Express Tumor Associated Antigens Research Articles

Abstract 1831: Affinity tuned XmAb®2+1 GPC3 x CD3 bispecific antibodies demonstrate selective activity in liver cancer models

Abstract Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Glypican 3 (GPC3), a lipid-anchored cell surface protein, is over-expressed in the majority of hepatocellular carcinoma (HCC) and a smaller subset of lung squamous cell carcinoma. It is considered an ideal target because its expression is not detected in adult tissues, and its function can promote tumor growth through the Wnt/beta-catenin pathway. Although GPC3 demonstrates good differential expression, bispecific T cell engagers are powerful immunomodulatory agents and careful tuning can improve the therapeutic window on all targets. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for GPC3 and monovalent for CD3. Reducing the affinity of GPC3 encouraged avid binding and strong killing activity only on high GPC3 expressing cell lines while minimizing reactivity on low expressing cell lines. We found modulating the CD3 affinity, either directly through mutation or indirectly by the molecular format, contributes to the selectivity of the bispecific antibodies. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding GPC3 antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Nargess Hassanzadeh-Kiabi, Kendra N. Avery, Rumana Rashid, Juan E. Diaz, Umesh S. Muchhal, Seung Y. Chu, Gregory L. Moore, Katrina Bykova, John R. Desjarlais. Affinity tuned XmAb®2+1 GPC3 x CD3 bispecific antibodies demonstrate selective activity in liver cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1831.

Tumor Associated Antigen Specific T Cells Given in Combination with Nivolumab for the Treatment of Hodgkin Lymphoma

Background: Hodgkin Lymphoma (HL) Reed Sternberg cells express tumor associated antigens (TAA) that are potential targets for cellular therapies. We recently demonstrated that TAA specific T cells (TAA-T) targeting WT1, PRAME and Survivin were safe and associated with prolonged time to progression in solid tumors (Hont JCO 2019). Hence, we evaluated whether TAA-T cells are safe and elicit anti-tumor effects in patients with relapsed/refractory (rel/ref) HL. We further evaluated the safety of Nivolumab following the TAA-T infusion and its effect on the persistence of the TAA-T cells in vivo. Methods: TAA-T products were generated from patients or healthy donors on 2 trials (NCT02203903; NCT03843294). Thirteen patients underwent procurement for product generation and 10 patients (2 allogeneic; 8 autologous) were infused TAA-T for rel/ref HL or as consolidation after autologous hematopoietic stem cell transplant (HSCT) at cumulative doses ranging from 0.5 X107 to 4 X107cells/m2. Patients were monitored for six weeks for safety and for response until disease progression. Seven patients received Nivolumab starting at 8 weeks after the first TAA-T infusion until disease progression or unacceptable toxicity. Results: TAA-T products (n=10) were polyclonal CD3+ T cells (Median 97%; 80.9-99.5%), comprised predominantly of CD4+ helper T cells (Median 10.5%; 1.74-20%) and CD8+ cytotoxic T cells (Median 70%; 29.3-87.5%). Specificity of TAA-T products was tested using Interferon-ϒ(INFϒ)-enzyme-linked immunospot (ELIspot) assay and defined as ≥ 2x spot-forming cells (SFC)/2.5X105cells against the tumor antigen as compared to irrelevant control antigen Actin(Figure 1). The median TAA specificity of the products was 2 antigens (range 0-3). All products were polyfunctional secreting INF-ϒ and TNF-α upon restimulation with tumor antigens (Fig 1). Median age of patients was 36yrs (range16-53). Patients had received a median 6 lines of therapy including HSCT prior to receiving TAA-T. Median follow-up post TAA-T#1 was 6 months (range 32 days-2.5yrs). There were no dose limiting toxicities observed within the 6 week safety monitoring period. In patients receiving Nivolumab post TAA-T, there were no increased immune related events over expected. One patient had Grade 3 seizures, possibly related to Nivolumab, 2 patients developed hypothyroidism requiring thyroid supplements and one patient developed myositis and discontinued Nivolumab after 5 months. The 2 patients who received TAA-T (1 donor derived and one autologous) as consolidation post HSCT achieved a continued complete remission (CCR) for 2+ years. Of the 8 patients with rel/ref HL at the time of infusion, 1 had disease progression at 6 weeks. He then received Nivolumab off protocol and achieved complete remission (CR) but developed Grade 4 GVHD. The remaining 7 patients had stable disease (SD) at 6 weeks. At a median follow-up of 6 months (32 days-2.5 years), 1 patient had progressive disease(PD) at 3 months, 1 patient had a complete metabolic response at 6 months and proceeded to allogeneic HSCT for definitive cure. 2 patients had PD at 6 months and the other 2 patients continue with SD at 6 months and remain on Nivolumab (Fig 1). All patients with objective responses (stable disease or better) recovered functional TAA-T cells in the peripheral blood at 3 months as detected by anti-Interferon-ϒ ELISPOT and reported as mean SFC/1 X105 cells for WT1(14±SD18.1); PRAME (17.4±15.3) and Survivin (4.5±7) compared to those with progressive disease with mean SFC/1 X105 cells for WT1 1.4(±2.3); PRAME (6.7±15.5) and Survivin (0.8±1.2). To evaluate TAA-T persistence, unique T cell receptor clonotypes defined in the TAA-T product and not present at baseline were detected in the peripheral blood 6 weeks post TAA-T, long-term persistence data and evaluating the effect on the TCR repertoire when adding nivolumab are pending. Conclusion: TAA-T cells given in combination with Nivolumab were safe when administered to patients with rel/ref HL with prolonged clinical responses (ranging from SD to CCR) observed in multiply relapsed patients. Disclosures Glenn: Genentech: Research Funding. Hanley:Mana Therapeutics: Honoraria, Other: Board Member; Cellevolve: Honoraria, Other: Board(Scientific Advisory Board). Bollard:Mana Therapeutics: Other: IP.

