In vitro induction of a bladder cancer-specific T-cell response by mRNA-transfected dendritic cells.

PURPOSE: To design a tumor-specific immunotherapeutic strategy for treating tumors for which no specific antigens are described (such as bladder urothelial carcinoma), we attempted to activate tumor-specific T-cells by dendritic cells transfected with tumor-derived mRNA. METHODS: Dendritic cells were generated from a patient's peripheral blood and loaded with mRNA derived from the urothelial carcinoma tissue of the same patient. Autologous T-cells were incubated twice on these dendritic cells and tested for their ability to lyse tumor cells. RESULTS: Dendritic cells transfected with tumor-derived mRNA were able to activate T-cells that recognized autologous tumor cells. Cytotoxicity was around 26% for an effector:target ratio of 50:1. Tumor-infiltrating lymphocytes did not kill the autologous tumor cells in vitro, but after a single stimulation with the transfected dendritic cells, they induced tumor cell lysis of 35.7% at an effector:target ratio of 50:1. CONCLUSIONS: These results indicate that dendritic cells transfected with tumor mRNA containing messages for one or more tumor antigens could serve for the ex vivo activation of effector T-cells or directly as vaccines for a wide range of human neoplasias.

Tumour-induced suppression of immune response and its correction.

Immunosuppressive features of tumour cells are a major obstacle for immunotherapy of cancer. We recently noted that RENCA cells effectively interfere with the in vivo activation of RENCA-specific T cells. To unravel the underlying mechanism, we evaluated the influence of RENCA cells on a mixed-lymphocyte/ tumour reaction as well as an allogeneic mixed-lymphocyte reaction. We observed that RENCA cells were not directly immunosuppressive. Instead, they initiated deviation of an immune response in at least two independent directions: (i) expansion of a population of NK1.1+/CD3+ cells, which was accompanied by elimination of mainly CD4+ lymphocytes, and (ii) production of a leukocyte-derived inhibitory factor. Expression of the costimulatory molecule B7.1 by RENCA cells prevented induction of anergy, while expression of MHC class II molecules prevented expansion of NK1.1+ cells, which was accompanied by a significant decrease in cell death. Hence, an unimpaired response was observed only when RENCA cells expressed B7.1 plus MHC class II molecules. Thus, even if a tumour itself is not immunosuppressive, it can induce a strong deviation of the immune response. It is concluded that the first contact between elements of the immune system and the tumour cell can confer a severe bias on immunoregulatory circuits.

Recombinant chimeric OKT3 scFv IgM antibodies mediate immune suppression while reducing T cell activation in vitro.

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), is a potent immunosuppressive agent used in clinical transplantation to treat allograft rejection. Two major drawbacks of this therapy are the systemic release of several cytokines due to cross-linking mediated by the mAb between T cells and FcgammaR-bearing cells and the human anti-mouse antibody (HAMA) response. To overcome these side effects, three chimeric OKT3 single chain variable fragment (scFv) IgM antibodies, scOKT3-gamma DeltaIgM wt, scOKT3-gamma DeltaIgM C575S and scOKT3-gamma DeltaIgM VAEVD, were generated. They consist of the light and heavy variable binding domains of OKT3 mAb as well as the CH3 and CH4 domains of different human IgM variants linked with a human IgG3 hinge region to provide more flexibility and stability. Like the native IgM, scOKT3-gamma DeltaIgM antibodies are able to form polymeric structures, which lead to an increase in binding affinity and immunosuppressive potential compared with the parental OKT3 mAb. However, independently of their polymerization, all scOKT3-gamma DeltaIgM constructs do not induce any significant T cell proliferation or cytokine release (IL-2, TNF-alpha and IFN-gamma) in in vitro assays, while their CD3-modulating properties are retained. These results suggest that the use of scOKT3-gamma DeltaIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.

A cell surface-displayed anti-c-myc single-chain antibody: new perspectives for the genetic improvement of cellular tumor vaccines.

