Adoptive Cell Therapy Of Cancer Research Articles

A Novel NKp44-Based Chimeric Antigen Receptor That Targets Multiple Types of Cancer

Genetic modification of T cells with an artificial tumor-targeting chimeric antigen receptor (CAR) is a new approach for adoptive cell therapy for cancer. Defining cell surface molecules that are both selectively expressed on cancer cells and can be safely targeted with T cells or NK cells is a significant challenge in this research field.NKp44 is a member of the natural cytotoxicity receptor (NCR) families and also known as NCR2. Expression of NKp44 is limited to activating NK cells, which leads to a marked increase in cytotoxicity against tumors. The receptor contains one extracellular immunoglobulin domain, type I transmembrane (TM) domain, and intracellular (IC) domain, and its surface expression seems to require binding of the TM domain to adaptor molecules of DAP12 accessory protein that contains ITAMs. The ligand for NKp44 is considered damage-associated molecular pattern molecules, which have been reported to be expressed by various types of cancer cells but not by healthy cells. Therefore, a wide range of cancer cells may be safely targeted if the ligand-binding domain of this receptor is used in a construction of a chimeric antigen receptor (CAR) as an antigen recognition site, instead of using single chain variable domains derived from monoclonal antibody.We created several NKp44-based CAR constructs, which shares the extracellular NKp44 IG domain as a ligand-binding domain. Surface expression levels and subsequent functional properties can differ among T cells or NK cells transduced with novel CARs with different structural characteristics. We thus tested whether swapping the domains other than the antigen-binding domain affected expression and function. The CAR genes were retrovirally transduced into human primary T cells according to a standard method. We also transduced human primary NK cells with NKp44-based CARs, by a previously reported method (Imai C, et al. Blood 2005), to compare the expression pattern of the CAR in NK cells with that in human T cells.Retroviral transfer of wild type NKp44 gene and a construct harboring IC(p44) both did not induce NKp44 surface expression (Fig 1A,B). By sharp contrast, primary NK cells were able to express the CAR protein on the cell surface after transfer of these two genes. Removal of the IC(p44) [EH(p44)-TM(p44)-IC(CD3z)] allowed slight surface expression in T cells (Fig1C). The replacement of TM(p44) with TM(CD8a) resulted in higher surface expression in T cells (Fig 1D). These observations indicated the presence of IC(p44) as well as TM(p44) in the CAR constructs hampered surface expression in T cells most likely due to the lack of DAP12 expression. In addition to TM replacement, replacement of EH(p44) with EH(CD8a) markedly increased surface expression of the CAR (Fig 1E). Similarly, we tested use of CD28 domains instead of CD8a. Surprisingly, as different from the case of CD8a, the construct EH(p44)-TM(CD28)-IC(CD3z) yielded highest surface expression among the all CAR constructs created in this study in T cells as well as in NK cells (Fig1F), while the replacement of EH(p44) of the abovementioned CAR with EH(CD28) resulted in marked reduction of the CAR expression (Fig 1G).We confirmed surface expression of NKp44 ligand with flow cytometric analysis using recombinant human NKp44 Fc chimera protein (R&D Systems, McKinley Place, Minneapolis, USA) on various tumor cell lines including myeloid leukemia (K562, THP-1, U937, KY821, HL60), T-cell leukemia (PEER, MOLT4, HSB2), Burkitt lymphoma (Raji), BCR-ABL-positive B-ALL (OP-1), osteosarcoma (MG63, NOS1, NOS2, NOS10, U2OS, SaOS2), rhabdomyosarcoma (Rh28, RMS-YM), neuroblastoma (SK-N-SH, NB1, NB16, IMR32), and cervical carcinoma (Hela). Function of the best construct [EH(p44)-TM(CD28)-IC(CD3z)] was further evaluated. Primary T cells transduced with this NKp44-based CAR exerted powerful cytotoxicity against tumor cell lines tested and produced interferon-g and granzyme B, while GFP-transduced T cells and control T cells transduced with truncated NKp44-based CAR did not.In conclusion, we have created a novel CAR based on the antigen-binding property derived from NKp44 receptor immunoglobulin domain. This CAR should be effective to redirect T cells as well as NK cells against various types of cancer including hematological malignancies. [Display omitted] DisclosuresImai:Juno Therapeutics: Patents & Royalties.