Abstract 5663: Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models

Abstract Bispecific antibodies enable therapeutic modalities inaccessible to traditional mAbs, such as T cell engagers that bind both CD3 on T cells and a target antigen on tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Prostate cancer (PC) is one of the most prevalent cancers in men, and end stage (castration-resistant prostate cancer) has no curative treatment option. Prostate specific membrane antigen (PSMA), a type II transmembrane protein with a large extracellular domain, has long generated interest as a therapeutic target. It is highly overexpressed in PC compared to normal tissue, and its expression has been shown to correlate with malignancy. Previous attempts to target PSMA include antibody-based radiotherapy and antibody drug conjugates, which have shown some success but can be hampered by the inherent toxicity of the modality. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for PSMA and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong activity on high PSMA expressing cell lines while minimizing reactivity on low expressing cell lines, matching PSMA's differential expression pattern. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding PSMA antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Matthew Dragovich, Erik W. Pong, Veronica Zeng, Michael Hedvat, Christine Bonzon, Kendra Avery, Rumana Rashid, Connie Ardila, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5663.

Abstract 2286: XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies

Abstract Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. One underexplored TAA is ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) for renal cell carcinoma (RCC). An activation marker for basophils, ENPP3 is a type II transmembrane protein that can promiscuously hydrolyze a large variety of nucleotide-containing molecules. Although its specific function in the tumor microenvironment is unknown, bulk RNAseq data from the TCGA describes broad overexpression of ENPP3 in clear cell and papillary RCC and less frequently in hepatocellular carcinoma (HCC). This pattern of expression has been confirmed by IHC and makes ENPP3 a potential target for a CD3 bispecific approach. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for ENPP3 and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong potency on high ENPP3 expressing cell lines while minimizing reactivity on low expressing cell lines, matching ENPP3's differential expression pattern. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. In vivo potency was evaluated by xenograft tumor models in human PBMC-engrafted NSG mice. In a KU812 tumor model, the lead candidate, XmAb30819, exhibited complete responses both as a single agent and in combination with checkpoint blockade. When tested against RXF393, an RCC cell line that overexpresses ENPP3 upon tumor engraftment, XmAb30819 demonstrated complete responses at all tested doses. Tolerability in non-human primates was evaluated in an escalating dose model and then confirmed in an expansion cohort. Dose-dependent T cell margination was observed, and doses that were active in the mouse tumor studies were well-tolerated. Preclinical studies have demonstrated the safety and efficacy of XmAb30819, an XmAb 2+1 ENPP3 x CD3 bispecific antibody for RCC. Citation Format: Alex Nisthal, Sung-Hyung Lee, Yoon Kyung Kim, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Liz Bogaert, Connie Ardila, Irene W. Leung, Nicole Rodriguez, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2286.

