Efficient transfer of PSA and PSMA cDNAs into DCs generates antibody and T cell antitumor responses in vivo

Gene therapy for prostate cancer may be realized through transduction of whole genes, such as PSA or PSMA, into immunotherapeutic dendritic cells (DCs). An oncoretroviral vector encoding human PSMA and a bicistronic oncoretroviral vector encoding human PSA and cell surface CD25 cDNAs were constructed. Remarkably, transfer of PSA/CD25 or PSMA cDNA during murine hematopoietic cell differentiation into DCs occurred with approximately 80% efficiency. In vitro, transduced DCs retained allostimulatory function and primed syngeneic T cells for tumor antigen-specific IFN-gamma secretion. In test experiments designed to elucidate mechanisms in vivo, syngeneic recipients of transduced DCs had increased anti-human PSA antibody titers and tumor-specific CD8(+) T cell IFN-gamma secretion with no detectable immune response to CD25. Gene-modified DC recipients had increased protection from specific tumor challenge for at least 18 weeks post-vaccination. DC vaccination also protected both male and female recipients. Gene-modified DC vaccination mediated regression of established, specific gene-expressing, TRAMP-C1 prostate cancer cell tumors. These findings indicate that antibody and cellular responses generated through PSA and PSMA gene transfer into DC yielded protective immunity, thereby providing further preclinical support for the implementation of immuno-gene therapy approaches for prostate cancer.     

Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer.

PURPOSE: Many patients with clinically localized prostate cancer develop biochemical failure despite excellent local therapy perhaps due to occult metastatic disease. One potential solution is the utilization of a well-tolerated systemic therapy (e.g., vaccine) in concert with local therapy. EXPERIMENTAL DESIGN: We present a randomized phase II clinical trial designed to determine if a poxviral vaccine encoding prostate-specific antigen (PSA) can induce a PSA-specific T-cell response when combined with radiotherapy in patients with clinically localized prostate cancer. Thirty patients were randomized in a 2:1 ratio into vaccine plus radiotherapy or radiotherapy-only arms. Those patients in the combination arm received a "priming" vaccine with recombinant vaccinia (rV) PSA plus r V containing the T-cell costimulatory molecule B7.1 (rV-B7.1) followed by monthly booster vaccines with recombinant fowlpox PSA. The vaccines were given with local granulocyte-macrophage colony-stimulating factor and low-dose systemic interleukin-2. Standard external beam radiation therapy was given between the fourth and the sixth vaccinations. RESULTS: Seventeen of 19 patients in the combination arm completed all eight vaccinations and 13 of these 17 patients had increases in PSA-specific T cells of at least 3-fold versus no detectable increases in the radiotherapy-only arm (P < 0.0005). There was also evidence of de novo generation of T cells to well-described prostate-associated antigens not found in the vaccine, providing indirect evidence of immune-mediated tumor killing. The vaccine was well tolerated. CONCLUSION: This vaccine regimen can be safely given in patients undergoing radiation therapy for localized prostate cancer, with the majority of patients generating a PSA-specific cellular immune response to vaccine.     

Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (alvac) cytokine gene delivery induces destruction of established prostate tumors