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal genetransfer.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19+ target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo. We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity.

Abstract A171: Long-term episomal gene transfer for safe engineering of T cells for adoptive cell therapy of cancer

Abstract Success in T-cell therapy of cancer is currently relying on long-term gene expression owing to retrovirus/lentivirus (RV/LV) integration. However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal and long-term cell engineering method using non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element for either over-expression or down-regulation of genes. T-cells engineered with a NILV-S/MAR(CD19CAR) vector have similar levels of CAR expression as T-cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV-S/MAR-engineered CD19CAR-T-cells exhibited similar cytotoxic capacity upon CD19+ target cells recognition as LV-engineered T-cells. We propose that NILV-S/MAR vectors are superior to current options for enabling long-term gene expression without the risk of insertional mutagenesis. Citation Format: Chuan Jin, Mohanraj Ramachandran, Grammatiki Fotaki, Berith Nilsson, Magnus Essand, Di Yu. Long-term episomal gene transfer for safe engineering of T cells for adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A171.

Abstract A095: New genes for enhancing T cell function in adoptive cell therapy of cancer

Abstract A growing number of clinical trials point to the exquisite ability of adoptively transferred T cells to eradicate vast masses of tumor cells, including aggressive metastatic tumors. In one therapeutic approach T cells are derived from tumor biopsies (tumor-infiltrating lymphocytes, or TILs) and express endogenous TCRs. In others T cells are expanded from the peripheral blood lymphocyte pool and redirected to recognize tumor antigens by virtue of exogenous TCR or chimeric antigen receptor (CAR) genes. Although reports on enduring clinical responses are encouraging, a significant portion of patients who do not respond, or respond only partially and transiently, underscore the critical challenge of improving T cell function and survival at the tumor site. In attempt to improve the clinical efficacy of adoptive cell therapy we created three classes of genetic adjuvants designed to operate autonomously in gene-modified T cells. One class encodes constitutively-active (ca) toll-like receptors devoid of their ligand-binding domain, the other produces membrane-anchored immunostimulatory cytokines and the third encodes a novel homo-oligomerizing configuration of tumor necrosis factor receptors, inducing constitutive ligand-free activation. For gene delivery into human TILs or peripheral blood T cells we have been using electroporation of in-vitro-transcribed mRNA. Here we present data obtained in preliminary ex-vivo experiments designed to assess the function of caTLR4, membrane IL-2, IL-12 (single chain) and IL-15 and trimeric caCD40 and their level of cooperation. The mere expression of caTLR4 mRNA in polyclonal human CD8 and CD4 T cells induced the production of IFN-γ, triggered the surface expression of CD25, CD69 and 4-1BB and upregulated a panel of cytokines and chemokines. In all samples of anti-melanoma TILs tested, caTLR4 induced a burst of IFN-γ secretion, which usually ceased within 24 hours post-transfection. Furthermore, caTLR4 enhanced the cytolytic activity of TILs and augmented the secretion of IFN-γ, TNF-α and GM-CSF in the presence of autologous, but not mismatched, melanoma for at least 4 days post-transfection. The membrane cytokines were found to act mainly in-cis and each could support the proliferation of human CD8 and CD4 T cells for at least 6 days post-transfection, at a magnitude comparable to a saturating dose of soluble IL-2. Following mRNA electroporation of anti-melanoma TILs, membrane cytokines cooperated with caTLR4 in inducing IFN-γ secretion and upregulating the activation and costimulatory molecules CD25, CD69, 4-1BB and OX40. Different combinations of caTLR4, membrane cytokines and caCD40 mRNA induced spontaneous cytokine secretion in mRNA-transfected TILs, typically acting in a synergistic manner. Even when the spontaneous, initial effect waned, a considerably higher fraction of mRNA-transfected, compared to control-transfected TILs could still respond robustly to autologous, but not allogeneic, melanoma. In the presence of the three classes of adjuvants, transfected short-term cultured ‘young’ TILs revealed exceptional upregulation of 4-1BB, OX40, CD25 and CD28 and an increase in IFN-γ production and no, or only minimal change in the inhibitory receptors PD-1 and CTLA-4. Here, too, the different adjuvants displayed marked synergy, with caCD40 often exerting a prominent effect even when delivered as a single gene. Enhanced killing of autologous melanoma cells by electroporated young TILs was observed up to ten days post-electroporation. Our findings suggest that this new panel of genetic adjuvants can substantially improve the functional properties of antitumor T cells for extended periods, offering a new tool in all approaches for cancer immunotherapy employing adoptive T cell transfer. Citation Format: Gideon Gross, Aviad Pato, Hadas Weinstein-Marom, Noam Levin, Alon Margalit, Orit Itzhaki, Michal Besser, Tamar Peretz, Arthur Machlenkin, Michal Lotem. New genes for enhancing T cell function in adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A095.