Combined hybrid structure of siRNA tailed IVT mRNA (ChriST mRNA) for enhancing DC maturation and subsequent anticancer T cell immunity

RNA therapeutics have received much attention in the development of anti-cancer therapies. Among them, synthetic mRNA (IVT mRNA) was investigated for cancer immunotherapy due to its abilities to express tumor associated antigens with stimulation of immune responses in dendritic cells (DCs). Despite of its great potential, several hurdles were remained such as insufficient immune stimulation and DC maturation. In this study, we aimed to present a novel IVT mRNA that can simultaneously express tumor associated antigens while suppress STAT3 proteins. Combined functions of siRNA and IVT mRNA were investigated and the hybrid structure of siRNA tailed mRNA (ChriST mRNA) was developed. We prepared the ChriST mRNA by employing polyA tail structures with RNAi sequences at the 3′ end of mRNA. Complementary strands were annealed to form duplex siRNA structure to induce STAT3 gene silencing. In addition, a hybrid structure of DNA/RNA was introduced into the ChriST mRNA between polyA tail and RNAi sequences. It was expected that DNA/RNA duplex would be readily cleaved by RNase H in the intracellular environment. After the cleavage, ChriST mRNA was fully functionalized in cells and exhibited enhanced tumor specific DC maturation.

418. Effective Treatment of Oligometastatic Melanoma Using Combination VSV Viroimmunotherapy and Stereotactic Radiotherapy

Oligometastatic (OM) disease represents an intermediate stage of progression between fully systemic metastasized disease and localized tumor burden. Clinical data is emerging that patients with OM are potentially curable, in part because of advances in the precision of radiation therapy treatment, and in part because of advances in the efficacy of immunotherapy on low burden systemic disease. We developed a model of OM melanoma by challenging mice both locally (subcutaneous injection) and systemically (intravenous injection) with B16 melanoma cells, resulting in both local tumor burden and a small number of prominent, systemically distributed lung metastases. We used this model to investigate whether systemic viroimmunotherapy, using Vesicular Stomatitis Virus (VSV) expressing tumor associated antigens to treat low level metastatic tumor burden, could be combined with clinically relevant stereotactic body radiation therapy (SBRT) to treat advanced local disease. In this respect, mice treated with only one treatment modality (systemic VSV-TAA, or local SBRT) needed to be sacrificed reproducibly due to progression of the untreated disease (local tumor or systemic metastatic disease, respectively). In contrast, mice treated with a combination of VSV-TAA and SBRT entered long-term remission resulting in apparent cure of tumor in up to 100% of mice depending upon the experiment. Increased lymphocyte, and CD45+ leukocyte infiltration was observed in tumors following both SBRT and VSV therapy 6-10 days following therapy. Experiments to delineate the role of immune cell subsets in the successful therapy are underway. In a significant proportion of mice, systemic VSV-TAA therapy was able to control local tumor, provided it was small at the start of therapy. SBRT was rarely able to have an impact upon the treatment of metastatic disease suggesting that, in this model, radiotherapy was not the dominant immunotherapy. However, treating mice with VSV-TAA was able to stimulate a detectable anti-tumor T-cell IFN-γ memory response. We plan to further investigate the immunogenicity of VSV-TAA treatment in the context of SBRT as part of a prime-boost immunotherapy strategy for OM disease. Therefore, we have developed a successful therapeutic regime to treat oligometastic disease that combines both radiotherapy to treat local disease, and systemic viroimmunotherapy to treat low-level metastatic disease and further studies to potentiate the anti-tumor effector response in vivo are in progress.

