Long-term outcomes of patients with recurrent ovarian cancer treated with a polyvalent vaccine with bevacizumab combination.

BACKGROUND: To characterize the safety, immunogenicity, and outcomes of patients with high-grade serous ovarian cancer (HGSOC) in second or greater remission treated with a polyvalent antigen-KLH plus OPT-821 vaccine construct and bevacizumab. METHODS: Patients with recurrent HGSOC were treated with the vaccine plus bevacizumab at our institution from 01/05/2011 to 03/20/2012. Follow-up continued until 03/2021. Blood/urine samples were collected. "Responders" had an immunogenic response to ≥ 3 antigens; "non-responders" to ≤ 2 antigens. RESULTS: Twenty-one patients were treated on study. One developed a dose-limiting toxicity (grade 4 fever). Two (10%) experienced bevacizumab-related grade 3 hypertension. Thirteen (68%) and 16 (84%) of 19 responded to ≥ 3 and ≥ 2 antigens, respectively (Globo-H, GM2, TF cluster Tn, MUC-1). Four of 21 patients were alive > 5 years post-treatment. Responders and non-responders had a median PFS of 4.9 months (95% CI: 2.8-8.1) and 5.0 months (95% CI: 0.7-cannot estimate), respectively; median OS was 30.7 months (95% CI: 16.9-52.0) and 34.2 months (95% CI: 12.8-cannot estimate), respectively. On two-timepoint analysis (baseline, week 17), increased IL-8 exhibited improved PFS (HR as 10-unit increase, 0.43; p = 0.04); increased PDGF exhibited worse OS (HR as 10-unit increase, 1.01; p = 0.02). CONCLUSIONS: This is the longest follow-up of vaccine administration with bevacizumab in patients with ovarian cancer. The vaccine was well tolerated with bevacizumab. Response was not associated with improved survival. On two-timepoint analysis, increased IL-8 was associated with significant improvement in PFS; increased PDGF with significantly worse OS. For all timepoint measurements, cytokine levels were not significantly associated with survival. TRIAL REGISTRATION: NCT01223235.

Effect of Oral ß-Glucan on Antibody Response to Ganglioside Vaccine in Patients With High-Risk Neuroblastoma: A Phase 2 Randomized Clinical Trial.

Importance: Among patients with high-risk relapsed metastatic neuroblastoma, oral ß-glucan adjuvant during GD2/GD3 ganglioside vaccine boost has stimulated IgG antibody response, which was associated with improved survival; however, the effectiveness of oral ß-glucan during the vaccine priming phase remains unproven. Objective: To isolate the adjuvant effect of oral ß-glucan on antibody response to GD2/GD3 ganglioside vaccine in patients with high-risk neuroblastoma. Design, Setting, and Participants: In this phase 2 randomized clinical trial, enrolled patients with high-risk neuroblastoma were randomized to 2 groups to receive the GD2/GD3 vaccine at a large cancer center in a major metropolitan area from October 2018 to September 2020. Data were analyzed from October 7, 2021, to February 28, 2022. Interventions: Eligible patients receiving GD2/GD3 vaccine were randomly assigned to group 1 (n = 54) to receive no ß-glucan or group 2 (n = 53) to receive an oral ß-glucan regimen during the first 5 weeks of vaccine priming. From week 6 onwards, all 107 patients received oral ß-glucan during vaccine boost for 1 year or until disease progression. Main Outcomes and Measures: Primary end point was comparison of anti-GD2 IgG1 response before vaccine injection 6 (week 32) in group 1 vs group 2. Seroconversion rate and the association of antibody titer with ß-glucan receptor dectin-1 single nucleotide polymorphism (SNP) rs3901533 were also assessed. Results: In all, 107 patients with high-risk neuroblastoma were randomized to the 2 groups: 54 patients (median [range] age, 5.2 [1.0-17.3] years; 28 [52%] male and 26 [48%] female) in group 1; and 53 patients (median [range] age, 6.2 [1.9-18.4] years; 25 [47%] male and 28 [53%] female) in group 2; both groups were also comparable in their first remission status at study entry (70% vs 70%). Adding oral ß-glucan during the first 5 weeks of vaccine priming elicited a higher anti-GD2 IgG1 antibody response in group 2 (1.80; 90% CI, 0.12-3.39; P = .08; planned type I error, 0.10). Anti-GD2 IgG1 titer of 230 ng/mL or greater by week 8 was associated with statistically favorable PFS. Antibody titer correlated significantly with dectin-1 SNP. The genotype frequency, seroconversion rates, and vaccine-related toxic effects were similar in the 2 groups. Conclusions and Relevance: This phase 2 randomized clinical trial found that adding oral ß-glucan during vaccine priming increased anti-GD2 IgG1 titer among genetic responders without added toxic effects. Because responder dectin-1 SNP was identical in the 2 randomized groups, no difference was detected in seroconversion rates. Alternative or additional adjuvants may be needed to enhance seroconversion. Trial Registration: ClinicalTrials.gov Identifier: NCT00911560.

