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Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.

Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.

Six short term-cultured melanoma cell lines and one small cell lung cancer cell line were treated in vitro with the alkylating agent mafosfamide. The sensitivity of the surviving cells to in vitro lysis by recombinant interleukin 2-activated autologous and allogeneic lymphocytes was then investigated. In no case did chemo-surviving tumor cells appear less sensitive to lymphocyte-mediated lysis than untreated counterparts. In three of seven cases (two of which were derived from the same patient), chemo-selected cells were even more sensitive to cytotoxic lymphocytes, a difference not explained by a different distribution of neoplastic cells in the various cell cycle phases. We also studied the inhibitory activity of activated lymphocytes on the clonogenic potential of chemo-surviving tumor cells by the human tumor clonogenic assay. Inhibitions of tumor cell growth in the two patients tested were 100 and 94%, respectively; the activity of lymphocytes was dependent on the coculture time and the effector/target cell ratio. These data indicate that in vitro treatment with mafosfamide does not select cells resistant to the action of activated lymphocytes and that, given the right experimental conditions, these immune effectors can completely lyse tumor cells.

The sensitivity of three different human and murine doxorubicin (Dx)-sensitive or -resistant pairs of tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes was studied. In two pairs of these sublines (LoVo human colon carcinoma and B16 mouse melanoma sublines), resistance to Dx was induced in vitro, while in the third pair (9229 human metastatic melanoma clones), Dx resistance was spontaneously present in clone 9229.24. Dx-resistant cells were efficiently lysed by rIL2-activated lymphocytes in a short-term 51Cr release assay; in some experiments a trend toward higher lysis of Dx-resistant cells was present. We then tested the tumor cell growth-inhibitory activity of rIL2-activated lymphocytes in the human tumor clonogenic assay after lymphocyte-tumor coculture. Complete inhibition of tumor cell growth was obtained with five of six sublines or clones (both Dx sensitive and resistant) after 3 to 6 days of coculture at effector lymphocyte/target tumor cell ratios of 5 to 50/1; a maximum 99% inhibition was observed with the melanoma clone 9229.4 even after coculture for 6 days at an effector lymphocyte/target tumor cell ratio of 50/1. By using lower effector lymphocyte/target tumor cell coculture ratios (1, 5, 25/1), it was shown that all the three Dx-resistant cell types were significantly more affected by activated lymphocytes than their Dx-sensitive counterparts. The LoVo/DX subline was also more lysed than its Dx-sensitive counterpart LoVo/H subline by an antitumor monoclonal antibody in a complement-mediated cytotoxicity assay, despite the fact that both sublines expressed a similar amount of antigen on the cell surface. These data indicate that Dx-resistant cancer cells are more susceptible to the lysis by rIL2-activated lymphocytes than their Dx-sensitive counterparts and that a complete inhibition of their clonogenic potential can be obtained in vitro.

In this study 15 consecutive melanoma patients were treated with two courses of bolus recombinant interleukin 2 (rIL2) and rIL2 plus in vitro-generated lymphokine-activated killers (LAK), respectively. The immunological monitoring performed after 4 days of rIL2 or rIL2 plus LAK, indicate that the in vivo peripheral blood lymphocyte (PBL), activation (spontaneous proliferation, tumor cytotoxicity, number of DR+ PBL, obtained after the second cycle of rIL2 plus LAK is significantly higher than after the first cycle of rIL2 alone. During the 5-day interval between the two courses, PBL activation returns to baseline levels and no evidence for increased sensitivity of PBL to rIL2 is present. To further confirm this, two additional patients were studied, in whom rIL2 was administered by continuous i.v. infusion. In these two patients the in vitro versus in vivo PBL activation could be directly and simultaneously compared by using in vitro the same concentration of rIL2 reached and maintained in the patients' sera. The PBL activation induced in vivo by a cycle of rIL2 alone was significantly less (about 10 times) than that obtained in vitro with a comparable rIL2 concentration. Thus, the infusion of in vitro highly activated PBL could explain the increased in vivo lymphocyte activation of the second cycle of rIL2 plus LAK over the first cycle of rIL2 alone.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy—that is, the genetic induction of a conditional suicide phenotype into donor T cells—allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk+ T cells contained HA-1– and H-Y–specific CD8+ cytotoxic T cells (CTL) precursors. Thymidine kinase–positive HA-1– and H-Y–specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (Delta LNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.

International audience; From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 x 10(9) infectious particles per milliliter were obtained, generating up to 6 x 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.

In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT. In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT.

In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.