EXOSOMES DERIVED FROM THE ASCITIC FLUID OF MICE BEARING A T-CELL LYMPHOMA EXPRESS TUMOR ASSOCIATED ANTIGENS

EXOSOMES DERIVED FROM THE ASCITIC FLUID OF MICE BEARING A T-CELL LYMPHOMA EXPRESS TUMOR ASSOCIATED ANTIGENS

Abstract 1500: Modulation of recombinant vaccinia virus vector immunogenicity

Abstract Recombinant poxviruses expressing tumor associated antigens (TAAs) are evaluated since 20 years as immunogenic vaccine vector in clinical trials. Possible limitation to recombinant viral vector is due to either prior systemic immunity to poxviruses or immunodominance of viral antigens which may reduce the induction of immune response against weaker tumor antigens. To address this issue, we developed a recombinant Vaccinia virus expressing HSV type I protein ICP47. This protein down-regulates MHC class-I antigen presentation by blocking the transporter associated with antigen processing (TAP), which translocates peptides, generated by proteasomal protein degradation, into the endoplasmic reticulum for loading onto MHC class I molecule. Herpes simplex virus (HSV) US12 gene, coding for infected cell protein 47 (ICP47) was introduced into wild type vaccinia virus and into a r.VV expressing MART-1/Melan-A27–35 HLA-A201 endoplasmic reticulum (ER)-targeted epitope. Following infection with non-replicating recombinant virus, effect of ICP47 expression on cell surface MHC-class-I, MHC class-II and co-stimulatory molecules was characterized by antibody staining and FACS analysis. Human T-lymphocytes were stimulated in vitro with autologous CD14+ cells infected with r.VV-US12, r.VV-Mart-US12 or control virus. Responsiveness of specific CD8+ and CD4+ to viral proteins and recombinant epitopes were monitored by MHC-multimer staining and interferon gamma (IFNg) expression analysis. Cells infected with HSV-US12-r.VV, demonstrated a decreased ability of presenting MHC class-I antigens to CD8+ T cells whereas MHC-class-II restricted presentation to CD4+ T cells remained unaffected. Co-expression of ER-Melan-A/Mart-126–35 appeared to partially compensate for the ICP47 related surface MHC class-I molecule down-regulation and preserve a strong capacity to induce CTL response against the TAA derived peptide. Thus, viral vectors expressing ICP47 confirmed a diminished TAP-dependant processing of endogenous class-I restricted epitopes while the immunogenic potential of recombinant epitopes directly targeted to the ER was enhanced. Such reagents could become of high relevance especially in multiple-boost vaccine protocol required in cancer immunotherapy. 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 1500.

Acute lymphoblastic leukemia-derived dendritic cells express tumor associated antigens: PNPT1, PMPCB, RHAMM, BSG and ERCC1.

In all types of leukemia both in children and adults there is a need for novel therapies that could reduce the risk of relapse after standard treatment. Acute lymphoblastic leukemia (ALL) cells are ineffective antigen presenting cells, but as shown by many authors including results from our laboratory, stimulation with CD40L restores their antigen expressing capacity. The development of T-cell therapies for leukemic patients can be based on discovery of leukemia-associated antigens (LAA) which could be recognized by the host immune system. The aim of our present study was to test the hypothesis that leukemia-derived dendritic cells maintain the expression of tumor associated antigens. Twenty five children with B-cell precursor ALL were prospectively enrolled into the study. The mononuclear cells from peripheral blood or bone marrow were cultured and stimulated (or not) with CD40L and IL-4. The assessment of costimulatory/adhesion molecules with the use of flow cytometry and real-time RT PCR were used to confirm the possibility of turning ALL cells into dendritic-like cells. Additionally 22 tumor associated antigens mRNA levels were determined by real-time PCR technique with the TaqMan chemistry using ready-to-use Low Density Arrays for Gene Expression. The results of the study showed maintained expression and even up-regulation of some (PNPT1, PMPCB, HMMR/RHAMM, BSG and ERCC1) tumor associated antigens in CD40-activated leukemic cells. CD40L stimulation leading to the differentiation of leukemic cells into DCs which combine both antigen presenting function and expression of tumor associated antigens represents an interesting approach in cancer immunotherapy.

Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines

Cancers express tumor associated antigens that should elicit immune attack, but spontaneous immune rejection of established cancer is rare. Recent data demonstrate that specific and active tumor-mediated mechanisms hinder host anti-tumor immunity. CD4+CD25+ T regulatory cells (Tregs) are important mediators of active immune evasion in cancer. Disrupting tumor-mediated mechanisms hindering host immunity is a novel approach to tumor immunotherapy. Treg depletion improves endogenous anti-tumor immunity and the efficacy of active immunotherapy in animal models for cancer, suggesting that inhibiting Treg function could also improve the limited successes of human cancer immunotherapy. We have identified five strategies to block Treg activity: depletion, interference with trafficking, inhibition of differentiation, blockade of function or raising the effector T cell threshold for suppression. Discovery of additional regulatory cell populations expands the potential targets for these approaches. The fusion toxin denileukin diftitox (Ontak) reduces Treg numbers and function in the blood of some patients with cancer. We discuss specific strategies to block Treg activity and present some of our preliminary data in this area. Combining Treg depletion with active vaccination and other approaches poses additional challenges that are discussed.