A randomised phase II trial of a trivalent ganglioside vaccine targeting GM2, GD2 and GD3 combined with immunological adjuvant OPT-821 versus OPT-821 alone in metastatic sarcoma patients rendered disease-free by surgery.

BACKGROUND: Recurrence after resection of metastatic sarcoma is common. The gangliosides GM2, GD2 and GD3 are strongly expressed across sarcoma subtypes. We hypothesised that generation of anti-ganglioside antibodies would control micrometastases and improve outcomes in sarcoma patients who were disease-free after metastasectomy. METHODS: We conducted a randomised phase II trial of the immunological adjuvant OPT-821 with a KLH-conjugated ganglioside vaccine targeting GM2, GD2 and GD3, versus OPT-821 alone in patients with metastatic sarcoma following complete metastasectomy. Patients received 10 subcutaneous injections at Weeks 1, 2, 3, 8, 16, 28, 40, 52, 68 and 84 and were followed for evidence of recurrent disease. The primary end-point was relapse-free survival. Secondary end-points included overall survival and serologic response. RESULTS: A total of 136 patients were randomised, 68 to each arm. The mean age was 51.2, 52.2% were male, 90.4% had relapsed disease, 86.8% had high-grade tumours and 14% had ≥4 metastases resected. Histologies included leiomyosarcoma (33%), spindle cell sarcoma (14%), undifferentiated pleomorphic sarcoma (13%), osteosarcoma (10%), synovial sarcoma (9%), liposarcoma (9%) and others (12%). Most adverse events were Grade ≤2 (83.8% and 70.6% in the vaccine and adjuvant arms, respectively). The most common (≥20% of patients) were injection site reaction (89.7%), fatigue (44.1%) and pyrexia (27.9%) on the vaccine arm, and injection site reaction (69.1%) on the adjuvant only arm. The 1-year relapse-free survival rate (34.5% and 34.8% in the vaccine and OPT-821 monotherapy arm, respectively) did not differ between arms (P = 0.725). One-year overall survival rates were 93.1% and 91.5% in the vaccine and OPT-821 monotherapy arm, respectively (P = 0.578). Serologic responses at week 9 were more frequent on the vaccine arm (96.5% of patients) than in the adjuvant arm (32.8%), and the difference between groups was durable. CONCLUSIONS: A sustained serologic response to vaccination was induced with the vaccine, but no difference in recurrence-free or overall survival was observed between treatment arms. CLINICALTRIALS: gov identifier: NCT01141491.

Phase I Trial of Oral Yeast-Derived ß-Glucan to Enhance Anti-GD2 Immunotherapy of Resistant High-Risk Neuroblastoma.

