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Abstract:  Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers [10, 2]. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL17-1A-VH17-1A-VHCD3-VLCD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkably, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor α, interleukin-6 and interferon γ. Maximal cytotoxicity (51Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8+ as well as CD4+ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8+ cells induced high 51Cr release within 4 h while CD4+ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in virto.

MT103 is a bispecific single-chain (anti-CD19/anti-CD3) antibody derivate designed to link B-cells and T-cells. Preclinical studies have shown that MT103 can activate human T cells resulting in complete lysis of CD19-positive human B cells at very low E:T ratios. The primary objective of this study was to evaluate the safety and tolerability of a continuous intravenous infusion of MT103 at different dose levels in patients with relapsed NHL requiring therapy and not eligible for curative therapy. Secondary objectives were to assess PK, pharmacodynamics and anti-tumor activity of MT103. A classical phase I dose escalation design was used with an initial MT103 dose of 0.5 μg/m2/24h and assessment of dose limiting toxicity (DLT) during the first 2 weeks of infusion. An additional treatment cycle of MT103 for four weeks was offered to patients if they showed stable disease (SD) or response at the first tumor assessment after 4 weeks. Up to now, 21 patients with relapsed NHL, who had previously received a median of four different regimens of chemo- and/or immune therapy, have been included. From Dose Level (DL)1 up to DL3 (5 μg/m2/24h) no DLT was observed. At DL4 (5 μg/m2/24h on day 1, 15 μg/m2/24h maintenance dose), 7 patients were treated, of whom 2 received less than 14 days of treatment. One patient experienced elevation of liver enzymes up to CTC grade 3 after 2 weeks, which recurred upon re-administration of MT103; and 1 patient experienced confusion/disorientation on the 2nd treatment day, which was assessed as DLT. Based on pharmacodynamic data the dose step from 5 to 15 μg/m2/24h (DL4) was modified to a continuous dose increase. So far, 2 patients have passed the DLT period of this modified administration of DL4. Overall, the most frequent related AE’s in DL1-4 were lymphopenia in 11 patients, leukopenia in 10 patients, elevation of liver enzymes and fever in 7 patients The most frequent ≥ Grade 3 AE’s were lymphopenia in 11 patients and leukopenia in 6 patients. There has been no evidence for dose-related toxicity so far. Depletion of circulating B cells was observed in 9 of 15 evaluable patients (treatment for >2 weeks and detectable peripheral B cells prior to treatment) that reached full depletion in 100% of patients at DL4. At DL4, 3 patients had bone marrow infiltration (>10%). One patient showed reduction of and 2 patients complete disappearance of lymphoma cells from bone marrow. Best overall tumour response in the 17 evaluable patients (treatment >2 weeks and assessment of all involved areas) was 1 CR (at DL4), 2 PR’s (at DL4), 1 MR, 10 SD’s and 3 PD’s. Four patients had a stabilisation over at least 6 months as assessed per follow-up questionnaire with one patient at DL2 having ongoing SD for 17.5 months up to now. Conclusions: The current data suggest that MT103 administered as continuous 4 to 8 week infusion appears to be well tolerated by patients with relapsed NHL. MT103 exerts biological activity as demonstrated by B-cell depletion, T-cell activation, and clinical activity as demonstrated by objective responses. The optimal treatment schedule and the optimal biological dose will be further assessed on the basis of (clinical) response and pharmacodynamic data from the ongoing dose escalation.

MT103 is a bispecific single-chain (anti-CD19/anti-CD3) antibody derivate designed to link B-cells and T-cells. Preclinical studies have shown that MT103 can activate human T cells resulting in complete lysis of CD19-positive human B cells at very low E:T ratios. The primary objective of this study was to evaluate the safety and tolerability of a continuous intravenous infusion of MT103 at different dose levels in patients with relapsed NHL requiring therapy and not eligible for curative therapy. Secondary objectives were to assess PK, pharmacodynamics and anti-tumor activity of MT103. A classical phase I dose escalation design was used with an initial MT103 dose of 0.5 μg/m2/24h and assessment of dose limiting toxicity (DLT) during the first 2 weeks of infusion. An additional treatment cycle of MT103 for four weeks was offered to patients if they showed stable disease (SD) or response at the first tumor assessment after 4 weeks. Up to now, 21 patients with relapsed NHL, who had previously received a median of four different regimens of chemo- and/or immune therapy, have been included. From Dose Level (DL)1 up to DL3 (5 μg/m2/24h) no DLT was observed. At DL4 (5 μg/m2/24h on day 1, 15 μg/m2/24h maintenance dose), 7 patients were treated, of whom 2 received less than 14 days of treatment. One patient experienced elevation of liver enzymes up to CTC grade 3 after 2 weeks, which recurred upon re-administration of MT103; and 1 patient experienced confusion/disorientation on the 2ndtreatment day, which was assessed as DLT. Based on pharmacodynamic data the dose step from 5 to 15 μg/m2/24h (DL4) was modified to a continuous dose increase. So far, 2 patients have passed the DLT period of this modified administration of DL4. Overall, the most frequent related AE's in DL1-4 were lymphopenia in 11 patients, leukopenia in 10 patients, elevation of liver enzymes and fever in 7 patients The most frequent ≥ Grade 3 AE's were lymphopenia in 11 patients and leukopenia in 6 patients. There has been no evidence for dose-related toxicity so far. Depletion of circulating B cells was observed in 9 of 15 evaluable patients (treatment for >2 weeks and detectable peripheral B cells prior to treatment) that reached full depletion in 100% of patients at DL4. At DL4, 3 patients had bone marrow infiltration (>10%). One patient showed reduction of and 2 patients complete disappearance of lymphoma cells from bone marrow. Best overall tumour response in the 17 evaluable patients (treatment >2 weeks and assessment of all involved areas) was 1 CR (at DL4), 2 PR's (at DL4), 1 MR, 10 SD's and 3 PD's. Four patients had a stabilisation over at least 6 months as assessed per follow-up questionnaire with one patient at DL2 having ongoing SD for 17.5 months up to now.