Gabriela R. Rossi

Effective Treatment of Preexisting Melanoma with Whole Cell Vaccines Expressing α(1,3)-Galactosyl Epitopes

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.c. and pulmonary alphaGal-negative melanoma (B16Null) tumors in the alpha(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16alphaGal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-alpha. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16alphaGal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing alphaGal epitopes. Vaccination with B16alphagal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.

Allogeneic Melanoma Vaccine Expressing αGal Epitopes Induces Antitumor Immunity to Autologous Antigens in Mice Without Signs of Toxicity

Owing to the absence of αGal epitopes in human cells and constant stimulation of the immune system by the symbiotic bacterial flora, humans develop high titers of natural antibodies against these epitopes. It has been demonstrated that syngeneic whole cell vaccines modified to express αGal epitopes could be used to generate a potent anticancer vaccine. In this study, we tested whether allogeneic whole cell cancer vaccines modified to express αGal epitopes would be effective for the treatment of murine melanoma. The α(1,3)galactosyltransferase (αGT) knockout mice (H-2b/b) with preexisting subcutaneous and pulmonary tumors [αGal(−) B16, H-2b/b] received therapeutic vaccinations with S91M3αGal(+) (H-2d/d) whole cell allogeneic vaccines. These mice had better survival and reduced pulmonary metastasis burden compared with control mice treated with S91M3 vaccine cells. Vaccination with S91M3αGal-induced cytotoxic CD8+ T cells recognizing the syngeneic αGal(−) B16 tumors measured by adoptive transfer to recipients bearing pulmonary metastases. The presence of allo-antigens did not dominate the induction of immunity to “cryptic” tumor antigens and had helped in the generation of a more efficient vaccine to treat preexisting tumors when compared with classic autologous vaccines. Vaccination with allogeneic αGal(+) vaccines did not induce signs of toxicity including changes in weight, hematology, chemistry, and histopathology of major perfused organs or autoimmunity in long-term murine models for breast, lung, and melanoma. This study established the safety and efficacy data of allogeneic αGal(+) whole cell vaccines and constituted the basis for the initiation of human clinical trials to treat human malignancies.

Cellular immunotherapy study of prostate cancer patients and resulting IgG responses to peptide epitopes predicted from prostate tumor-associated autoantigens.

The immunogenicity of a cellular immunotherapy using genetically modified vaccines to express &agr;(1,3)galactosyl epitopes (&agr;Gal) was evaluated in advanced prostate cancer (PC) patients. In this dose escalation phase I study, we report safety, feasibility, and immunologic data of an immunotherapy composed of 2 human PC cell lines engineered to express &agr;Gal epitopes (HyperAcute-Prostate, HAP, NewLink Genetics). Eight patients received up to 12 biweekly vaccinations with HAP. Enrolled patients (aged range, 53–85 y) had American Joint Committee on Cancer stage IV, any T, any N, M1, Eastern Cooperative Oncology Group PS⩽2, at least 1 prior hormonal treatment and <3 prior chemotherapies, adequate bone marrow and organ function, and albumin ≥3.0 g/dL. Serum IgG antibodies to synthetic peptides overexpressed in PC were determined by enzyme-linked immunosorbent assay. Results indicate that HAP immunotherapy induced humoral immune responses to autoantigens in 2 of 8 patients. These patients developed IgG antibody to multiple epitopes overexpressed in PC after immunization. These responding patients received higher doses of the immunotherapy suggesting a dose response. Two immunogenic proteins (prostate-specific membrane antigen, hepsin) belong to the extracellular molecules family participating in malignant cell invasion. Median overall survival for patients was 25.1 months with 1 patient surviving over 70 months with stable PSA and bone metastasis before expiring of other causes. Three of 8 patients showed PSA stabilization (>100 d). In conclusion, HAP immunotherapy induces IgG responses to epitopes from autoantigens overexpressed in PC suggesting dose-dependent effect. HAP represents a viable immunotherapy approach to induce immune responses against tumor cells and may provide clinical benefit with minimal toxicity.