Identification and Ex Vivo Expansion of a Circulating NK Cell Progenitor Population That Leads to Sustained Production of CD56+ NK Cells

Natural Killer (NK) cells are a component of the innate immune system that can lyse virally infected cells and tumor cells. Although IL-2 activated and ex vivo expanded NK cells are being developed for adoptive cell therapy of cancer, recent data suggests significant heterogeneity exists within these NK cell populations as defined phenotypically, functionally and developmentally. In order to maximize the efficacy of adoptive NK cell therapy, further insights into the ontogeny and behavior of various NK subsets are needed.NK cells have been historically defined as lymphocytes expressing CD56 but lacking CD3. However, we now report the identification of a population of human peripheral blood mononuclear cells that lack expression of the canonical NK markers CD56 and CD16, but otherwise share phenotypic and functional properties with NK cells, which we term "double negative (DN)" killer cells. Besides CD56 and CD16, these cells lack other standard lymphocyte markers including CD3, CD14, CD19, CD20, and CD33, although the majority of these DN cells express surface markers considered to be NK-specific, including CD8α, CD94, CD224, NKp30, CD127, NKp46, NKG2D, and NKG2A.We sorted CD3- CD19- CD14- CD20- human PBMNCs into CD16- CD56Bright, CD16+ CD56dim, CD16+ CD56- and CD16-CD56-(DN) cellular subsets by flow cytometry, then expanded these subsets in vitro by co-culture with irradiated EBV-transformed LCL feeder cells and IL-2 (500IU/mL). DN cells expanded 40-60 fold after 14 days in culture, with 55% of cells retaining the DN phenotype while 40% of cells acquired expression of CD56 but not CD16 (termed CD56SP). Generation of CD56SP cells from DN cells cultured in vitro suggests that circulating DN cells contain NK cell precursors. When DN cells were expanded for 14 days in culture and the residual remaining DN cells were then resorted and re-expanded for an additional 14 days, exponential cellular growth continued. After this addition 14 days of culture, 10-40% of cells became CD56SP NK cells, while the remainder remained DN. Repeated cycles of resorting residual DN cells demonstrated that the DN subset could continually regenerate additional DN cells as well as produce CD56SP NK cells for at least 45 days, suggesting unique proliferative and differentiative properties of this cell population.CD56SP NK cells generated in a 14 day cell culture from DN cells had up-regulation of a variety of cell surface receptors including CD30 (+57%), CD127 (+0.7%), CD161 (+69%), CD57 (+5.7%), CD8α (+40%), CD244 (+99%), NKp30 (+79%), NKp44 (+53%), CD25 (+3.5%), CD69 (+95%), and had higher intracellular IFN-γ (+35%), and perforin (+60%), suggesting both NK differentiation and education occurred in these progeny (figure). In contrast, expression of inhibitory receptors in the DN population, such as CD94 (88%), CD158e (12%) and NKG2A (24%) showed minimal change in expression levels in their progeny DN cells or CD 56SP NK cells at culture-day 14, consistent with prior studies showing inhibitory receptors are less environmentally influenced.CD56SP cells generated from the DN cells also had higher levels of bone marrow and lymph node homing receptors CXCR4 and CCR7 and possessed superior degranulation capacity against K562 cells compared to pre or post expanded DN or pre-expanded CD56SP cells (figure). Further, gene expression analysis comparing DN vs. CD56SP cells at day 0 and DN and CD56SP cells generated from DN cells at day 14, showed DN cells had a gene expression profile with enhanced expression of genes involved in processes such as cell division and replication, while CD56SP cells generated from DN cells had a more cytotoxic or "active phenotype".Conclusion: CD56- 16- DN cells share many characteristics with canonical NK cells, have long-term in vitro differentiation and proliferation capacity, and may represent a cell population that is a self-renewing progenitor for highly activated and cytotoxic CD56SP NK cells. Isolation and expansion of a progenitor population with long-term NK cell repopulating potential in adoptive NK cell immunotherapy trials could offer advantages over trials transferring more differentiated NK cells with limited in vivo proliferative capacity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