We have shown recently that cell surface-bound, single-chain Fv antibodies (scFv) are a powerful tool for the improvement of cellular tumor vaccines. To simplify this approach and to develop a general tool for the generation and improvement of cellular tumor vaccines, we chose an scFv against a peptide from the human proto-oncogene c-myc that could anchor any c-myc-tagged protein to the cell surface. The retroviral vector p50-Mx-neo (pMESV) was used to express scFv on the surface of the human melanoma line SkMel63. The cell-bound anti-c-myc scFv bound specifically to a soluble purified anti-CD28 scFv carrying a c-myc peptide-tag at its C terminus. Proof of principle was determined by incubating human peripheral blood lymphocytes with a mixture of (a) anti-c-myc-transfected SkMel63 cells binding the anti-CD28 scFv and (b) SkMel63 cells transfected with an anti-CD3 scFv. A clear synergistic effect on T-cell activation was observed that was comparable with that obtained in previous studies using SkMel63 cells transfected with the gene for the anti-CD28 scFv. As the cell surface-displayed anti-c-myc scFv can bind any c-myc-tagged protein of interest, this technique facilitates the genetic engineering of cellular vaccines for the therapy of virtually all human neoplasias.

In vitro and in vivo induction of a Th cell response toward peptides of the melanoma-associated glycoprotein 100 protein selected by the TEPITOPE program.

The melanoma-associated Ag glycoprotein 100 was analyzed by the T cell epitope prediction software TEPITOPE. Seven HLA-DR promiscuous peptides predicted with a stringent threshold were used to load dendritic cells (DC), and induction of a proliferative response was monitored. PBMC of all nine donors including two patients with malignant melanoma responded to at least one of the peptides. The proliferative response was defined as a Th response by the selective expansion of CD4(+) cells, up-regulation of CD25 and CD40L, and IL-2 and IFN-gamma expression. Peptide-loaded DC also initiated a T helper response in vivo (i.e., tumor growth in the SCID mouse was significantly retarded by the transfer of PBMC together with peptide-loaded DC). Because the use of the TEPITOPE program allows for a prediction of T cell epitopes; because the predicted peptides can be rapidly confirmed by inducing a Th response in the individual patient; and because application of peptide-loaded DC suffices for the in vivo activation of helper cells, vaccination with MHC class II-binding peptides of tumor-associated Ags becomes a feasible and likely powerful tool in the immunotherapy of cancer.

Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation.

To increase the valency, stability, and therapeutic potential of bispecific antibodies, we have constructed a tetravalent tandem diabody (Tandab) that is specific to both human CD3 (T-cell antigen) and CD19 (B-cell marker; S. M. Kipriyanov et al., J. Mol. Biol., 293: 41-56, 1999). It was generated by the functional dimerization of a single chain molecule that contained four antibody variable domains (V(H) and V(L)) in an orientation that prevented intramolecular pairing. Compared with a previously constructed heterodimeric CD3 x CD19 diabody, the Tandab exhibited a higher apparent affinity to both CD3+ and CD19+ cells and longer blood retention when injected into mice. Biodistribution studies in mice bearing Burkitt's lymphoma xenografts demonstrated specific accumulation of the radioiodinated Tandab in a tumor site with tumor-to-blood ratios of 1.5, 8.1, and 13.3 at 3, 18, and 24 h, respectively. Treatment of severe combined immunodeficiency mice bearing established Burkitt's lymphoma (5 mm in diameter) with human peripheral blood lymphocytes, Tandab, and anti-CD28 MAbs resulted in the complete elimination of tumors in all of the animals within 10 days. In contrast, mice receiving human peripheral blood lymphocytes in combination with either the diabody alone or the diabody plus anti-CD28 MAbs showed only partial tumor regression. These data demonstrate that the CD3 x CD19 Tandab may be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.

Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells.

The use of anti-CD3 x antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 x CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 x CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

Recombinant gp100 protein presented by dendritic cells elicits a T-helper-cell response in vitro and in vivo.