The “Side-Population” of Human Lymphoma Cells Have Increased Chemo-Resistance, Stem-Cell Like Properties and Are Potential Targets for Immunotherapy

We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that:is least sensitive to chemotherapy andhas tumor repopulating potential.In this study we characterized SP cells in ten human lymphoma cell lines. Five of the 11 lymphoma cell lines tested (HDLM2, L428, Ramos, Toledo, Karpas) had a distinct SP ranging from 0.8–2% of total cells. We subsequently found SP cells with tumor associated markers in biopsy samples from patients with Hodgkin's Lymphoma, T-cell Lymphoma, and Follicular lymphoma. Culture of the sorted cell lines show that SP but not the non-SP cells produced progeny that were both SP and non-SP cells. Gene expression analysis of the SP showed increased expression of ABCG2 and other ABC transporters that mediate both the transport of Hoechst dye and selected chemotherapeutic agents out of the cell. We evaluated whether lymphoma cell lines with an SP are resistant to the chemotherapeutic drug gemcitabine. The viability of lymphoma lines containing SP cells was significantly higher (mean 59.8%; range 37.4–87.8%%) than the viability of lines without an SP (mean 9.3% range 3.0%–12.2%) when cultured with 10nM gemcitabine for 3 days. The SP component of the lymphoma cells became enriched 10 fold when cultured with gemcitabine. Furthermore, when equal numbers of SP and non-SP cells were grown in separate fractions with gemcitabine, there was reduced viability of the non-SP (2,247 +/−294 mean counts per minute (cpm)) by thymidine assay. In contrast, the viability of the SP was preserved (13,200 +/− 7500 mean cpm). There was no difference in the viability between the untreated SP and Non SP, so that the gemcitabine rather than the Hoechst dye accounts for the differences in viability. Although lymphoma SP cells are resistant to chemotherapeutic agents, they also express higher levels of tumor associated antigens that are known targets for cytotoxic T cells. Hence RT-PCR and immunohistochemistry both show that SP cells are HLA positive and have increased expression of MAGE-4, SSX2, Survivin and NY-ESO transcripts and protein compared to non-SP cells from the same tumor. In IFNγ ELISPOT assays, we have shown that T cells secrete IFNγ in response to SP targets. Hence, lymphoma cell lines and primary lymphoma tissue contain chemotherapy-resistant SP cells which express tumor associated antigens and may be targeted by specific T cells.

Vaccination Strategies for Patients with B-CLL.