Prostate‐specific antigen (PSA) is expressed by prostate epithelial cells and has a highly restricted tissue distribution. Prostatic malignancies in 95% of patients continue to express PSA, making this antigen a good candidate for targeted immunotherapy. The goals of our studies are to generate a recombinant PSA adenovirus type 5 (Ad5‐PSA) that is safe and effectively activates a PSA‐specific T‐cell response capable of eliminating prostate cancer cells, and to characterize the immunologic basis for this rejection. Here we show that immunization of mice with Ad5‐PSA induced PSA‐specific cellular and humoral immunity that was protective against a subcutaneous challenge with RM11 prostate cancer cells expressing PSA (RM11psa), but not mock‐transfected RM11 tumor cells (RM11neo). Mice immunized with recombinant adenovirus type 5 encoding β‐galactosidase (Ad5‐lacZ) did not generate protective immunity. Antitumor activity was predominantly mediated by CD8⁺ T lymphocytes. Although Ad5‐PSA immunization prior to RM11psa challenge was protective, Ad5‐PSA immunization alone was not able to control the growth of existing RM11psa tumors. In contrast, established RM11psa tumors ranging in size from 500 to 1,000 mm³ were efficiently eliminated if Ad5‐PSA priming was followed 7 days later by intratumoral injection of recombinant canarypox viruses (ALVAC) encoding interleukin‐12 (IL‐12), IL‐2, and tumor necrosis factor‐α. In this case, antitumor immunity was still dominated by CD8⁺ T lymphocytes, but natural killer cells became necessary for a maximal response. These data provide information on the effector cell populations in a protective immune response to prostate cancer and demonstrate the utility of an Ad5‐PSA vaccine combined with cytokine gene delivery to eliminate large established tumors that are refractory to other interventional methods. © 2001 Wiley‐Liss, Inc.     

Preoperative prediction of extracapsular tumor extension at radical retropubic prostatectomy in Taiwanese patients with T1c prostate cancer

BACKGROUND: To find a predictor for extracapsular tumor extension at radical retropubic prostatectomy (RRP) in Taiwanese patients with stage T1c prostate cancer (PC), preoperative transrectal sonoguiding prostate biopsy outcomes and clinicopathological data obtained from these patients were reviewed. METHODS: Fifty-five consecutive men who underwent radical retropubic prostatectomy for stage T1c PC were included. Preoperative sextant needle biopsies of the prostate were performed and whole-mount prostatectomy specimens were processed. The pathological end point was tumor capsular perforation extending entirely through the prostate capsule. Preoperative prostate-specific antigen (PSA), free-to-total PSA ratio, prostate volume, PSA density, Gleason score, number of positive biopsy cores, percentage cancer of sextant biopsies, percentage cancer of one lobe and percentage cancer of one core were analyzed for their ability to predict extracapsular tumor extension at RRP. RESULTS: Eighteen of the 55 specimens showed evidence of tumor capsular perforation. Those with extracapsular tumor extension (ECE) had higher PSA than organ-confined disease (OCD) (18.4 vs 8.3 ng/ml, P < 0.01). The ECE had a higher PSA density than OCD (0.556 vs 0.226, P < 0.01). The percentage of cancer in biopsies, percentage cancer of one lobe and percentage cancer of one core were all higher in ECE than OCD (P < 0.05). The ECE had a higher biopsy Gleason score than OCD (5.6 vs 4.5, P < 0.01). CONCLUSIONS: The four strongest predictors for extracapsular tumor extension of patients with T1c PC were PSA density >or=0.35, biopsy Gleason score >or=6, >or=20% cancer in biopsies and PSA >or=10 ng/ml.     

Prostate-specific antigen induction by a steroid hormone in T47D cells growing in SCID mice

Previous studies revealed that prostate-specific antigen (PSA) is present in > 30% of human breast tumor cytosols. Survival analysis showed that patients with PSA-producing tumors have a reduced risk for relapse, suggesting PSA to be an independent favorable prognostic marker for a large subset of breast cancer patients. The present investigation established an in vivo model for the induction of PSA in human breast cancer tumors growing as xenografts in severe combined immunodeficient (SCID) mice. The human mammary cancer cell-line T47D was grown i.m. in female mice. When the tumor and leg diameter reached 10 mm, the mice were stimulated daily with norgestrel for either 5 or 7 days to produce PSA, and sacrificed on day 8. The prostate cancer cell-line LNCaP was grown in male mice and functioned as a positive control for PSA production. After T47D and LNCaP mice were sacrificed, a highly sensitive immunofluorometric assay was used to analyze the PSA concentration in the tumor, muscle, liver, and kidney cytosols. Norgestrel-stimulated T47D mice showed significantly more PSA in the tumors compared to tumors of the control mice. However, PSA levels in tumors of the stimulated mice were significantly lower than those in the LNCaP xenografts. No PSA levels above background were present in the blood and normal tissue of the norgestrel-stimulated or control T47D xenografts. This mouse model will be a valuable tool for investigating and screening new therapies for a subgroup of breast cancer patients who have significant PSA concentrations in their tumors.     