Beta glucans, complex polysaccharides, prime leukocyte dectin-1 and CR3-receptors and enhance anti-tumor cytotoxicity of complement-activating monoclonal antibodies. We conducted a phase I study (clinicaltrials.gov NCT00492167) to determine the safety of the combination of yeast-derived beta glucan (BG) and anti-GD2 murine monoclonal antibody 3F8 in patients with relapsed or refractory high-risk neuroblastoma. Patients received intravenous 3F8 (fixed dose of 10 mg/m2/day × 10 days) and oral BG (dose-escalated from 10-200 mg/kg/day × 17 days in cohorts of 3-6 patients each). Forty-four patients completed 141 cycles. One patient developed DLT: transient self-limiting hepatic transaminase elevation 5 days after starting BG (120 mg/kg/day). Overall, 1, 3, 12 and 24 evaluable patients had complete response, partial response, stable and progressive disease, respectively, at the end of treatment. Positive human anti-mouse antibody response and dectin-1 rs3901533 polymorphism were associated with better overall survival. BG dose level and serum BG levels did not correlate with response. Progression-free and overall survival at 2 years were 28% and 61%, respectively. BG lacked major toxicity. Treatment with 3F8 plus BG was associated with anti-neuroblastoma responses in patients with resistant disease. Although the maximal tolerated dose for yeast BG was not reached, considering the large volume of oral BG, we recommended 40 mg/kg/day as the phase II dose.

Survival Impact of Anti-GD2 Antibody Response in a Phase II Ganglioside Vaccine Trial Among Patients With High-Risk Neuroblastoma With Prior Disease Progression.

PURPOSE: Anti-GD2 monoclonal antibody (mAb) has proven efficacy in high-risk neuroblastoma (HR-NB). A small phase I GD2/GD3 vaccine trial (n = 15) described long-term survival and a favorable safety profile among patients with a history of disease progression (PD). The kinetics of mounting antibody response to vaccine and its prognostic impact on survival are now investigated in a phase II study (ClinicalTrials.gov identifier: NCT00911560). PATIENTS AND METHODS: One hundred two patients with HR-NB who achieved remission after salvage therapies were enrolled in this trial. They received seven subcutaneous injections of GD2/GD3 vaccine spanning 1 year plus oral ß-glucan starting at week 6 after the third dose of vaccine. Serum anti-vaccine antibody titers were quantified by enzyme-linked immunosorbent assay. Single nucleotide polymorphisms (SNPs) were determined by quantitative polymerase chain reaction. Kaplan-Meier and landmark Cox Regression models were used for survival estimates. RESULTS: Patients had a history of one (63%), two (21%), or three to six (16%) episodes of PD. 82% of them progressed following anti-GD2 mAb (m3F8/dinutuximab/naxitamab) therapy. Vaccine-related toxicities were self-limited injection-associated local reactions and fever without any > grade 3 toxicities. The progression-free survival (PFS) was 32% ± 6%, and the overall survival (OS) was 71% ± 7% at 5 years. Serum anti-GD2 (immunoglobulin G1 [IgG1] and IgM) and anti-GD3 (IgG1) titers showed notable increases following the initiation of ß-glucan at week 6. There was an association between IgG1 titer and SNP rs3901533 of dectin-1, the ß-glucan receptor. Multivariable analyses showed that anti-GD2-IgG1 titer ≥ 150 ng/mL by week 8 was associated with favorable PFS and OS, while having prior episodes of PD and the time from last PD to vaccine were associated with PFS. CONCLUSION: GD2/GD3 vaccine plus ß-glucan elicited robust antibody responses in patients with HR-NB with prior PD. Higher anti-GD2-IgG1 titer was associated with improved survival.

Pharmacokinetics and Biodistribution of a [89Zr]Zr-DFO-MSTP2109A Anti-STEAP1 Antibody in Metastatic Castration-Resistant Prostate Cancer Patients.

