Björn Cochlovius

Treatment of Human B Cell Lymphoma Xenografts with a CD3 × CD19 Diabody and T Cells

The use of anti-CD3 × 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 CD3ε 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 × 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 × CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma.

To target NK cells against non-Hodgkin’s lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcγRIII (CD16). Bacterially produced CD19 × CD16 BsDb specifically interacted with both CD19+ and CD16+ cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 × CD16 BsDb with a previously described CD19 × CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt’s lymphoma (5 mm in diameter) with human PBLs, CD19 × CD16 BsDb, CD19 × CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.

Effect of Domain Order on the Activity of Bacterially Produced Bispecific Single-chain Fv Antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.

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 patients 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.

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-γ 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.

Oral DNA vaccination: antigen uptake and presentation by dendritic cells elicits protective immunity

Melanoma differentiation antigens, such as glycoprotein 100 (gp100), have been shown to induce both cellular and humoral immune responses against melanoma in mouse and man. They are therefore considered as potential targets for melanoma immunotherapy. In this study, we have used the attenuated auxotrophic mutant strain SL7207 of Salmonella typhimurium as vehicle for a human gp100 (hgp100) DNA vaccine against melanoma. In vitro studies indicate that Salmonella/pCMV-hgp100 is efficiently scavenged by dendritic cells, resulting in the expression of the hgp100 transcription unit in the DC. In addition, oral administration of Salmonella/pCMV-hgp100 results in the expression of hgp100 RNA and protein by cells exhibiting DC-morphology in mesenteric lymph nodes as soon as 3 days after vaccination. Analysis of the efficacy of the Salmonella/pCMV-hgp100 vaccine in the B16/hgp100 model demonstrated the induction of strong anti-hgp100 CTL responses and protective immunity in 70% of the vaccinated mice, but not in control mice. Based on these data, we consider S. typhimurium as a useful vehicle for the design of recombinant DNA based anti-cancer vaccines.

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 FcγR‐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‐γ ΔIgM wt, scOKT3‐γ ΔIgM C575S and scOKT3‐γ ΔIgM 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‐γ ΔIgM 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‐γ ΔIgM constructs do not induce any significant T cell proliferation or cytokine release (IL‐2, TNF‐α and IFN‐γ) in in vitro assays, while their CD3‐modulating properties are retained. These results suggest that the use of scOKT3‐γ ΔIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.

Tumour-induced suppression of immune response and its correction.

Abstract 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.

Combined effect of recombinant CD19 x CD16 diabody and thalidomide in a preclinical model of human B cell lymphoma.

Combining different treatment strategies offers the possibility of improving treatment results for cancer patients. The aim of our study was therefore to investigate the combination of treatment of established s.c. human B non-Hodgkins lymphoma in severe immune deficient mice using a recombinant bispecific CD19×CD16 diabody (targeting natural killer cells to CD19+ cells) and the angiogenesis inhibitor thalidomide. Monotherapy with either thalidomide or diabody caused an approximate 50% reduction in tumor growth rate. The combined treatment showed evidence for a synergistic effect resulting in a 74% reduction in median tumor size. In the combined treatment group, two of five animals had complete remissions of their s.c. tumor. These results suggest that a combination treatment with recombinant diabodies and angiogenesis inhibition represents a useful approach in cancer therapy.

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. Int. J. Cancer 77:114–122, 1998.© 1998 Wiley‐Liss, Inc.