88%), reducing to 44% (95% CI of 29% to 67%) at 3 years. According to stage, relapse free survival at 1 year was 70% (III) and 63% (IV), and at 3 years was 54% (III), and 13% (IV). Among the 21 patients completing at least 1 year of therapy, relapse free survival at 3 years was 52%. Seventeen patients relapsed (III/IV: 10/7). Six of these patients were retreated and only two relapsed. Toxicities were grade 1 with most common being injection site rash (23%), asthenia (17%), myalgia (17%), and fever (7%). One patient required dose modification for elevated liver function tests. Conclusions: Although limited in scope, our data further support a potential beneficial effect of adjuvant GM-CSF in resected stage III/IV melanoma. Recurrences in the treated population were often localized and amenable to further surgical treatment. The clinical relevance of this strategy is currently being prospectively tested (E4697, now closed to accrual). No significant financial relationships to disclose.

Successful Topical Respiratory Tract Immunization of Primates against Ebola Virus

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.

The use of adjuvant granulocyte-macrophage colony-stimulating factor in HIV-related disseminated atypical mycobacterial infection

Mycobacterium avium complex (MAC) continues to be a challenging problem in some patients with advanced human immunodeficiency virus (HIV) infection despite the availability of potent chemotherapeutic agents. The use of adjunctive immunomodulatory therapy has shown promise in vitro, but there is currently limited supportive clinical evidence [Kemper CA, Bermudez LE, Derenski SC. Immunomodulatory treatment of Mycobacterium avium complex bacteraemia in patients with AIDS by use of recombinant granulocyte-macrophage colony-stimulating factor. J Infect Dis 1998;177:914-20; Kedzierska K, Johnson M, Mijch A, Cooke I, Rainbird M, Roberts S, et al. Granulocyte-macrophage colony-stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type-1 infected monocytes/macrophages in vitro and in vivo. J Infect Dis 2000;181:390-94]. We report the resolution of MAC disease resistant to traditional therapy, in an HIV-infected individual, following the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF).

Addition of GM-CSF to a peptide/KLH vaccine results in increased frequencies of CXCR3-expressing KLH-specific T cells.

T-cell trafficking is determined by expression patterns of chemokine receptors. The chemokine receptor CXCR3 is expressed on a subpopulation of type 1 T cells and plays an important role for migration of T cells into inflamed and tumor tissues. Here, we studied the chemokine receptor expression on specific T cells generated against the neoantigen keyhole limpet hemocyanin (KLH) in patients who had been immunized in the context of a tumor peptide vaccination trial with or without the adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF). In patients immunized in the presence of GM-CSF the fraction of CXCR3(+) KLH-specific T cells was significantly higher than in patients immunized in the absence of GM-CSF (median 45 vs. 20%, P = 0.001). In contrast, the chemokine receptor CCR4, associated with migration to the skin was found in both cohorts on less than 10% of KLH-specific T cells. These results show that CXCR3 expression on vaccine-induced T cells can be modulated by modifying the local vaccine milieu.

Recombinant, tumour-derived idiotype vaccination for indolent B cell non-Hodgkin’s lymphomas: a focus on FavId™

FavId™ (Favrille, Inc., San Diego, CA, USA) is a personalised therapeutic vaccine product for B cell non-Hodgkin’s lymphoma, custom-manufactured from individual patient’s tumour cells. This investigational agent consists of recombinant tumour-specific immunoglobulin (idiotype [Id]) chemically conjugated to the highly immunogenic carrier protein keyhole limpet haemocyanin (Id–KLH). The vaccine product is administered by subcutaneous co-injection with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF) with the goal of stimulating tumour-specific T cell and humoral immunity. Therapeutic Id vaccines have shown promising results in early phase clinical trials in follicular lymphoma, and several Phase III trials are ongoing. FavId’s advantages over other Id vaccine formulations include its rapid and efficient manufacturing technology utilising recombinant baculovirus, with a production time of only 8 – 12 weeks. In Phase II studies, FavId Id–KLH plus GM-CSF vaccines have been found to be safe, immunogenic and clinically active in follicular lymphoma. At present, FavId is being tested in a randomised, placebo-controlled Phase III trial in follicular lymphoma, aimed at improving the time to disease progression when administered following cytoreduction with rituximab. If found to be efficacious in this pivotal trial, FavId would represent a tumour-selective immunotherapy for lymphoma with little toxicity and a novel mechanism of action.

Immunotherapy of melanoma.

Over the past decade, tumour immunology has progressed enormously, as a result of the application of a range of new technologies. Screening of tumour gene expression libraries using cancer patients' sera and cancer-specific T lymphocytes has defined a large number of tumour-specific antigenic proteins. The repertoire of known tumour antigens is rapidly expanding as a consequence of these advances. In addition, over the last few years the analysis of human T-cell responses specific for several tumour antigens in cancer patients has been made possible by the use of novel staining reagents (i.e. tetramers). This has allowed the characterization and monitoring of specific tumour responses, and has provided an opportunity to greatly accelerate the development of new anticancer vaccines. Recent results have demonstrated that the immune system can detect the presence of malignant cells in cancer patients, and have raised hopes for the use of antigenic cancer proteins as vaccines to induce tumour-specific cell-mediated immunity. Tumour immunology is now on solid technical and conceptual footing. However, the challenge remains to apply to patients the results obtained in the laboratories.