A six-transmembrane epithelial antigen of prostate-1 (STEAP1) is a newly identified target in prostate cancer. The use of radio-labeled STEAP1-targeting antibodies with positron emission tomography (PET) may allow for detection of sites of metastatic prostate cancer and may refine patient selection for antigen-directed therapies. This was a prospective study in seven patients with metastatic castration-resistant prostate cancer who had at least one archival biopsy that was STEAP1-positive by immunohistochemistry. Patients received intravenous injections of ∼185 MBq and 10 mg of [89Zr]Zr-DFO-MSTP2109A, a humanized IgG1 monoclonal antibody directed against STEAP1. PET/CT images, blood samples, and whole-body counts were monitored longitudinally in six patients. Here, we report on safety, biodistribution, pharmacokinetics, dose estimates to normal tissues, and initial tumor targeting for this group of patients. There was no significant acute or subacute toxicity. Favorable biodistribution and enhanced lesion uptake (in both bone and soft tissue) were observed on imaging using a mass of 10 mg of DFO-MSTP2109A. The best lesion discrimination was seen at the latest imaging time, a median of 6 days postadministration. Pharmacokinetics showed a median serum T1/2 ß of 198 h, volume of central compartment of 3.54 L (similar to plasma volume), and clearance of 19.7 mL/h. The median biologic T1/2 for whole-body retention was 469 h. The highest mean absorbed doses to normal organs (mGy/MBq) were 1.18, 1.11, 0.78, 0.73, and 0.71 for liver, heart wall, lung, kidney, and spleen, respectively. Excellent targeting of metastatic prostate sites in both bone and soft tissue was observed, with an optimal imaging time of 6 days postadministration. The liver and heart were the normal organs that experienced the highest absorbed doses. The pharmacokinetics were similar to other antibodies without major cross-reactivity with normal tissues. A more detailed analysis of lesion targeting in a larger patient population with correlation to immunohistology and standard imaging modalities has been reported.

Imaging Patients with Metastatic Castration-Resistant Prostate Cancer Using 89Zr-DFO-MSTP2109A Anti-STEAP1 Antibody.

Six-transmembrane epithelial antigen of prostate-1 (STEAP1) is a relatively newly identified target in prostate cancer. We evaluated the ability of PET/CT with 89Zr-DFO-MSTP2109A, an antibody that recognizes STEAP1, to detect lesions in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods: Nineteen mCRPC patients were prospectively imaged using approximately 185 MBq/10 mg of 89Zr-DFO-MSTP2109A. 89Zr-DFO-MSTP2109A PET/CT images obtained 4-7 d after injection were compared with bone and CT scans. Uptake in lesions was measured. Fifteen patients were treated with an antibody-drug conjugate (ADC) based on MSTP2109A; ADC treatment-related data were correlated with tumor uptake by PET imaging. Bone or soft-tissue biopsy samples were evaluated. Results: No significant toxicity occurred. Excellent uptake was observed in bone and soft-tissue disease. Median SUVmax was 20.6 in bone and 16.8 in soft tissue. Sixteen of 17 lesions biopsied were positive on 89Zr-DFO-MSTP2109A, and all sites were histologically positive (1 on repeat biopsy). Bayesian analysis resulted in a best estimate of 86% of histologically positive lesions being true-positive on imaging (95% confidence interval, 75%-100%). There was no correlation between SUVmax tumor uptake and STEAP1 immunohistochemistry, survival after ADC treatment, number of ADC treatment cycles, or change in prostate-specific antigen level. Conclusion:89Zr-DFO-MSTP2109A is well tolerated and shows localization in mCRPC sites in bone and soft tissue. Given the high SUV in tumor and localization of a large number of lesions, this reagent warrants further exploration as a companion diagnostic in patients undergoing STEAP1-directed therapy.

A Phase I Study of Unimolecular Pentavalent (Globo-H-GM2-sTn-TF-Tn) Immunization of Patients with Epithelial Ovarian, Fallopian Tube, or Peritoneal Cancer in First Remission.

