Torsten Dreier

Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody.

A recent study reported on an anti‐CD19/anti‐CD3 single‐chain bispecific antibody (bscCD19xCD3) exhibiting high activity against human B lymphoma cell lines (Löffler et al., Blood 2000;95:2098–103). In the present study, we have explored in detail the in vitro efficacy, T‐cell donor variability, binding characteristics, specificity, kinetics and interleukin‐2 (IL‐2) dependence of bscCD19xCD3. We found that a majority of human donor T cells tested (n = 86) gave half‐maximal B‐lymphoma cell lysis (ED50) within a range of 10–50 pg/ml bscCD19xCD3, corresponding to sub‐picomolar concentrations of the bispecific antibody. Under identical experimental conditions, the anti‐CD20 monoclonal antibody rituximab had an at least 100,000‐fold lower in vitro efficacy. The extreme potency of bscCD19xCD3 was in sharp contrast to the relatively low affinity of the anti‐CD3 and anti‐CD19 single‐chain Fv portions in KD ranges of 10−7 and 10−9 M, respectively. Cell lysis by bscCD19xCD3 was predominantly mediated by the population of CD8/CD45RO‐positive T cells. Both immortalized CD4‐ and CD8‐positive human T‐cell clones were highly active effector cells as well. Cell lysis by bscCD19xCD3 was rapid and specific. The respective parental monoclonal antibodies inhibited cell lysis and CD19‐negative cells were not harmed by T cells in the presence of high amounts of bscCD19xCD3. The potent T‐cell stimulus IL‐2 could not markedly augment the activity of bscCD19xCD3‐stimulated T cells. In conclusion, bscCD19xCD3 could redirect unstimulated cytotoxic T cells against CD19‐positive cells in an unexpectedly potent, rapid and specific fashion.

T Cell Costimulus-Independent and Very Efficacious Inhibition of Tumor Growth in Mice Bearing Subcutaneous or Leukemic Human B Cell Lymphoma Xenografts by a CD19-/CD3- Bispecific Single-Chain Antibody Construct

We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.

In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment

In our study, a novel, fully human, recombinant monoclonal antibody of the IgG1 isotype, called MT201, was characterized for its binding properties, complement‐dependent (CDC) and antibody‐dependent cellular cytotoxicity (ADCC), as well as for its in vivo antitumor activity in a nude mouse model. MT201 was found to bind its target, the epithelial cell adhesion molecule (Ep‐CAM; also called 17‐1A antigen, KSA, EGP‐2, GA733‐2), with low affinity in a range similar to that of the clinically validated, murine monoclonal IgG2a antibody edrecolomab (Panorex®). MT201 exhibited Ep‐CAM‐specific CDC with a potency similar to that of edrecolomab. However, the efficacy of ADCC of MT201, as mediated by human immune effector cells, was by 2 orders of magnitude higher than that of edrecolomab. Addition of human serum reduced the ADCC of MT201 while it essentially abolished ADCC of edrecolomab within the concentration range tested. In a nude mouse xenograft model, growth of tumors derived from the human colon carcinoma line HT‐29 was significantly and comparably suppressed by MT201 and edrecolomab. The fully human nature and the improved ADCC of MT201 with human effector cells will make MT201 a promising candidate for the clinical development of a novel pan‐carcinoma antibody that is superior to edrecolomab.

EFFICIENT TUMOR CELL LYSIS BY AUTOLOGOUS, TUMOR-RESIDENT T LYMPHOCYTES IN PRIMARY OVARIAN CANCER SAMPLES BY AN EP-CAM-/CD3-BISPECIFIC ANTIBODY

The epithelial cell adhesion molecule (Ep‐CAM) is expressed on the surface of most human carcinomas, including ovarian, breast, lung, prostate and colorectal carcinoma. Ep‐CAM was shown to be a valid target for monoclonal antibody‐based therapies. We have investigated whether an Ep‐CAM‐/CD3‐bispecific single‐chain antibody called bscEp‐CAM × CD3 is effective in tumor cell elimination within the cellular microenvironment of primary ovarian cancer tissue. The ex vivo elimination of ovarian cancer cells in tumor preparations from 21 patients was monitored by flow cytometry using Ep‐CAM/CA‐125 double‐labeling or Ep‐CAM single‐labeling combined with propidium iodide uptake of cells. Methodology was established by the ovarian cancer cell line OvCAR. A total of 17 (81%) patient samples showed a dose‐dependent tumor cell elimination by bscEp‐CAM × CD3. High and specific tumor cell lysis was seen at bscEp‐CAM × CD3 concentrations as low as 1 ng/ml, at very low effector:target ratios and in the absence of T cell costimulation. The high efficacy of the bispecific antibody may be due to the non‐restricted activation of tumor‐resident cytotoxic T lymphocytes. In clinical trials, the ex vivo data with the T cell‐recruiting bispecific antibody bscEp‐CAM × CD3 may translate into a high response rate and efficacy of tumor cell elimination.

CTL-Assays for Functional Testing of Bispecific Antibody Fragments

Bispecific antibody fragments, designed as therapeutic agents for the treatment of neoplastic diseases, usually combine the binding regions of antibodies against a specific antigen on the target cell with an antibody binding site against cell surface molecules on effector cells of the immune system such as NK cells or T lymphocytes. The establishment of appropriate in vitro assays for the determination of target cell lysis by the effector cells, activated by these molecules, are absolutely crucial for the functional testing. The choice of this assay mainly depends on the nature and availability of the target cells or the technical equipment available (e.g. the opportunity to work with radioactively labelled material).