Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model.

BACKGROUND: Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model. METHODS: We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated. RESULTS: Despite its low affinity of approximately 10(7) M(-1) for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC(50) of 70 ng/ml [0.47 nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4(+) and CD8(+) T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced. CONCLUSION: Our data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.

Cancer therapy with trifunctional antibodies: linking innate and adaptive immunity.

Trifunctional antibodies (trAbs) are promising novel anticancer biologics with a particular mode of action capable of linking innate with adaptive immunity. Based on their unique structure, trifunctional IgG-like heterodimeric antibodies, consisting of nonhuman mouse and rat immunoglobulin halves are able to redirect T lymphocytes, as well as accessory cells, to the tumor site. This recruitment of immune cells is accompanied by cellular activation events elicited by anti-CD3, as well as Fcγ-receptor engagement of trAbs supported by a proinflammatory Th1-biased cytokine milieu. All necessary immunological factors required for long-term vaccination-like effects are stimulated along trAb-mediated therapeutic interventions. Thus, the concerted interplay of antibody-dependent cellular cytotoxicity plus the polyclonal T-cell cytotoxicity and Fcγ-receptor-driven induction of long-lasting immune responses after the initial tumor cell elimination represent the major hallmarks of trAb-mediated treatment of malignant diseases.

Immunomonitoring results of a phase II/III study of malignant ascites patients treated with the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3).

Patients with malignant ascites secondary to primary carcinomas benefit from intraperitoneal therapy with the trifunctional antibody catumaxomab (anti-EpCAM × anti-CD3). Here, we report the analysis of peritoneal fluid samples from 258 patients with malignant ascites randomized to catumaxomab or control groups to investigate the molecular effects of catumaxomab treatment. In the catumaxomab group, tumor cell numbers and peritoneal levels of VEGF decreased, whereas the activation status of CD4(+) and CD8(+) T-cell populations increased more than two-fold after treatment. Notably, CD133(+)/EpCAM(+) cancer stem cells vanished from the catumaxomab samples but not from the control samples. In vitro investigations indicated that catumaxomab eliminated tumor cells in a manner associated with release of proinflammatory Th1 cytokines. Together, our findings show that catumaxomab therapy activates peritoneal T cells and eliminates EpCAM(+) tumor cells, establishing a molecular and cellular basis to understand in vivo efficacy within the immunosuppressed malignant ascites tissue microenvironment.

Structural and functional characterization of the trifunctional antibody catumaxomab.

The Triomab family of trifunctional, bispecific antibodies that maintain an IgG-like shape are novel tumor targeting agents. These chimeras consist of two half antibodies, each with one light and one heavy chain, that originate from parental mouse IgG2a and rat IgG2b isotypes. This combination allows cost-effective biopharmaceutical manufacturing at an industrial scale since this specific mouse/rat isotype combination favors matching of corresponding antibody halves during production by means of quadroma technology. Whereas every Triomab family member is composed of an anti-CD3 rat IgG2b half antibody for T cell recognition, the antigen binding site presented by the mouse IgG2a isotype is exchangeable. Several Triomab antibodies have been generated that bind to tumor-associated antigens, e.g., EpCAM (catumaxomab), HER2/neu (ertumaxomab), CD20 (FBTA05), gangliosides GD2/GD3 (Ektomun), on appropriate tumor target cells associated with carcinomas, lymphomas or melanomas. Catumaxomab (Removab) was launched in Europe for treatment of malignant ascites in April 2009. Here, we report the structural and functional characterization of this product. Mass spectrometry revealed an intact mass of 150511 Dalton (Da) and 23717 Da, 24716 Da, 51957 Da and 52019 Da of the reduced and alkylated rat light chain, mouse light chain, rat heavy chain, mouse heavy chain chains, respectively. The observed masses were in agreement with the expected masses based on the amino acid sequence obtained from cDNA sequencing. The glycosylation profile was similar to other human IgG consisting of biantennary oligosaccharides with different numbers of terminal galactose. CD spectroscopy showed mainly beta-sheets secondary structure that is typical for IgG antibodies. Binding measurement revealed the unique trifunctional features of catumaxomab. Other analytical tools were used to evaluate characteristics of catumaxomab preparations, including the presence of isoforms and aggregates.

Advanced antigen delivery of murine survivin: chimeric virus-like particles in cancer vaccine research.

Success in cancer immunotherapy depends on the identification and efficient targeting of specific tumor-associated antigens. Two pivotal strategies to prime patients' immune system against malignant cells are tumor-specific adoptive T-cell therapy and tumor-specific vaccination. Here, we will focus on immunotherapeutic vaccination and discuss the advantages and disadvantages of different strategies to deliver tumor-specific T-cell epitopes. A particular focus will be put on virus-like particles (VLPs) as vehicle to deliver tumor-specific epitopes in the context of full-length proteins, as multi-epitope constructs or as individual tumor-associated T-cell epitopes. VLPs represent non-infectious and non-replicating antigen delivery systems devoid of any nucleic acid. They constitute innovative immunotherapeutic agents against cancer due to their superior, adjuvant-like antigenicity. We will present various tumor-associated antigens currently in different stages of development including survivin, as promising candidates for targeted tumor therapies.