We conducted a phase I study in ovarian cancer patients to evaluate the safety and immunogenicity of a synthetic unimolecular pentavalent carbohydrate vaccine (Globo-H, GM2, sTn, TF, and Tn) supported on a peptide backbone, conjugated to keyhole limpet haemocyanin (KLH), and mixed with immunological adjuvant QS-21. Twenty-four advanced-stage, poor-risk, first-remission ovarian cancer patients were enrolled from January 2011-Septermber 2013. Three dose levels were planned (25, 50, 100 mcg) with three cohorts of six patients each, with an additional 6-patient expansion cohort at the MTD. ELISA serologic IgM and IgG responses for each antigen was defined as positive response if antibody titers were ≥1:80 over the respective patient's pre-vaccination serum. The study would be considered positive if at least four of 12 patients treated at the MTD showed immune responses for at least three of the five antigens. Twenty-four patients (median age, 54 years [range, 36-68]) were included in the safety analysis. Histology was high-grade serous in 22 patients (92%); 18 had stage III and six stage IV disease. The vaccine was well-tolerated at all doses, with no DLTs. At the highest treated dose, IgG and/or IgM responses were recorded against ≥3 antigens in 9/12 patients (75%), ≥4 in 7/12 (58%), and 5 in 3/12 (25%). With a median follow-up of 19 months (range, 2-39), 20 patients (83%) recurred and six (25%) died. The unimolecular pentavalent vaccine construct was shown to be safe and immunogenic. Such a construct greatly simplifies regulatory requirements and manufacturing, facilitates scalability, and provides adaptability.

Molecular basis of antibody binding to mucin glycopeptides in lung cancer.

Glycopeptides bearing Tn epitopes are emerging targets for cancer diagnosis and immunotherapy. In this study, we analyzed membrane proteins containing O-glycosylated tandem repeat (TR) sequences in lung cancer patients of different types and stages, using gene microarray data in public domain. The expression of Tn and glycopeptide epitopes on the surface of lung cancer cell lines were studied by monoclonal IgG antibodies 14A, 16A, and B72.3. The binding of mAbs to synthetic glycopeptides were studied by surface plasmon resonance. Nine mucin mRNAs were found to be expressed in lung cancer patients but at similar level to healthy individuals. At protein level, a glycopeptide epitope on cancer cell surface is preferably recognized by mAb 16A, as compared to peptide-alone (14A) or sugar-alone epitopes (B72.3). 14A and 16A favor clustered TR containing more than three TR sequences, with 10-fold lower Kd than two consecutive TR. B72.3 preferrably recognized clustered sialyl-Tn displayed on MUC1 but not other O-glycoproteins, with 100-fold stronger binding when MUC1 is transfected as a sugar carrier, while the total sugar epitopes remain unchanged. These findings indicate that clusters of both TR backbones and sugars are essential for mAb binding to mucin glycopeptides. Three rules of antibody binding to mucin glycopeptides at molecular level are presented here: first, the peptide backbone of a glycopeptide is preferentially recognized by B cells through mutations in complementarity determining regions (CDRs) of B cell receptor, and the sugar-binding specificity is acquired through mutations in frame work of heavy chain; secondly, consecutive tandem repeats (TR) of peptides and glycopeptides are preferentially recognized by B cells, which favor clustered TR containing more than three TR sequences; thirdly, certain sugar-specific B cells recognize and accommodate clustered Tn and sialyl-Tn displayed on the surface of a mucin but not other membrane proteins.

Phase I trial of a bivalent gangliosides vaccine in combination with ß-glucan for high-risk neuroblastoma in second or later remission.

PURPOSE: To report on a phase I trial designed to find the maximally tolerated dose in children of the immunologic adjuvant OPT-821 in a vaccine containing neuroblastoma-associated antigens (GD2 and GD3; Clinicaltrials.gov NCT00911560). Secondary objectives were to obtain preliminary data on immune response and activity against minimal residual disease (MRD). Treatment also included the immunostimulant ß-glucan. EXPERIMENTAL DESIGN: Patients with neuroblastoma in ≥2nd complete/very good partial remission received vaccine subcutaneously (weeks 1-2-3-8-20-32-52). Vaccine contained 30 µg each of GD2 and GD3 stabilized as lactones and conjugated to the immunologic carrier protein keyhole limpet hemocyanin; and OPT-821, which was dose escalated as 50, 75, 100, and 150 µg/m(2) per injection. Oral ß-glucan (40 mg/kg/day, 14 days on/14 days off) started week 6. RESULTS: The study was completed with 15 patients because there was no dose-limiting toxicity at 150 µg/m(2) of OPT-821 (the dosing used in adults). Thirteen of fifteen patients received the entire protocol treatment, including 12 who remain relapse-free at 24+ to 39+ (median 32+) months and 1 who relapsed (single node) at 21 months. Relapse-free survival was 80% ± 10% at 24 months. Vaccine and ß-glucan were well tolerated. Twelve of fifteen patients had antibody responses against GD2 and/or GD3. Disappearance of MRD was documented in 6 of 10 patients assessable for response. CONCLUSIONS: This immunotherapy program lacks major toxicity and is transportable to any outpatient clinic. Patient outcome is encouraging but the efficacy is uncertain because of the complexity and heterogeneity of prior therapies. A larger phase II trial is underway.