Senescent cells re‐engineered to express soluble programmed death receptor‐1 for inhibiting programmed death receptor‐1/programmed death ligand‐1 as a vaccination approach against breast cancer

Various types of vaccines have been proposed as approaches for prevention or delay of the onset of cancer by boosting the endogenous immune system. We previously developed a senescent‐cell‐based vaccine, induced by radiation and veliparib, as a preventive and therapeutic tool against triple‐negative breast cancer. However, the programmed death receptor‐1/programmed death ligand‐1 (PD‐1/PD‐L1) pathway was found to play an important role in vaccine failure. Hence, we further developed soluble programmed death receptor‐1 (sPD1)‐expressing senescent cells to overcome PD‐L1/PD‐1‐mediated immune suppression while vaccinating to promote dendritic cell (DC) maturity, thereby amplifying T‐cell activation. In the present study, sPD1‐expressing senescent cells showed a particularly active status characterized by growth arrest and modified immunostimulatory cytokine secretion in vitro. As expected, sPD1‐expressing senescent tumor cell vaccine (STCV/sPD‐1) treatment attracted more mature DC and fewer exhausted‐PD1⁺ T cells in vivo. During the course of the vaccine studies, we observed greater safety and efficacy for STCV/sPD‐1 than for control treatments. STCV/sPD‐1 pre‐injections provided complete protection from 4T1 tumor challenge in mice. Additionally, the in vivo therapeutic study of mice with s.c. 4T1 tumor showed that STCV/sPD‐1 vaccination delayed tumorigenesis and suppressed tumor progression at early stages. These results showed that STCV/sPD‐1 effectively induced a strong antitumor immune response against cancer and suggested that it might be a potential strategy for TNBC prevention.     

Delayed disease progression after allogeneic cell vaccination in hormone-resistant prostate cancer and correlation with immunologic variables.

PURPOSE: There are a significant number of patients with asymptomatic hormone-resistant prostate cancer who have increasing prostate-specific antigen (PSA) levels but little or no evaluable disease. The immunogenicity and minimal toxicity associated with cell-based vaccine therapy makes this approach attractive for these patients. EXPERIMENTAL DESIGN: We have evaluated a vaccine comprising monthly intradermal injection of three irradiated allogeneic prostate cell lines (8 x 10(6) cells each) over 1 year. The first two doses were supplemented with bacille Calmette-Guérin as vaccine adjuvant. Twenty-eight hormone-resistant prostate cancer patients were enrolled. Patients were assessed clinically and PSA levels were measured monthly. Radiologic scans (X-ray, computed tomography, and bone scan) were taken at baseline and at intervals throughout the treatment period. Comprehensive monthly immunologic monitoring was undertaken including proliferation studies, activation markers, cytokine protein expression, and gene copy number. This longitudinal data was analyzed through predictive modeling using artificial neural network feed-forward/back-propagation algorithms with multilayer perceptron architecture.RESULTS: Eleven of the 26 patients showed statistically significant, prolonged decreases in their PSA velocity (PSAV). None experienced any significant toxicity. Median time to disease progression was 58 weeks, compared with recent studies of other agents and historical control values of around 28 weeks. PSAV-responding patients showed a titratable T(H)1 cytokine release profile in response to restimulation with a vaccine lysate, while nonresponders showed a mixed T(H)1 and T(H)2 response. Furthermore, immunologic profile correlated with PSAV response by artificial neural network analysis. We found predictive power not only in expression of cytokines after maximal stimulation with phorbol 12-myristate 13-acetate, but also the method of analysis (qPCR measurement of IFN-gamma > qPCR measurement tumor necrosis factor-alpha > protein expression of IFN-gamma > protein expression of interleukin 2). CONCLUSIONS: Whole cell allogeneic vaccination in hormone-resistant prostate cancer is nontoxic and improves the natural history of the disease. Longitudinal changes in immunologic function in vaccinated patients may be better interpreted through predictive modeling using tools such as the artificial neural network rather than periodic "snapshot" readouts.     