Murine polyomavirus-like particles induce maturation of bone marrow-derived dendritic cells and proliferation of T cells.

We analysed the effects of murine polyomavirus-like particles (PLPs) on bone marrow-derived dendritic cells (BMDCs) and T cells in vitro. BMDCs activated with PLPs up-regulated CD40, CD80, CD86 and major histocompatibility complex (MHC) class II surface markers and produced proinflammatory cytokines. Chimeric PLPs [expressing the ovalbumin (OVA)-peptides OVA(257-264) or OVA(323-339)], but not wildtype PLPs, activated OVA-specific CD8 T cells and OVA-specific CD4 T cells, respectively, indicating both MHC class I and II presentation of the peptides by antigen-presenting cells. Our results suggest that PLPs may be used as vaccine adjuvants priming dendritic cells to induce potent T cell responses.

Development of a vaccine marker technology: display of B cell epitopes on the surface of recombinant polyomavirus-like pentamers and capsoids induces peptide-specific antibodies in piglets after vaccination.

Highly immunogenic capsomers (pentamers) and virus-like particles (VLPs) were generated through insertion of foreign B cell epitopes into the surface-exposed loops of the VP1 protein of murine polyomavirus and via heterologous expression of the recombinant fusion proteins in E. coli. Usually, complex proteins like the keyhole limpet hemocyanin (KLH) act as standard carrier devices for the display of such immunogenic peptides after chemical linkage. Here, a comparative analysis revealed that antibody responses raised against the carrier entities, KLH or VP1 pentamers, did not significantly differ up to 18 weeks, demonstrating the highly immunogenic nature of VP1-based particulate structures. The carrier-specific antibody response was reproducibly detected in the meat juice after processing. More importantly, chimeric VP1 pentamers and VLPs carrying peptides of 12 and 14 amino acids in length, inserted into the BC2 loop, induced a strong and long-lasting humoral immune response against VP1 and the inserted foreign epitope. Remarkably, the epitope-specific antibody response was only moderately decreased when VP1 pentamers were used instead of VLPs. In conclusion, we identified polyomavirus VP1-based structures displaying surface-exposed immunodominant B cell epitopes as being an efficient carrier system for the induction of potent peptide-specific antibodies. The application of this approach in vaccine marker technology in livestock holding and the meat production chain is discussed.

Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma.

Polyomavirus-like-particles (PLPs) are empty, non-replicative, non-infectious particles that represent a potent antigen-delivery system against malignant disease. Protective anti-tumour immunity can be induced under therapy conditions by subcutaneous (s.c.) treatment with particulate antigenic structures like chimerical polyomavirus-pentamers (PPs). These PPs displaying an immunodominant H-2Kb-restricted ovalbumin (OVA)257-264 epitope evoked nearly complete tumour remission in MO5 (B16-OVA) melanoma-bearing C57BL/6 mice by two s.c. applications in a weekly interval. The immunotherapeutic intervention started at day 4 after melanoma implant. Furthermore, 40% of melanoma-bearing mice vaccinated with heterologous PPs carrying a H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitope derived from of tyrosinase-related protein 2 (TRP2) survived similar treatment conditions. However, a late immunotherapeutic onset at day 10 post melanoma inoculation revealed no significant differences between the therapeutic values (40-60% survival) of VP1-OVA252-270 and VP1-TRP2180-192 PPs, respectively. These experiments underlined the capacity of PPs to break T cell tolerance against a differentially expressed self-antigen. As a correlate for preventive and therapeutic immunity against MO5 melanoma the number of OVA257-264- or TRP2180-188-specific CD8 T cells were significantly increased within the splenocyte population of treated mice as measured by H-2Kb-OVA257-264-PE tetramer staining or appropriate ELISPOT assays, respectively. These results reveal that heterologous PLPs and even chimerical PPs represent highly efficient antigen carriers for inducing CTL responses underlining their potential as immunotherapeutics against cancer.

Blending of a conventional Mycoplasma hyopneumoniae vaccine with a positive marker: tracking of immunised pigs by peptide-specific antibodies raised to the marker component.

Highly immunodominant marker antigens simply blended to existing veterinary vaccines may represent a smart approach for addressing the still open issue of vaccination compliance. This approach was evaluated by blending a widely deployed Mycoplasma hyopneumoniae vaccine with a peptide-KLH (Keyhole Limpet Hemocyanin) conjugate as marker. Piglets were vaccinated twice with: (i) a combination of the M. hyopneumoniae-specific vaccine and the marker, (ii) M. hyopneumoniae-specific vaccine, (iii) marker alone or (iv) placebo dose only. All piglets which received the M. hyopneumoniae-specific vaccine/marker formulation or, as control, the marker blended with Montanide IMS1313 adjuvant responded to the respective immunisation from day 21 to 77 post vaccination as seropositive for the appropriate peptide and KLH. However, the responder rate to M. hyopneumoniae of piglets administered with M. hyopneumoniae-specific vaccine/marker was slightly reduced at day 35 and 49 post immunisation in comparison with piglets vaccinated with M. hyopneumoniae-specific vaccine alone. Accordingly, we conclude that this marker technology could be successfully applied to label a whole set of vaccines prevented that the blending process will be optimised.