Applying PET to broaden the diagnostic utility of the clinically validated CA19.9 serum biomarker for oncology.

UNLABELLED: Despite their considerable advantages, many circulating biomarkers have well-documented limitations. One prominent shortcoming in oncology is a high frequency of false-positive indications for malignant disease in upfront diagnosis. Because one common cause of false positivism is biomarker production from benign disorders in unrelated host tissues, we hypothesized that probing the sites of biomarker secretion with an imaging tool could be a broadly useful strategy to deconvolute the meaning of foreboding but inconclusive circulating biomarker levels. METHODS: In preparation to address this hypothesis clinically, we developed (89)Zr-5B1, a fully human, antibody-based radiotracer targeting tumor-associated CA19.9 in the preclinical setting. RESULTS: (89)Zr-5B1 localized to multiple tumor models representing diseases with undetectable and supraphysiologic serum CA19.9 levels. Among these, (89)Zr-5B1 detected orthotopic models of pancreatic ductal adenocarcinoma, an elusive cancer for which the serum assay is measured in humans but with limited specificity in part because of the frequency of CA19.9 secretion from benign hepatic pathologies. CONCLUSION: In this report, a general strategy to supplement some of the shortcomings of otherwise highly useful circulating biomarkers with immunoPET is described. To expedite the clinical validation of this model, a human monoclonal antibody to CA19.9 (a highly visible but partially flawed serum biomarker for several cancers) was radiolabeled and evaluated, and the compelling preclinical evidence suggests that the radiotracer may enhance the fidelity of diagnosis and staging of pancreatic ductal adenocarcinoma, a notoriously occult cancer.

Accelerated tumor growth mediated by sublytic levels of antibody-induced complement activation is associated with activation of the PI3K/AKT survival pathway.

PURPOSE: We addressed the possibility that low levels of tumor cell-bound antibodies targeting gangliosides might accelerate tumor growth. EXPERIMENTAL DESIGN: To test this hypothesis, we treated mice with a range of monoclonal antibody (mAb) doses against GM2, GD2, GD3, and CD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in vitro. We also explored the mechanisms behind the complement-mediated tumor growth acceleration that we observed and an approach to overcome it. RESULTS: Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growth of high GM2 expressing cell lines both in vitro and in a SCID mouse model, whereas very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgM mAb targeting GM2 but consistent against an IgG mAb targeting GD3 as well. These findings were mirrored by in vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the phosphoinositide 3-kinase (PI3K)/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K activation and accelerated tumor growth in vitro and in vivo are eliminated by PI3K inhibitors NVP-BEZ235 and Wortmannin. These PI3K inhibitors also significantly increased efficacy of high doses of these four mAbs. CONCLUSION: Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced antibodies targeting a variety of tumor cell surface antigens.

Synthesis and preclinical evaluation of QS-21 variants leading to simplified vaccine adjuvants and mechanistic probes.

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

Immunization with N-propionyl polysialic acid-KLH conjugate in patients with small cell lung cancer is safe and induces IgM antibodies reactive with SCLC cells and bactericidal against group B meningococci.