Algenpantucel-L immunotherapy in pancreatic adenocarcinoma

Pancreatic adenocarcinoma is the 4th leading cause of cancer death in the USA and the EU. A minority of patients presents with surgically resectable and potentially curable disease, but among these, 80% are destined to relapse and overall survival rates with adjuvant chemotherapy average 24 months. Immunotherapy is a promising therapeutic option and a potential paradigm shift in the treatment of patients with pancreatic cancer, and may be particularly effective when used early in the disease course to prevent metastatic spread. Algenpantucel-L (HyperAcute Pancreas, NewLink Genetics, Ames, IA, USA) is a whole-cell immunotherapy consisting of irradiated allogeneic pancreatic cancer cells genetically engineered to express the murine enzyme α-GT, which results in hyperacute rejection of the tumor cells with complement- and antibody-dependent cytotoxicity. Phase II clinical trial data has been encouraging, particularly for patients who demonstrated humoral immunologic responses. Here, we report preliminary results and biomarkers correlations with clinical activity of algenpantucel-L in pancreatic cancer.

High‐throughput fluorescent screening of transgenic animals: Phenotyping and haplotyping

Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high‐throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples.

Pancreatic cancer: Update on Immunotherapies and Algenpantucel-L

ABSTRACT Pancreatic adenocarcinoma is notoriously lethal, and despite improvements in systemic chemotherapy approaches bringing survival rates for metastatic disease to almost 1 year, by 2030 it is expected to become the second leading cause of cancer death. Pancreatic cancer (PC) prognosis has been associated with both the presence of intratumoral helper and cytotoxic T lymphocytes, as well as humoral immune responses to tumor associated antigens like mesothelin. It is well described that the PC microenvironment is characterized by a fibroinflammatory and immunosuppressive stroma. On these premises several immune-targeted strategies have been developed to harness the adaptable immune system with a goal of improving survival with little toxicity. Cancer vaccines involve the administration of tumor-associated antigens with the goal of inducing an endogenous anti-tumor response. Among several strategies discussed, we will focus on the algenpantucel-L (HyperAcute™ Pancreas) immunotherapy. Algenpantucel-L is a whole cell immunotherapy consisting of irradiated allogeneic PC cells genetically engineered to express the murine enzyme α(1,3)-galactosyltransferase (αGT), which ultimately leads to hyperacute rejection with complement- and antibody-dependent cytotoxicity. While phase III data in the adjuvant treatment of pancreatic cancer are pending, phase II results have been encouraging, particularly for patients who demonstrated humoral immunologic responses. Novel strategies using immune checkpoint inhibitors, costimulatory antibodies, and combinations with cancer vaccines may overcome immunotolerance and improve treatment success.

Chapter 29 – HyperAcute Vaccines: A Novel Cancer Immunotherapy

The hyperacute rejection of a xenotransplant is characterized by a complement-antibody mediated immune response dependent on αGal epitopes. Animal studies confirm that αGal epitopes expressed on allogeneic tumor vaccines elicit a potent T-cell-dependent antitumor immunity. Based on these immunologic reactions, we hypothesized that the hyperacute rejection mechanism could be exploited to alter antigen processing resulting in a novel therapeutic approach to treat human malignancies. Clinical trials data confirm that an immediate hypersensitivity response directed toward a vaccine composed of genetically modified allogeneic tumor cells expressing the xenoantigen αGal (HyperAcute vaccines) constitutes a polyvalent tumor cell vaccine with signs of clinical efficacy, concomitant to eliciting both a humoral IgG response as well as T-cell-mediated antitumor immunity. This conceptually innovative immunotherapy degrades tumoral immune escape and portends a promising genetic engineering tactic for the cost-effective development of a generally applicable human cancer vaccine principle with minimal toxicity. Encouraging results support additional clinical immunotherapy studies using HyperAcute vaccines.