Predicting prostate specific antigen failure after radical retropubic prostatectomy for T1c prostate cancer

PURPOSE: Clinicopathological data were reviewed to find a predictor of prostate specific antigen (PSA) failure in Taiwanese patients who had received radical retropubic prostatectomy (RRP) for stage T1c prostate cancer (PC). METHODS: Fifty-five consecutive men who underwent RRP for stage T1c PC were included. The clinical end point was PSA failure (PSA >0.2 ng/ml). Preoperative PSA, free-to-total PSA ratio, prostate volume, PSA density, transrectal sextant biopsy and whole mount of RRP parameters were analyzed for their ability to predict postoperative PSA failure. RESULTS: Fifteen of the 55 patients developed PSA failure during the follow-up period. Those with PSA failure had higher PSA, higher percentage of cancer in biopsies and higher biopsy Gleason score than the freedom from PSA failure group (all P < 0.05). The PSA failure group had higher pathology Gleason score and a higher incidence of extracapsular tumor extension than the freedom from PSA failure group (all P < 0.05). The PSA failure group had a larger tumor volume and higher incidence of combined peripheral lobe with transitional lobe involvement than the freedom from PSA failure group (all P < 0.05). Multivariate analysis revealed that the predictors for PSA failure after RRP were biopsy Gleason score > or =6, tumor volume > or =2.5 ml and PSA > or =10 ng/ml. CONCLUSION: The single most significant predictor for PSA failure in T1c PC patients after RRP was tumor volume > or =2.5 ml.     

O-glycosylation regulates LNCaP prostate cancer cell susceptibility to apoptosis induced by galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.     

Enhancement of immunogenicity of a therapeutic cervical cancer DNA‐based vaccine by co‐application of sequence‐optimized genetic adjuvants

Treatment of patients with cervical cancer by conventional methods (mainly surgery, but also radiotherapy and chemotherapy) results in a significant loss in quality of life. A therapeutic DNA vaccine directed to tumor‐specific antigens of the human papilloma virus (HPV) could be an attractive treatment option. We have developed a nontransforming HPV‐16 E7‐based DNA vaccine containing all putative T cell epitopes (HPV‐16 E7SH). DNA vaccines, however, are less immunogenic than protein‐ or peptide‐based vaccines in larger animals and humans. In this study, we have investigated an adjuvant gene support of the HPV‐16 E7SH therapeutic cervical cancer vaccine. DNA encoded cytokines (IL‐2, IL‐12, GM‐CSF, IFN‐γ) and the chemokine MIP1‐α were co‐applied either simultaneously or at different time points pre‐ or post‐E7SH vaccination. In addition, sequence‐optimized adjuvant genes were compared to wild type genes. Three combinations investigated lead to an enhanced IFN‐γ response of the induced T cells in mice. Interestingly, IFN‐γ secretion of splenocytes did not strictly correlate with tumor response in tumor regression experiments. Gene‐encoded MIP‐1α applied 5 days prior to E7SH‐immunization combined with IFN‐γ or IL‐12 (3 days) or IL‐2 (5 days) postimmunization lead to a significantly enhanced tumor response that was clearly associated with granzyme B secretion and target cells lysis. Our results suggest that a conditioning application and combination with adjuvant genes may be a promising strategy to enhance synergistically immune responses by DNA immunization for the treatment of cervical cancer. © 2009 UICC     

Dendritic cells reconstituted with a human heparanase gene induce potent cytotoxic T-cell responses against gastric tumor cells in vitro.