PURPOSE: Polysialic acid (polySA) is a polymer side chain bound to the neural cell adhesion molecule that is extensively expressed on the surface of small cell lung cancer (SCLC) cells. In our previous study, a robust antibody response was noted in patients with SCLC after vaccination with 30 µg of keyhole limpet hemocyanin (KLH)-conjugated N-propionylated (NP-) polySA, but peripheral neuropathy and ataxia were detected in several vaccinated patients. The objectives of the current trial were to establish the lowest optimal dose and to confirm the safety of the induction of antibodies against polySA with the NP-polySA vaccine. EXPERIMENTAL DESIGN: Patients with SCLC who completed initial treatment and had no evidence of disease progression were injected with either 10 or 3 µg of NP-polySA conjugated to KLH and mixed with 100 µg of immunologic adjuvant (QS-21) at weeks 1, 2, 3, 4, 8, and 16. RESULTS: Nine patients were enrolled at each of the two dose levels. Prior to vaccination, one patient in each group had low-titer antibodies against polysialic acid. All patients at the 10 µg vaccine dose level responded to vaccination with IgM antibody titers against polysialic acid (median titer 1/1,280 by ELISA), and all but one patient made IgM and IgG antibodies against the artificial vaccine immunogen, NP-polysialic acid (median titer 1/10,240). The antibody responses at the 3 µg vaccine dose level were lower; six of nine patients developed antibodies against polysialic acid (median titer 1/160). Post-vaccination sera from 6/9 and 3/9 patients in the 10 and 3 µg groups reacted strongly with human SCLC cells by fluorescent-activated cell sorting (FACS). Sera from all patients in the 10 µg dose group also had bactericidal activity against group B meningococci with rabbit complement. Self-limited grade 3 ataxia of unclear etiology was seen in 1 of 18 patients. CONCLUSIONS: Vaccination with NP-polySA-KLH resulted in consistent high-titer antibody responses, with the 10 µg dose significantly more immunogenic than the 3 µg dose. This study establishes the lowest optimally immunogenic dose of NP-polysialic acid in this NP-polysialic acid-KLH conjugate vaccine to be at least 10 µg, and it establishes the vaccine's safety. We plan to incorporate NP-polySA into a polyvalent vaccine against SCLC with four glycolipid antigens also widely expressed in SCLC-GD2, GD3, fucosylated GM1, and globo H.

Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer.

One of the most widely used and potent immunological adjuvants is a mixture of soluble triterpene glycosides purified from the soap bark tree (Quillaja saponaria). Despite challenges in production, quality control, stability and toxicity, the QS-21 fraction from this extract has exhibited exceptional adjuvant properties for a range of antigens. It possesses an ability to augment clinically significant antibody and T-cell responses to vaccine antigens against a variety of infectious diseases, degenerative disorders and cancers. The recent synthesis of active molecules of QS-21 has provided a robust method to produce this leading vaccine adjuvant in high purity as well as to produce novel synthetic QS-21 congeners designed to induce increased immune responsiveness and decreased toxicity.

Human monoclonal antibodies to sialyl-Lewis (CA19.9) with potent CDC, ADCC, and antitumor activity.

PURPOSE: The carbohydrate antigen sialyl-Lewis(a) (sLe(a)), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract and breast and on small-cell lung cancers. Since overexpression of sLe(a) appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe(a) is an attractive molecular target for tumor therapy. EXPERIMENTAL DESIGN: We generated and characterized fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe(a)-KLH vaccine. RESULTS: Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe(a) (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2-3Galß1-3(Fucα1-4)GlcNAcß as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC(50) 0.1 µg/mL vs. 1.7 µg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells engrafted into SCID (severe combined immunodeficient mice) mice. Treatment during the first 21 days with four doses of r5B1 (100 µg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. CONCLUSION: On the basis of the potential of sLe(a) as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility.

Impact of minimal tumor burden on antibody response to vaccination.