4PD - A universal killer T-cell for adoptive cell therapy of cancer

4PD - A universal killer T-cell for adoptive cell therapy of cancer

New horizons in target antigen selection in adoptive cell therapy of cancer

New horizons in target antigen selection in adoptive cell therapy of cancer

A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor.

Chimeric antigen receptors (CARs), which combine an antibody-derived binding domain (single chain fragment variable) with T-cell-activating signaling domains, have become a promising tool in the adoptive cellular therapy of cancer. Retro- and lenti-viral transductions are currently the standard methods to equip T cells with a CAR; permanent CAR expression, however, harbors several risks like uncontrolled auto-reactivity. Modification of T cells by electroporation with CAR-encoding RNA to achieve transient expression likely circumvents these difficulties. We here present a GMP-compliant protocol to activate and expand T cells for clinical application. The protocol is optimized in particular to produce CAR-modified T cells in clinically sufficient numbers under full GMP-compliance from late-stage cancer patients. This protocol allows the generation of 6.7 × 10(8) CAR-expressing T cells from one patient leukapheresis. The CAR-engineered T cells produced pro-inflammatory cytokines after stimulation with antigen-bearing tumor cells and lysed tumor cells in an antigen-specific manner. This functional capacity was maintained after cryopreservation. Taken together, we provide a clinically applicable protocol to transiently engineer sufficient numbers of antigen-specific patient T cells for use in adoptive cell therapy of cancer.

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T(H) lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.

T cell receptor (TCR)-transgenic CD8 lymphocytes rendered insensitive to transforming growth factor beta (TGFβ) signaling mediate superior tumor regression in an animal model of adoptive cell therapy

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.

29. Redirected Allogeneic T Cells for Adoptive Cell Therapy of Cancer

29. Redirected Allogeneic T Cells for Adoptive Cell Therapy of Cancer

231. Development of Genetically Engineered T Cells Expressing a Chimeric Antigen Receptor Against Prostate Stem Cell Antigen for the Adoptive Cell Therapy of Cancer

231. Development of Genetically Engineered T Cells Expressing a Chimeric Antigen Receptor Against Prostate Stem Cell Antigen for the Adoptive Cell Therapy of Cancer

White Paper on Adoptive Cell Therapy for Cancer with Tumor-Infiltrating Lymphocytes: A Report of the CTEP Subcommittee on Adoptive Cell Therapy

Adoptive T-cell therapy (ACT) using expanded autologous tumor-infiltrating lymphocytes (TIL) and tumor antigen-specific T cell expanded from peripheral blood are complex but powerful immunotherapies directed against metastatic melanoma. A number of nonrandomized clinical trials using TIL combined with high-dose interleukin-2 (IL-2) have consistently found clinical response rates of 50% or more in metastatic melanoma patients accompanied by long progression-free survival. Recent studies have also established practical methods for the expansion of TIL from melanoma tumors with high success rates. These results have set the stage for randomized phase II/III clinical trials to determine whether ACT provides benefit in stage IV melanoma. Here, we provide an overview of the current state-of-the art in T-cell-based therapies for melanoma focusing on ACT using expanded TIL and address some of the key unanswered biological and clinical questions in the field. Different phase II/III randomized clinical trial scenarios comparing the efficacy of TIL therapy to high-dose IL-2 alone are described. Finally, we provide a roadmap describing the critical steps required to test TIL therapy in a randomized multicenter setting. We suggest an approach using centralized cell expansion facilities that will receive specimens and ship expanded TIL infusion products to participating centers to ensure maximal yield and product consistency. If successful, this approach will definitively answer the question of whether ACT can enter mainstream treatment for cancer.