Effect of cellular vaccination of prostate cancer patients on IgG responses to peptide epitopes predicted from prostate tumor–associated autoantigens.

2558 Background: Tumor cell-based vaccines against prostate cancer (PC) represent a minimally toxic approach to treat the disease and prevent recurrence. Pre-clinical studies in rodent models of cancers have demonstrated the antitumor effectiveness of a vaccine composed of tumor cells transduced with the α-1,3-galactosyltransferase gene encoding the αGal epitope. We have completed a Phase I study for safety and feasibility of a prostate vaccine composed of two irradiated human PC cell lines engineered to express membrane-associated αGal epitopes (NLG-11928 NewLink Genetics, Inc.) The primary goals of this dose escalation study were to determine safety and vaccine-induced humoral immune responses to autoantigens frequently overexpressed in human prostate tumors. METHODS Eight patients received up to 12 biweekly vaccinations with NLG-11928 Plasma samples were collected from 8 patients with AJCC stage IV, Any T, Any N, M1, ECOG PS < 2, at least one prior hormonal treatment and < 3 prior chemotherapies, adequate bone marrow and organ function, and albumin >3.0 g/dl, Median age 70 (range 53-85). Specific IgG in plasma was determined by direct ELISA to 50 synthetic peptides representative of proteins commonly overexpressed in prostate tumors. RESULTS Median survival was 25.1 months (range 5 - 60 months) with one treated patient remaining alive at 60 months with stable PSA and bone metastasis since 2007. NLG-11928 induced IgG responses to synthetic peptides in a dose dependent manner. In 5 of 8 patients IgG responses were seen against at least one synthetic peptide predicted from PSMA, hepsin, EZH2, prostein, PARIS-1, GRP78, survivin, and SOX2. Furthermore, in 3 of these 5 patients, reactivity against a total of 19 synthetic peptide fragments was observed. CONCLUSIONS Vaccination with NLG-11928 induces dose-dependent IgG responses to epitopes from autoantigens frequently overexpressed in PC. Some of these proteins (PSMA, hepsin) belong to the extracellular molecules participating in malignant cell invasion. NLG-11928 may represent a viable approach to induce immune response against tumor cells bearing these proteins and provide a clinical benefit.

Abstract 5507: Immunological findings in a phase II immunotherapy study using allogeneic lung cancer cells modified to express alpha(1,3)galactosyltransferase in advanced non-small cell lung cancer (NSCLC)

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Alpha(1,3)galactose carbohydrate (αGal) epitopes are highly immunogenic in man. In animal models, vaccination with αGal expressing tumor cells induced rejection of tumor and improved survival. In a phase II anti-tumor immunotherapy study, we evaluated the safety, anticancer activity and immunogenicity of allogeneic human lung cancer cells expressing αGal epitopes HyperAcute-Lung (HAL) vaccines. Methods: Patients with advanced NSCLC received intradermal injections of 300 million HAL vaccine cells every 2 weeks for up to eight doses. Anti-αGal (IgG) and anti-CEA antibodies (Ab) were monitored by ELISA. Cellular immune responses were evaluated by ELISPOT assay (IFN-γ and IL-5). Serum was collected before and at several time points post-immunization. PBMC were collected prior to immunization, after the 4th vaccination and after 8 vaccinations. PBMC were cultured with autologous dendritic cells (DC) pulsed with one of 4 irradiated NSCLC cell lines, three of which were the parental (wild type) cell lines used to generate the vaccine. The fourth was a NSCLC line that was not a component of the vaccine. We compared the reactivity of PBMC stimulated with DC pulsed with these tumor cells pre- and post-immunization in 18 patients with available samples. Results: All patients demonstrated anti-αGal Ab prior to immunization and all responded to vaccination by increasing anti-αGal Ab 2 to 100-fold. Thirty-two patients were tested for anti-CEA Ab. Twenty patients showed significant increases in anti-CEA Ab after immunization. IFN-γ release was detected in 10 of 18 patients evaluated. Patients demonstrating IFN-γ release by ELISPOT after immunization had a median overall survival of 92 weeks (range 40 to 157) comparing favorably with 33.5 weeks (range, 12 to 200) in patients that showed little IFN-γ response. The majority of patients tested showed IL-5 induction after vaccination. In both assays, the HAL1 and HAL2 vaccine cell lines induced greater reactivity on ELISPOT. Patients responding with higher levels of IFN-γ and IL-5 also showed reactivity to the NSCLC cell line that was not a component of the vaccine suggesting that reactivity and possibly cross-priming to shared tumor antigens is induced after vaccination with HyperAcute immunotherapy. Conclusion: Hyperacute anti-tumor immunotherapy induced humoral and cellular responses. Cytokine expression, especially IFN-γ, might be predictive for increased survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5507. doi:10.1158/1538-7445.AM2011-5507