Four randomized phase III trials conducted recently in melanoma patients in the adjuvant setting have been based in part on the correlation between antibody responses in immunized patients and improved survival. Each of these randomized trials demonstrated no clinical benefit, although again there was a significant correlation between antibody response after vaccination and disease free and overall survival. To better understand this paradox, we established a surgical adjuvant model targeting GD2 ganglioside on EL4 lymphoma cells injected into the foot pad followed by amputation at variable intervals. Our findings are (1) comparable strong therapeutic benefit resulted from treatment of mice after amputation with a GD2-KLH conjugate vaccine or with anti-GD2 monoclonal antibody 3F8. (2) The strongest correlation was between antibody induction in response to vaccination and prolonged survival. (3) Antibody titers in response to vaccination in tumor challenged mice as compared to unchallenged mice were far lower despite the absence of detectable recurrences at the time. (4) The half life of administered 3F8 monoclonal antibody (but not control antibody) in challenged mice administered was significantly shorter than the half life of 3F8 antibody in unchallenged controls. The correlation between vaccine-induced antibody titers and prolonged survival may reflect, at least in part, increased tumor burden in antibody-negative mice. Absorption of vaccine-induced antibodies by increased, although not detected tumor burden may also explain the correlation between vaccine-induced antibody titers and survival in the adjuvant clinical trials described above.

The known immunologically active components of Astragalus account for only a small proportion of the immunological adjuvant activity when combined with conjugate vaccines.

The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an immunological adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an immunological adjuvant in future studies. Several flavonoids also had significant adjuvant activity. However, when the activities of these known immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to adjuvant activity, or that the adjuvant activity of Astragalus is greater than the activity of the sum of its parts.

Preclinical evaluation of the synthetic adjuvant SQS-21 and its constituent isomeric saponins.

The saponin fraction QS-21 from Quillaja saponaria has been demonstrated to be a potent immunological adjuvant when mixed with keyhole limpet hemocyanin conjugate vaccines, as well as with other classes of subunit antigen vaccines. QS-21 adjuvant is composed of two isomers that include the apiose and xylose forms in a ratio of 65:35, respectively. The chemical syntheses of these two isomers in pure form have recently been disclosed. Herein we describe detailed in vivo immunological evaluations of these synthetic QS-21 isomeric constituents, employing the GD3-KLH melanoma antigen. With this vaccine construct, high antibody titers against GD3 ganglioside and KLH were elicited when GD3-KLH was co-administered with adjuvant, either as the individual separate synthetic QS-21 isomers (SQS-21-Api or SQS-21-Xyl), or as its reconstituted 65:35 isomeric mixture (SQS-21). These antibody titer levels were comparable to that elicited by vaccinations employing naturally derived QS-21 (PQS-21). Moreover, toxicities of the synthetic saponin adjuvants were also found to be comparable to that of naturally derived PQS-21. These findings demonstrate unequivocally that the adjuvant activity of QS-21 resides in these two principal isomeric forms, and not in trace contaminants within the natural extracts. This lays the foundation for future exploration of structure-function correlations to enable the discovery of novel saponins with increased potency, enhanced stability, and attenuated toxicity.

Cancer biomarkers defined by autoantibody signatures to aberrant O-glycopeptide epitopes.

Autoantibodies to cancer antigens hold promise as biomarkers for early detection of cancer. Proteins that are aberrantly processed in cancer cells are likely to present autoantibody targets. The extracellular mucin MUC1 is overexpressed and aberrantly glycosylated in many cancers; thus, we evaluated whether autoantibodies generated to aberrant O-glycoforms of MUC1 might serve as sensitive diagnostic biomarkers for cancer. Using an antibody-based glycoprofiling ELISA assay, we documented that aberrant truncated glycoforms were not detected in sera of cancer patients. An O-glycopeptide microarray was developed that detected IgG antibodies to aberrant O-glycopeptide epitopes in patients vaccinated with a keyhole limpet hemocyanin-conjugated truncated MUC1 peptide. We detected cancer-associated IgG autoantibodies in sera from breast, ovarian, and prostate cancer patients against different aberrent O-glycopeptide epitopes derived from MUC1. These autoantibodies represent a previously unaddressed source of sensitive biomarkers for early detection of cancer. The methods we have developed for chemoenzymatic synthesis of O-glycopeptides on microarrays may allow for broader mining of the entire cancer O-glycopeptidome.