BACKGROUND AND AIMS: Dendritic cell-based tumor vaccination is a promising approach in the treatment of cancer. Strategies to modify dendritic cells (DCs) with tumor-associated antigens (TAAs) can elicit specific immune responses against tumors. Heparanase is overexpressed in gastric cancer, especially in invasive and metastatic cells, but is downregulated in differential normal tissue. Therefore, heparanase is a potential target in immunotherapy for patients with advanced gastric cancer who are not candidates for surgery. The present paper was designed to investigate the immune response of a heparanase gene-modified DC-based vaccine against gastric cancer cell lines in vitro. METHODS: DCs from peripheral blood mononuclear cells of healthy HLA-A2-positive donors were transfected with recombinant adenovirus containing the full-length cDNA of heparanase (rAd-Hpa) to generate heparanase gene-modified DC vaccine. T lymphocytes from the same donors were repeatedly activated by genetically modified DC vaccine to generate heparanase-specific cytotoxicity T lymphocytes (CTLs). CTL-mediated cell lysis of gastric cancer cells lines (KATO-III and SGC-7901) was analyzed in vitro by a standard (51)Cr releasing assay. IFN-gamma secretion was measured by ELISA in heparanase-specific CTLs cocultured with those gastric cancer cell lines. RESULTS: Our results showed that the expression of heparanase in DCs transfected with rAd-Hpa was significantly increased. Furthermore, DCs transfected with rAd-Hpa could induce heparanase-specific CTLs against HLA-matched and heparanase-positive gastric cancer cells in vitro, while there were no killing effects on autologous lymphocytes. Meanwhile, these rAd-Hpa-modified DCs could increase IFN-gamma secretion of effector cells when cocultured with KATO-III cells. CONCLUSIONS: These findings demonstrate for the first time that the transduction of DCs with rAd-Hpa can induce CTLs that specifically lyse heparanase-positive gastric cancer cells and increase IFN-gamma secretion in an MHC-restricted fashion. Heparanase gene-modified DC vaccine offers a great opportunity for immunotherapy in patients with advanced gastric cancer and possibly also with other malignancies.     

Myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated tumor cell lysates enhance antitumor activity in murine lung cancer

The present study aimed to investigate the efficacy of a myeloid dendritic cell (mDCs) and plasmacytoid (p)DC combined vaccine loaded with heat-treated cancer cell lysates against lung cancer cells. The mDCs and pDCs were selected using magnetic bead sorting. Antigen loading was performed by adding heat-treated Lewis lung cancer cell lysates to mDC, pDC or mDC+pDC (1:1). Surface expression of CD80, CD86, CD40 and major histocompatibility complex (MHC)-II molecules were determined using flow cytometry, and the secretion of cytokines IL-12, IL-6 and TNF-α were assessed using ELISA assays. The effect of the mDC and pDC vaccine on cytotoxic T lymphocytes (CTLs) against tumor cells was investigated. Tumor-bearing nude mice were intravenously injected with the mDC and pDC combined vaccine. Tumor tissues were collected for hematoxylin and eosin and TUNEL staining. Loading with tumor cell lysate significantly upregulated the surface expression of costimulatory molecules MHC-II on DCs and enhanced secretions of IL-6, IL-12 and TNF-α by DCs. In addition, the tumor cell lysate-loaded mDC and pDC combined vaccine significantly promoted lymphocyte proliferation and enhanced CTL-mediated cytotoxicity against Lewis lung cancer cells compared with mDC or pDC treatment alone. Furthermore, intravenous injection of the mDC and pDC combined vaccine into tumor-bearing nude mice significantly inhibited subcutaneous tumor growth and induced necrosis and apoptosis within the tumor tissue. Overall, the pDC and mDC combination vaccine loaded with heat-treated Lewis lung cancer cell lysate had a synergistic effect on the induction of T lymphocyte proliferation and antitumor efficacy, which may be associated with the upregulation of co-stimulatory molecules and cytokine secretions.     