Abstract 2423: Phase I/II study of antitumor vaccination using lung cancer cells expressing murine α(1,3)galactosyltransferase (αGT) in non-small cell lung cancer (NSCLC)

Background: Alpha(1,3)-linked galactose (αGal) epitopes are immunogenic and mediate rejection of non-primate xenografts. Vaccination of αGal knockout mice with tumor expressing αGT stimulated immunity against αGal-negative tumor and induced rejection of tumor. We studied the safety and antitumor activity of a vaccine using allogeneic human NSCLC cells engineered to express murine αGT gene in patients with NSCLC. The vaccine is composed of equal numbers of three αGT transduced human NSCLC cell lines. Methods: Patients with metastatic or recurrent NSCLC, age ≥18, ECOG PS ≤2, ≤2 prior systemic therapies, adequate hematological, hepatic and renal function, and informed consent were eligible. In phase I cohorts of patients received intradermal injections totaling 3, 10, 30, or 100 × 10E+6 vaccine cells every 4-weeks x 4; or 500 × 10E+6 vaccine cells followed by 300 × 10E+6 vaccine cells every 2-weeks × 7. In Phase II, eligible patients received injections totaling 300 × 10E+6 vaccine cells every 2 weeks for 8 doses. Adverse events were assessed using CTC v3.0 and response was determined using modified RECIST criteria. Serum anti-αGal antibody titers were monitored and ELISPOT assays for induction of interferonγ and interleukin-5 by PBMC were examined pre- and post vaccination. Vaccine site biopsies were performed. Results: 17 patients were treated in phase I. No vaccine-related serious adverse events (AE) were observed. Related AE9s were ≤CTC grade 2 and included injection site pain/discomfort, local hyperpigmentation, skin reactions, local urticaria, arthralgias/myalgias, dyspnea, fatigue, herpes zoster, hypertension, lymphopenia and mild serum transaminitis. Six patients experienced stable disease for ≥4.0 months (median 8.0, range, 6.0-63.1 months). In phase II, 23 patients treated, 16 patients received one and five patients received two prior treatment regimens and two patients had no prior treatment (one of those had surgery only). Seven patients demonstrated SD ≥4.0 months. Median survival for the entire group was 11.0 months (range, 2.1-35.7 months) and 23.7 months (range, 11.0-35.7 months) for patients that achieved SD. Vaccine sites demonstrated infiltration of lymphocytes, granulocytes and eosinophils. Anti-αGal titers increased in all patients, and release of interferonγ and interleukin-5 during co-culture with vaccine parental and an unrelated NSCLC cell line was detected in some patients. Increases in anti-CEA antibodies were also detected. Conclusions: Antitumor vaccination using genetically altered human lung cancer cells expressing αGT is safe and feasible. A number of patients had prolonged SD and the median survival of vaccinated patients compared favorably to that reported in patients receiving 2nd line chemotherapy for relapsed or progressive advanced NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2423.