Induction of a helper T (TH)-cell response is a critical element in the generation of anti-tumor immunity. The majority of immunotherapeutic approaches have so far been concerned with the generation of cytotoxic T lymphocytes (CTLs). This also accounts for gp100, a melanoma-associated protein which induces a potent CTL response. Because of the high immunogenicity of gp100, we considered it of special interest to explore the feasibility of generating gp100-specific TH cells. Human dendritic cells (DCs) were loaded with recombinant gp100 protein, and the response of autologous TH cells was evaluated in vitro and in vivo. We have observed that gp100 peptides can be presented by DCs of certain MHC class II haplotypes, which led to proliferation and cytokine production of TH-1 cells in vitro. Furthermore, transfer of gp100 protein-loaded human DCs into SCID mice also induced proliferation of autologous, unprimed peripheral blood leukocytes (PBLs) and selective expansion of TH cells. When human T cells from the spleen of SCID mice were recovered and restimulated in vitro, they strongly proliferated in response to gp100-loaded DCs, while showing minimal proliferative activity in response to DCs loaded with a control antigen. Thus, it is possible to induce an efficient MHC class II-restricted TH response by in vitro stimulation or in vivo vaccination with DCs which have been loaded with a purified tumor-associated antigen.

Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (VHand VL) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3xCD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3(+)and CD19(+)cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19(+)cells in the presence of activated human PBL. Incubated in human serum at 37 degrees C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.

Apoptosis of a human melanoma cell line specifically induced by membrane-bound single-chain antibodies.

CD28 is a key regulatory molecule in T cell responses. Ag-TCR/CD3 interactions without costimulatory signals provided by the binding of B7 ligands to the CD28R appear to be inadequate for an effective T cell activation. Indeed, the absence of B7 on the tumor cell surface is probably one of the factors contributing to the escape of tumors from immunological control and destruction. Therefore, to increase the immunogenicity of tumor cell vaccines, we have expressed anti-CD3 and anti-CD28 single-chain Abs (scFv) separately on the surface of a human melanoma SkMel63 cell line (HLA-A*0201). A mixture of cells expressing anti-CD3 with cells expressing anti-CD28 resulted in a marked activation of allogeneic human PBL in vitro. The apparent induction of a Th1 differentiation pathway was accompanied by the proliferation of MHC-independent NK cells and MHC-dependent CD8+ T cells. PBL that had been cultured together with transfected SkMel63 tumor cells were able to specifically induce apoptosis in untransfected SkMel63 cells. In contrast, three other tumor cell lines expressing HLA-A*0201, including two melanoma cell lines, showed no significant apoptosis. These results provide valuable information for both adoptive immunotherapy and the generation of autologous tumor vaccines.

Human melanoma therapy in the SCID mouse: in vivo targeting and reactivation of melanoma-specific cytotoxic T cells by bi-specific antibody fragments.

The adoptive transfer of tumor-specific cytotoxic T cells (CTL) offers a promising perspective in cancer immunotherapy. However, the ex vivo-generated T lymphocytes are mostly IL-2-dependent. Here we explored the possibility of circumventing the requirement for IL-2, known for severe side effects in the patient, and of simultaneously targeting the CTL towards the tumor by the use of 2 bi-specific antibody fragments. As a model system, we used SCID mice bearing an s.c.-implanted human melanoma line (BLM-gp100) and in vitro-generated CTL specific for the gp100-derived immunogenic peptide YLEPGPVTA, which were injected i.v. with delay. To maintain the cytotoxic potential of the transferred CTL, 2 bi-specific antibody (biAb) fragments were generated which bound with one arm either CD3 or CD28, a combination known to support the activation of CTL. For targeting the CTL, both biAbs contained the F(ab') part of HD-Me13, an antibody recognizing p97, a non-immunogenic melanoma-associated surface molecule. In vitro and in vivo, the addition of the 2 biAbs increased the cytotoxic potential of the gp100-specific CTL and supported their clonal expansion in the absence of IL-2. Correspondingly, significantly higher numbers of CTL were recovered from melanoma-bearing SCID-mice that received the 2 biAb than from mice treated with the CTL only. In animals treated with CTL plus both biAbs, the primary tumor did not grow, and none of the mice developed metastases. Thus, this set of bi-specific antibody fragments was proved to target effector cells in the tumor-bearing host and to efficiently support in vivo clonal expansion and cytolytic activity of in vitro-generated CTL.