ErbB-2 signaling is involved in regulating PSA secretion in androgen-independent human prostate cancer LNCaP C-81 cells

The expression and secretion of prostate-specific antigen (PSA) are regulated by androgens in normal prostate secretory epithelial cells. In prostate cancer patients, the serum PSA level is usually elevated and cancer cells are initially responsive to androgens. However, those cancer cells become androgen-independent after androgen ablation therapy. In hormone-refractory cancer patients, even in an androgen-deprived environment, the circulation level of PSA rebounds and is constitutively elevated through a yet unknown mechanism. Tyrosine phosphorylation of ErbB-2 is involved in regulating the androgen-responsive phenotype of prostate cancer cells, and it is at least partly regulated by the cellular form of prostatic acid phosphatase (PAcP), a prostate-unique protein tyrosine phosphatase. We investigated the ErbB-2 signal pathway in androgen-independent PSA secretion. LNCaP C-81 cells, which are androgen-independent LNCaP cells lacking endogenous PAcP expression with a hypertyrosine phosphorylated ErbB-2, secreted a higher level of PSA in conditioned media than did androgen-sensitive LNCaP C-33 parental cells. A restored expression of cellular PAcP in C-81 cells was concurrent with a decrease in tyrophosphorylation of ErbB-2 and reduction of PSA secretion. Moreover, transient transfection of C-33 cells with the wild-type ErbB-2 or a constitutively active mutant of MEK1 cDNA resulted in an increased level of secreted PSA. The elevation of secreted PSA level by the forced expression of ErbB-2 was inhibited by an MEK inhibitor, PD98059. In C-81 cells, the expression of a dominant negative mutant of ErbB-2 reduced the secreted level of PSA. The inhibition of ErbB-2 or mitogen-activated protein (MAP) kinases by specific inhibitors AG879, AG825, or PD98059 led to a decrease in PSA secretion. Taken together, our data clearly indicate that the ErbB-2 signal pathway via MAP kinases (ERK1/2) is involved in regulating the secretion of PSA by androgen-independent human prostate cancer LNCaP C-81 cells in an androgen-depleted environment.     

Evaluation of the current incidence of nodal metastasis from prostate cancer

To determine the influences of transrectal ultrasonography, prostate-specific antigen (PSA), and heightened public awarencess of prostate cancer stage at diagnosis, we prospectively evaluated our most recent 173 patients who had a pelvic lymphadenectomy from 1987 to 1991. All patients had clinically localized prostate cancer and underwent bilateral limited pelvic lymph node dissections (N = 173); 19 (10.7%) were found to have nodal metastasis. Pathologic tumor stage and grade information was available for 168 patients who had a simultaneous radical prostatectomy. Clinical T-stage data revealed that only one patient had a T3 lesion. Pathologic T stage showed 7.1% to be T1a (12/168), 4.1% to be T1b (7/168), 13.7% to be T2a (23/168), 34.5% to be T2b (58/168), and 40.5% to be T3 lesions (68/168). Metastatic nodal involvement was not seen in any T1a, T1b, or T2a lesions. A Gleason's score of less than 5 lesions was predictive of no nodal metastasis. The clinical stage was upstaged pathologically in none of the T1a, 16.7% of the clinical T1b, 75% of the T2a, and 73% of the T2b, lesions. With regard to serum PSA, 27% of those patients with a level > 20 ng/ml had nodal metastasis (6/22) in this series. Although an elevated PSA was not predictive of tumor nodal metastasis, no patient with a normal PSA had nodal metastasis. Although the distribution of pathologic T stages is similar to that reported in the literature, our low incidence of nodal metastasis may suggest that prostate cancer is being diagnosed earlier. © 1993 Wiley-Liss, Inc.     