Development of Adoptive Cell Therapy for Cancer: A Clinical Perspective

Adoptive cellular therapy provides the promise of a potentially powerful general treatment for cancer. Although this is a complex and challenging field, there have been major advances in basic and translational research resulting in clinical trial activity that is now beginning to confirm this promise. However, these trials are also identifying new challenges and this review focuses on these clinical issues. For tumors such as melanoma, in which tumor-specific T cells can be readily identified and isolated, the adoptive transfer of "tumor-infiltrating lymphocytes" (TILs) already appears to offer significant patient benefit and this approach now warrants further development. Genetically engineered T cells offer a means to endow peripheral blood T cells with antitumor activity and in principle these techniques could allow the treatment of a wide range of cancers. Genetic engineering also offers the means to endow T cells with new properties and enhanced functions. There have been clear proof-of-principle trials showing responses with T cell receptor (TCR)-engineered T cells and this can be built on with further development. By contrast, other trials have produced significant toxicity related to expression of target antigen on normal tissue, particularly with enhanced receptors. The challenge ahead lies in understanding how to achieve the balance between targeted antitumor immune responses while avoiding toxicity associated with on-target destruction of antigen-expressing normal tissues. Cellular therapy of cancer will need continued preclinical evaluation as well as carefully designed clinical trials addressing the various issues. For these challenges to be fully assessed, and for progression to a widely used, effective and safe therapy, development as cooperative groups is an appropriate way forward.

Approach to Establish Long Term Anti-Acute Myeloid Leukemia (AML) Immune Responses by Inducing Central Memory AML Specific CD4 T Cells.

The efficacy of adoptive cell therapy of cancer and leukemia is often limited by the failure of cultured T cells, particularly cloned CD8+ T cells, to persist in vivo, and insight into the basis for the poor survival of the transferred cells is lacking. We previously reported a novel culture method that induces AML dendritic cell differentiation and primes in situ AML-reactive T cells (AMLDC culture) (Zhong et al. Exp Hematol 2008, 36:486). Highly reactive anti-AML T-cell lines were generated. These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. The culture procedure has been further optimized in this study. We found that by the addition of the TLR-4 agonist LPS,(1–100 ng/ml) in the last 24 hours of AMLDC culture (day 6), CD80, CD86, CD53, CD83 or HLA-Dr expression of AML cells pre-induced by cytokine combination GM-CSF/IL-4 or GM-CSF/IL4/IL2/IL7/IL12 could be significantly enhanced (n=6, P<0.005–0.04; n=8, P<0.0001–0.005 respectively). Addition of LPS increased the IFN-gamma secretion by T cells generated from AMLDC culture in response to autologous AML cells 4–10 fold (3 of 3; P<0.001). Addition of TNF-alpha, (10–20 ng/ml) in the last 24 hours of culture could also significantly enhance the expression of above surface molecules. However, LPS induced significantly higher expression of CD80 and CD86 on AMLDC compared with TNF-alpha. T cells generated from AMLDC culture with TNF-alpha led to differential effects on autologous AML reactivity. IFN-gamma secretion was enhanced in 4 of 7 studies and suppressed in 3 of 7. The significantly enhanced AML cell reactivity of autologous T cells generated from AMLDC culture by adding LPS was also demonstrated in limiting dilution AMLDC culture in 96 well plates. The majority of T cell lines selected and expanded from AMLDC culture with LPS were CD3+CD4+ (12 of 18), and 18 T cell lines tested expressed moderate to high level of CD62L, implying the central memory phenotype.Conclusions: Timely exposure of AMLDC culture to TLR-4 agonists, followed by T cell expansion, may promote the generation of AML-reactive T cells and differentiation toward the central memory phenotype. Theoretically, this should promote long-term maintenance and potential of regulation of both humoral and cellular immune responses against AML upon infusion of AML reactive autologous T cells derived from such cultures and may therefore enhance the therapeutic efficacy of these cells.