B-chronic lymphocytic leukemia (B-CLL) cells express tumor associated antigens that may generate a T cell mediated immune response, but present these antigens poorly. Moreover, patients with B-CLL often have poor immune function due to the disease or its treatment. We have shown that expression of transgenic CD40L increases the immunogenecity of human B-CLL cells ex vivo and in vivo, and that this effect can be potentiated by co-expression of transgenic IL2. Previous studies described outcomes when adenoviral vectors were used to obtain gene transfer, but because of the complexities and expense of manufacture of viral vectors, and their lingering safety concerns, we determined whether it was possible to use electroporation (with the MaxCyte device) as a physical means of transferring CD40L and IL2 plasmids to produce vaccines with similar biological properties in vitro and in vivo. Table 1 compares the phenotype of the vaccines using each vector.Table 1. Comparision of immunogenic characteristics and viability of the adenoviral and plasmid vaccinesType of VaccineCD40L (%)CD80 (%)CD86 (%)IL-2 (pg/ml/10e6 cells)Viability (%) IL2 CD40LAdenoviralPre0.2 ± 0.012.6 ± 2.47.5 ± 3.9Post66.1 ± 5.5*50.2 ± 7.8*69.5 ±11*253.5 ± 82.693.694.2PlasmidPre1.3 ± 0.8511.5 ± 6.219.7 ± 6.8Post55.5 ± 5.1*19.2 ± 9.326.4 ± 9.74806.6 ±1398.984.488.4All the values are given as mean ± SE. * P< 0.01, Paired Student's t test.Vaccines made by both approaches met the release criteria for CD40L and IL2 expression (CD40L ≥20% and IL-2 ≥ 150 pg/ml/1x10e6 cells ), but expression of IL2 was higher in the plasmid vaccines, expression of CD40L was equivalent in each and expression of the additional co-stimulatory molecules CD80 and CD86 (induced after CD40 activation by transgenic CD40L) was higher in the adenoviral vaccines. Fourteen patients were given adenoviral-vaccines and nine the plasmid transduced cells. Each of these patients received up to 18 s.c. injections of IL-2 secreting and CD40L expressing tumor cells. Both types of vaccine were well tolerated. Table 2 shows the results of culturing patient T cells with autologous B-CLL tumor cells.Table 2. Comparision of anti-B-CLL T cell responses induced by adenoviral and plasmid vaccinesType of VaccinePre-vaccineAfter 3rd vaccineAfter 6th vaccineAdenoviral307.3 ± 293.9375 ± 306.8656.8 ± 373.8IFN-γ spots/10e6 T cells IL-5 spots/10e6 T cells012.8 ± 7.95.8 ± 2.3Plasmid31.1 ± 14.838 ± 17.832.9 ± 19.5IFN-γ spots/10e6 T cells IL-5 spots/10e6 T cells4 ± 2.714 ± 10.2203.9 ± 156.3*All the values were are given as mean ± SE. *P<0.05, Wilcoxon Signed Ranks testAfter 3 and 6 injections, both the adenoviral and plasmid vaccines had induced a rise in spot forming cells (SFC) for IL5, a cytokine associated with Th2 cells, but the rise was greatest in the recipients of the electroporated plasmid vaccine. By contrast, only the adenoviral vaccine induced a rise in SFC that produced IFN-γ, a cytokine associated with Th1 cells. Studies using MHC class I and II blocking antibodies showed that the IL5 and IFN-γ responses to both types of vaccine were mediated by HLA restricted T lymphocytes. The 1-year progression-free survival rates (PFS) for adenoviral vaccine group and plasmid vector group were 43% and 22% respectively. Figure 1 shows 1-year PFS rates for each group. Hence electroporation provides a more rapid and simpler means of preparing IL2/CD40L expressing B-CLL vaccines, but the cells express higher levels of IL2 and lower levels of “secondary” co-stimulator molecules than adenoviral vaccines, and produce an anti-tumor immune response of different polarity. Currently, we are evaluating electroporation of mRNA encoded CD40L which appears to augment upregulation of additional costimulatory molecules. [Display omitted]

Immunogenicity and safety of a DNA prime/adenovirus boost vaccine against rhesus CEA in nonhuman primates

Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients.

Bioconjugated gold nanoparticles as a molecular based contrast agent: implications for imaging of deep tumors using optoacoustic tomography

Optoacoustic tomography (OAT) is a novel medical imaging method that uses optical illumination and ultrasonic detection to produce deep tissue images based on their light absorption. Abnormal angiogenesis in advanced tumors, that increases the blood content of the tumor, is an endogenous contrast agent for OAT. In early stages, however, angiogenesis is not sufficient to differentiate a tumor from normal tissue; justifying the application of an exogenous contrast agent. We have developed a molecular based contrast agent composed of gold nanoparticles conjugated to a monoclonal antibody that improves OAT imaging to potentiate its use in imaging deep tumors in early stages of cancer or metastatic lesions. Due to their strong optoacoustic signal, we used gold nanoparticles (NPs) as a contrast agent. To target NPs to breast cancer cells, we conjugated NPs to a monoclonal antibody that specifically binds cell surface receptors known to be overexpressed in human breast tumors. In a series of in vitro experiments, Herceptin (monoclonal antibody that binds HER2/neu) conjugated to 40 nm NPs (Mab/NPs) selectively targeted human SK-BR-3 breast cancer cells. The breast cancer cells were detected and imaged by OAT in a gelatin phantom that optically resembled breast tissue. Sensitivity experiments showed that a concentration as low as 10(9) NPs per ml were detectable at a depth of 6 cm. Experimental data together with theoretical analysis demonstrate the feasibility of detection of deeply seeded small tumors that express tumor associated antigens using targeted gold NPs and OAT.

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen

Abstract Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7–1 (CD80) and only a subset express B7–2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7–1 and B7–2 also induce alloantigen specific T- cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen

Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7-1 (CD80) and only a subset express B7-2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7-1 and B7-2 also induce alloantigen specific T-cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.