Combined adjuvants of poly(I:C) plus LAG‐3‐Ig improve antitumor effects of tumor‐specific T cells,preventing their exhaustion

Therapeutic cancer vaccines are designed to treat cancer by boosting the endogenous immune system to fight against the cancer. In the development of clinically effective cancer vaccines, one of the most practical objectives is to identify adjuvants that are capable of optimizing the vaccine effects. In this study, we explored the potential of polyinosinic–polycytidylic acid (poly(I:C)) and LAG‐3‐Ig (soluble recombinant protein of lymphocyte activation gene‐3 [LAG‐3] extracellular domain fused with human IgG Fc region) as adjuvants for P1A tumor antigen peptide vaccine in a pre‐established P815 mouse tumor model with a transfer of tumor‐specific T cells. Whereas the use of poly(I:C) or LAG‐3‐Ig as a signal adjuvant induced a slight enhancement of P1A vaccine effects compared to incomplete Freund's adjuvant, combined treatment with poly(I:C) plus LAG‐3‐Ig remarkably potentiated antitumor effects, leading to complete rejection of pre‐established tumor and long‐term survival of mice. The potent adjuvant effects of poly(I:C) plus LAG‐3‐Ig were associated with an enhanced infiltration of T cells in the tumor tissues, and an increased proliferation and Th1‐type cytokine production of tumor‐reactive T cells. Importantly, the combined adjuvant of poly(I:C) plus LAG‐3‐Ig downregulated expressions of PD‐1, LAG‐3, and TIGIT on P1A‐specific T cells, indicating prevention of T cell exhaustion. Taken together, the results of the current study show that the combined adjuvants of poly(I:C) plus LAG‐3‐Ig with tumor peptide vaccine induce profound antitumor effects by activating tumor‐specific T cells.     

An effective tumor vaccine optimized for costimulation via bispecific and trispecific fusion proteins

T cell costimulation has great therapeutic potential if it can be optimized and controlled. To achieve this, we engineered T cell-activating fusion proteins and immunocytokines that specifically attach to viral antigens of a virus-infected tumor vaccine. We employed the avian Newcastle Disease Virus because this agent is highly efficient for human tumor cell infection, and leads to introduction of viral hemagglutinin-neuraminidase (HN) molecules at the tumor cell surface. Here, we demonstrated the strong potentiation of the T cell stimulatory activity of such a vaccine upon attachment of bispecific or trispecific fusion proteins which bind with one arm to viral HN molecules of the vaccine, and with the other arm either to CD3 (signal 1), to CD28 (costimulatory signal 2a), or to interleukin-2 receptor (costimulatory signal 2b) on T cells. A vaccine with a combination of all three signals triggered the strongest activation of na?ve human T cells, thereby inducing the most durable bystander antitumor activity in vitro. Adoptive transfer of such polyclonally activated cells into immunodeficient mice bearing human breast carcinoma caused tumor regression. Furthermore, tumor-reactive memory T cells from draining lymph nodes of carcinoma patients could be efficiently reactivated in a short-term ELISpot assay using an autologous tumor vaccine with optimized signals 1 and 2, but not with a similarly modified vaccine from an unrelated tumor cell line. Our data describe new bioactive molecules which in combination with an established virus-modified tumor vaccine greatly augments the antitumor activity of T cells from healthy donors and cancer patients.     