Strategy to augment the efficacy of immunotherapy for refractory breast cancer using trastuzumab combined adoptive cell therapy

3062 Background: Antitumor monoclonal antibody (mAb) is able to make molecular change in cancer cells and may also induce immune response via its Fc domain. CTLs selectively kill autologous tumor cells (AuTCs) is powerful for adoptive cell therapy of cancer. To investigate the immunological activity and anti-cancer effect in the combination therapy using the anti-HER2 Mab (trastuzumab, Tr) with adoptive cell therapy, a pilot clinical trial was performed for patients (pts) with refractory breast cancer. Methods: Fourteen pts with recurrent breast cancer were enrolled, they all had failed the conventional chemoradiotherapy. MLTC was performed using PBMC and primary- cultured AuTCs for 7–14 days. The generated cells were analyzed by Flow Cytometry, and the cytokine production and killing activity were evaluated by the ELISA and 51Cr-release assay. The cells were administrated by intra-peritumoral injection biweekly and Tr (2mg/kg) was infused every week. The treatments were repeated for 6 to 22 injections, and the total numbers of administered T cells were reached to ∼5.6 x 109. To assess the mechanism of Tr-effects, the antigen-loaded dendritic cells (DC) were examined in the phenotype, cytokine productions, and the ability to induce HER2 specific T cells in vitro. Results: After ∼ 2 weeks of MLTC, the derived cells, containing a preponderance of CD3+CD4+ or CD8+ T cells and CD56+ NK cells, showed significant degrees of cytotoxicity and IFN-γ production against AuTCs and HLA-matched allogenic tumor cell lines in 4 cases tested. The clinical response showed PR/SD/PD was 1/6/7 and adverse effect was tolerable. The tumor marker proteins (CEA, etc) were decreased in 5 pts. The carcinomatous pleural effusion was well controlled in 6 pts. In DC experiments, Tr-opsonized antigen-loaded DC enhanced the ability to induce CD8+ T cells specific for HER2-peptides in IFN-γ ELISPOT assay. Conclusions: Clinical responses were observed in pts with refractory breast cancer after receiving the adoptive cell therapy plus Tr. Our results suggested that the efficacy of immunotherapy might be augmented by anti-Her2 mAb, and it might be in part due to the involvement of mAb in the ability of DC cross-presentation followed by the enhancement of antitumor cellular immunity. No significant financial relationships to disclose.

Combination of gemcitabine and adoptive cell therapy of autologous anti-tumor CTL induces clinical activities in patients with refractory lung cancer