Antigen loss variants of a murine renal cell carcinoma: implications for tumor vaccination.

Vaccination with tumour cells genetically modified to support induction of an immune response either by production of cytokines or expression of co-stimulatory molecules provides a promising therapeutic approach. We have evaluated the efficiency of tumour vaccination using RENCA cells, a renal cell carcinoma of the BALB/c strain, which were stably transfected with MHC class II, B7.1 or both. Tumour growth after vaccination with MHC class II and/or B7.1 transfected RENCA cells was extremely variable, with protection close to 100% after vaccination with some clones and no effect of vaccination with others. To unravel the underlying mechanism, untransfected RENCA cells were cloned, and individual clones were tested for immunogenicity; that cloned RENCA cells varied considerably in immunogenicity. Whereas all clones displayed comparable growth rates in nude mice, some grew very slowly in immunocompetent syngenetic hosts. Vaccination with rapidly growing clones was ineffective and, importantly, this feature remained unaltered by vaccination with MHC class II and/or B7.1 transfected clones. Instead, 8 of 10 mice rejected the parental line after immunisation with a pool of MHC class II and B7.1 transfected clones. Finally, by cloning RENCA cells, we obtained one highly immunogenic clone (P2). Vaccination with this clone led to an individual-specific response, which indicates that during the cloning procedure a new strongly immunogenic entity must have arisen. Taken together, our results indicate that vaccination with MHC II and/or B7.1 transfected tumour cells induces an efficient immune response, but only if the tumour is weakly immunogenic. Since tumours may be composed of clones displaying different antigenicities, it is mandatory to use bulk cell populations for transfection and vaccination.

Stable expression of a retrovirally transferred adhesion molecule in a human melanoma-specific cytotoxic T lymohocyte clone.

The adoptive transfer of in vitro generated tumor-specific cytotoxic T lymphocytes (CTL) is considered a promising perspective in cancer therapy. One possible drawback lies in the inappropriate homing of in vitro cultured lymphocytes, which could be circumvented by introducing the appropriate targeting molecules. Here we describe a protocol that allows a rapid and stable transfection of cytotoxic T cell clones. As a model system we used a CTL clone specific for the melanoma-associated antigen gp100 and a cDNA encoding for murine CD14 containing the variant exen v10 which is supposed to facilitate lymphocyte homing towards the skin. CD44v10 cDNA was ligated into the retroviral vector pMV-7, which was used to transfect the ecotropic GP-E-86 and the amphotropic PA317 cells. After several cycles of transduction to increase the viral titre, supernatants of the amphotropic PA317-CD44v10 line were used for transduction of CD44v10 into a human CTL clone. After three cycles of transduction at 12-h intervals, low but stable expression of CD44v10 was observed throughout the culture period of 10 weeks. The phenotype of the transduced CTL clone was unaltered and the cytotoxic potential was only slightly reduced as compared to the parental clone. The efficiency of stable transduction within a period of 1 week makes the protocol well suited for the in vivo transfer of transduced cells and, in the special case, should guarantee appropriate homing of the transduced CTL clone.

Human neoplasms elicit multiple specific immune responses in the autologous host.

Expression of cDNA libraries from human melanoma, renal cancer, astrocytoma, and Hodgkin disease in Escherichia coli and screening for clones reactive with high-titer IgG antibodies in autologous patient serum lead to the discovery of at least four antigens with a restricted expression pattern in each tumor. Besides antigens known to elicit T-cell responses, such as MAGE-1 and tyrosinase, numerous additional antigens that were overexpressed or specifically expressed in tumors of the same type were identified. Sequence analyses suggest that many of these molecules, besides being the target of a specific immune response, might be of relevance for tumor growth. Antibodies to a given antigen were usually confined to patients with the same tumor type. The unexpected frequency of human tumor antigens, which can be readily defined at the molecular level by the serological analysis of autologous tumor cDNA expression cloning, indicates that human neoplasms elicit multiple specific immune responses in the autologous host and provides diagnostic and therapeutic approaches to human cancer.