Induction of protective immunity to RM-1 prostate cancer cells with ALVAC-IL-2/IL-12/TNF-alpha combination therapy

Human prostate cancers characteristically express low levels of major histocompatibility complex (MHC) Class I, which makes it challenging to induce protective antitumor responses involving T cells. Here we demonstrate that a whole cell tumor vaccine can induce protective T cell immunity to a low MHC Class I-expressing mouse prostate cancer cell line, RM-1. ALVAC recombinant canarypox viruses encoding interleukin-2, interleukin-12 and tumor necrosis factor-alpha were used to create therapeutic vaccines in 2 different ways. The RM-1 cells were pre-infected in vitro with the viruses prior to injection (pre-infection vaccine) or the RM-1 cells were injected alone, followed by the viruses (separate injection vaccine). The vaccines were each tested subcutaneously or intradermally. The pre-infection vaccine resulted in 100% clearance of primary tumors, whereas intradermal delivery of the separate injection vaccine cleared 40-60% of primary tumors. Despite the highly efficient primary tumor clearance by the pre-infection vaccine, only the separate injection vaccine generated protection upon rechallenge. Tumor-free survival induced by the separate injection vaccine required natural killer (NK) cells, CD4(+), and CD8(+) T cells. None of these cells alone were sufficient to induce tumor-free survival to the primary challenge, demonstrating an important cooperativity between NK cells and T cells. Secondary clearance of tumors also required NK and CD8(+) T cells, but not CD4(+) T cells. We report for the first time the generation of T cell immunity to the RM-1 prostate cancer cell line, demonstrating that it is possible to generate protective T cell immunity to a MHC I-low expressing tumor.     

A phase 1 trial of NY-ESO-1-specific TCR-engineered T-cell therapy combined with a lymph node-targeting nanoparticulate peptide vaccine for the treatment of advanced soft tissue sarcoma

The efficacy of immune checkpoint inhibitors is limited in refractory solid tumors. T-cell receptor gene-modified T (TCR-T)-cell therapy has attracted attention as a new immunotherapy for refractory cold tumors. We first investigated the preclinical efficacy and mode of action of TCR-T cells combined with the pullulan nanogel:long peptide antigen (LPA) vaccine in a mouse sarcoma model that is resistant to immune checkpoint inhibition. Without lymphodepletion, the pullulan nanogel:LPA vaccine markedly increased the number of TCR-T cells in the draining lymph node and tumor tissue. This change was associated with enhanced CXCR3 expression in TCR-T cells in the draining lymph node. In the phase 1 trial, autologous New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific TCR-T cells were infused twice into HLA-matched patients with NY-ESO-1⁺ soft tissue sarcoma (STS). The pullulan nanogel:LPA vaccine contains an epitope recognized by TCR-T cells, and it was subcutaneously injected 1 day before and 7 days after the infusion of TCR-T cells. Lymphodepletion was not performed. Three patients with refractory synovial sarcoma (SS) were treated. Two out of the three patients developed cytokine release syndrome (CRS) with low-to-moderate cytokine level elevation. We found obvious tumor shrinkage lasting for more than 2 years by tumor imaging and long-term persistence of TCR-T cells in one patient. In conclusion, NY-ESO-1-specific TCR-T-cell therapy plus vaccination with the pullulan nanogel carrying an LPA containing the NY-ESO-1 epitope without lymphodepletion is feasible and can induce promising long-lasting therapeutic effects in refractory SS (Registration ID: JMA-IIA00346).     

Cytotoxic T cell responses are enhanced by antigen design involving the presentation of MUC1 peptide on cholera toxin B subunit

Induction of cytotoxic T lymphocytes (CTL) is critical to cancer vaccine based immunotherapy. Efforts to elicit CTLs against tumor MUC1 with peptide based vaccine have not been successful in clinical application. We have design a MUC1 vaccine by replacing B cell epitope of CTB with MUC1 VNTR peptide. Immunization with hybrid CTB-MUC1 plus aluminum hydroxide and CpG adujuvant (CTB-MUC1-Alum-CpG) induce MUC1-specific CTLs in mice. Moreover, this vaccination can prevent tumor growth and reduce tumor burden in MUC1⁺B16 mice model. Meanwhile, CTB-MUC1-Alum-CpG vaccination can promote Th1 cells and CD8+ T cells inflate to tumor tissue. Our approach might be applicable to other cancer vaccine design.