3053 Background: Cytotoxic T cells selectively kill autologous tumor cells is powerful for adoptive cell therapy of cancer. Gemcitabine (GEM) is able to induce molecular changes in cancer cells that make them to induce an antigen specific CTL response. This study was to evaluate the anti-tumor and the immunological activity using the CTL in combination with GEM. Methods: The 50Gy irradiated autologous tumor cells were cocultured with PBMCs and CTLs was developed with RPMI 1640 and rIL-2 (50 u/ml) for 7–14 days. Nine pts with non small cell lung cancer failed their prior chemotherapy were enrolled this pilot study. GEM regimen [intravenous (iv.) GEM (1000mg/m2) - day 1, 8 and 15 ] was started without CTL administration for 1st cycle. GEM was administered at least 2 cycles with a 1-week interval and combined with iv. CTL therapy (0.9 x 108 - 4.6 x 108 cells/injection + IL-2 0.4 MIU; biweekly for 6 to 12 injections) from 2nd cycle. The mean total administered T cells were reached to 3.9 x 109 - 5.6 x 109. PBMCs were analyzed their surface markers by Flow Cytometry and the cytokine productions of IFN-γ etc. in the serum were measured by ELISA before and after 1st cycle of GEM administration and 3rd cycles of CTL injection. Results: After finishing 2 cycles of GEM and 3 injections of CTL, the ratio of CD4/CD8 in PBMCs increased in 7/9 pts. In contrast, CD3/CD19 decreased in 6/9 pts. The cytokine production of IFN-γ in the serum revealed an increase after treatment, the levels of TGF-β were decreased simultaneously. There was no remarkable change in the levels of NKG2D in the PBMCs and MIG, IP10 in the serum. The clinical response showed PR/SD/PD was 2/5/2. The tumor marker proteins (CEA) were also decreased significantly in 4 of 9 pts. The adverse effects were tolerable with grade &lt;2 fever, nausea and fatigue and no bone marrow suppression was observed. Conclusions: These results suggested the synergistic enhancement of antitumor effect might be induced between CTLs and anti-cancer agent GEM. Marked clinical responses were observed in two pts after the treatment. Thus this chemo-immunotherapy will be applicable for the patients with refractory lung cancer. No significant financial relationships to disclose.

Ex vivo generation and expansion of autologous anti-tumor CTL lines for adoptive cell therapy in patients with solid tumors

12516 Background: Ex-vivo generation of cytotoxic T cell (CTL) lines able to selectively kill autologous tumor cells (TC) is crucial for adoptive cell therapy of cancer. We have described the ex vivo generation of anti-tumor HLA-restricted CTL using CD8-enriched blood mononuclear cells (PBMC) and dendritic cells, pulsed with apoptotic TC as source of tumor antigens (Montagna, Int J Cancer 2004). These cells possess a marked cytotoxicity against TC but very low levels of cytotoxicity against autologous non-malignant controls, suggesting their safe in vivo use. Aim of this study was to evaluate the possibility of generating a large number of anti-tumor CTL lines, in compliance with good manufacture procedure (GMP), to be utilized for adoptive cell therapy. Methods: Four patients with advanced, pretreated solid tumors have been evaluated: 2 sarcoma, 1 renal carcinoma and 1 ovarian carcinoma. Tumor sample was obtained during surgical procedures planned for therapeutic purposes, while PBMC were obtained by a single apheresis. All reagents employed for cultures were produced under GMP. Based on our reported method, the current protocol included two or three rounds of tumor-specific stimulation, followed by two rounds of antigen-independent expansion. Results: We have obtained, after the first round of expansion and in total, a range of 0.5–2×109 and 5–20×109 CTL, respectively. Such large amounts of anti-tumor CTL were generated even if a low number of viable TC was available. In 3 out of 4 anti-tumor CTL lines, &gt;85% of effector cells were CD3+/CD8+. Conclusions: We demonstrated the feasibility of obtaining, in GMP conditions, large quantities of anti-tumor specific CTL suitable for in vivo use. A phase I protocol of lymphoablative therapy followed by CTL transfer in vivo has been designed and submitted for approval to the competent regulatory agency. No significant financial relationships to disclose.

Effects of development of host immunity on the biodistribution of xenogeneic MHC non-restricted cytotoxic T cells: implications for adoptive cell therapy of cancer.

The human MHC non-restricted cytotoxic T cell line TALL-104 has potent anti-tumor effects in dogs with spontaneous tumors. This study was designed to examine the effects of the development of host immune responses on the baseline organ distribution of TALL-104 cells in healthy dogs. 111In-oxine labeled TALL-104 cells (107 cells/kg) were infused systemically in three dogs, either on day 1, 3, or 5 of a 5-day injection cycle; two dogs received two more injections of the labeled cells at monthly intervals, whereas the third dog received free 111In-oxine, 3 months after the first 5-day infusion. Analysis of blood and plasma cell clearances and imaging studies indicated a progressively faster clearance of the cells from the blood and organs after multiple daily injections as well as at the time of each monthly boost when host immune responses against the xenogeneic cells had developed. These findings have important therapeutic implications for the design of effective TALL-104 cell administration schedules in clinical trials.