Markus Jensen

Fludarabine in Combination With Alemtuzumab Is Effective and Feasible in Patients With Relapsed or Refractory B-Cell Chronic Lymphocytic Leukemia: Results of a Phase II Trial

PURPOSE To determine the efficacy and safety of a newly developed concomitant administration of fludarabine and alemtuzumab (FluCam) in patients with relapsed or refractory B-cell chronic lymphocytic leukemia (B-CLL). PATIENTS AND METHODS A total of 36 patients were treated in this phase II study (median age, 61.47 years; mean number of prior chemotherapies, 2.6; Binet stage C, n = 28). After an initial dose escalation of alemtuzumab over 3 days, alemtuzumab 30 mg and fludarabine 30 mg/m2 were administered on 3 consecutive days. Treatment was repeated after 28 days for up to six cycles. Restaging (following National Cancer Institute criteria) was carried out after cycles 2 and 4 and 1 month after the end of treatment. RESULTS The overall response rate was 83% (11 complete responses, 19 partial responses, one stable disease, and five progressive diseases). Two patients with progressive disease developed fungal pneumonias, and one patient died as a result of Escherichia coli sepsis. Two subclinical cytomegalovirus reactivations occurred. CONCLUSION The new FluCam regimen is effective and feasible in patients with relapsed and refractory B-CLL.

Rapid tumor lysis in a patient with B-cell chronic lymphocytic leukemia and lymphocytosis treated with an anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab)

SummaryIn this report we present a patient with B-cell chronic lymphocytic leukemia who developed an acute tumor lysis syndrome after administration of the human anti-CD20 antibody IDEC-C2B8 (RITUXIMAB) in standard dose of 375 mg/m2. IDEC-C2B8 has been demonstrated to have only mild and tolerable side effects in patients with follicular lymphoma. In these trials patients with lymphocytosis >5000/μl were excluded. Physicians must be aware of this hitherto unreported phenomenon in patients with high CD20-positive blood counts.

Specific activation of resting T cells against CA19-9+ tumor cells by an anti-CD3/CA19-9 bispecific antibody in combination with a costimulatory anti-CD28 antibody.

Summary Specific activation of resting lymphocytes for tumor targeting can be achieved by bispecific monoclonal antibodies (bi-mAb) with specificity for tumor antigens and T-cell-activating antigens, respectively, in combination with a costimulatory anti-CD28 antibody. We describe the generation and function of a bi-mAb with specificity for CD3 and for the tumor antigen CA19–9. The bi-mAb OKT3/NSI19–9 was generated by somatic fusion of two hybridoma lines secreting antibodies against CA19–9 and CD3, respectively. A hybrid/hybridoma was established, and its bi-mAb was characterized. In combination with a costimulatory anti-CD28 mAb resting peripheral lymphocytes could be activated specifically with T-cell proliferation and secretion of high amounts of interferon-γ. On specific T-cell activation, bi-mAb OKT3/NSI19–9 could also redirect the cytotoxic effects of these T cells toward CA19–9+ tumor cells in vitro. Our results indicate that specific activation of resting T cells with bi-mAb OKT3/NSI19–9 in combination with an anti-CD28 mAb can activate resting T cells specifically and leads to antigen-dependent bi-mAb-mediated cytotoxicity against CA19–9+ target cells. This approach may offer new perspectives for the specific immunotherapy of CA19–9+ tumors.

Specific activation of resting T cells against tumour cells by bispecific antibodies and CD28-mediated costimulation is accompanied by Th1 differentiation and recruitment of MHC-independent cytotoxicity

Specific activation of resting lymphocytes for tumour targeting can be achieved by bispecific monoclonal antibodies (bi‐MoAbs) with specificity for tumour antigens and T cell‐activating antigens in combination with a costimulatory anti‐CD28 antibody. In this study we focus on the immunomodulatory function of an anti‐CD3/CA19‐9 bi‐MoAb in combination with a costimulatory anti‐CD28 antibody which may result not only in antigen‐specific, T cell‐mediated tumour cell lysis but also in recruitment of other cellular effector functions. In combination with costimulatory anti‐CD28 antibodies, resting peripheral lymphocytes could be activated specifically to secrete high amounts of Th1 cytokines (IL‐2, interferon‐gamma (IFN‐γ)) characterizing a cellular immune response. In contrast, no IL‐4 and only low amounts of IL‐10 could be detected. Furthermore, bi‐MoAb‐mediated CA19‐9‐specific activation of T cells was accompanied by recruitment of MHC‐ and CA19‐9‐independent cytotoxicity, as was determined by lysis of different CA19‐9−cell lines. This MHC‐independent cytotxicity was mediated at least in part by activated natural killer (NK) cells, as depletion of CD16+ NK cells resulted in substantial decrease of cytotoxicity against CA19‐9− targets. Our results indicate that specific activation of resting T cells with CD3‐associated bi‐MoAbs in combination with an anti‐CD28 antibody leads to a Th1 differentiation pathway and is accompanied by recruitment of MHC‐independent lymphokine‐activated killer (LAK) cell cytotoxicity which can possibly be directed against a heterogeneous tumour.

The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis.

To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell‐based immunotherapy we have generated a bi‐specific CD3 × NCAM antibody (OE‐1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE‐1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)‐derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC‐ derived T cells were activated by OE‐1, NK cells were almost completely depleted, suggesting that T cells activated by OE‐1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7– effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour‐directed cytotoxicity was enhanced when NK cells were present during preactivation with OE‐1. These data strongly support a bi‐phasic therapeutic concept of primarily stimulating T cells with the bi‐specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis.

One step generation of fully chimeric antibodies using Cgamma1- and Ckappa mutant mice.

Humanized antibodies (Abs) are effective drugs against a variety of diseases such as cancer, autoimmune diseases, transplant rejection and others. The most powerful technology to develop humanized Abs is the use of mice that produce humanized Abs. By modifying the genetic background of F004 mice a new mouse substrain was developed for optimized “one step” generation of chimeric humanized monoclonal Abs. The new mice (F004-Jen) demonstrated improved fertility still expressing the human locus at the same level as the parental F004 mouse. The value of these mice for the generation of chimeric Abs was exemplified for a panel of chimeric Abs against the human neural cell adhesion molecule (NCAM): The fully chimeric human IgG1/κ Ab Ch.MK1 bound to NCAM expressing cells with a KD=4.3-8.7×10−8 M and was functionally active as demonstrated by depleting NCAM expressing cells. We also demonstrated that chimeric IgG1/κ Abs can be induced by hybridoma class switching of IgM producing hybridoma cells, providing an alternative way to chimeric Abs. The present data highlight F004-Jen mice as an efficient tool for “one step” generation of chimeric Abs.

The novel chimeric anti-NCAM (neural cell adhesion molecule) antibody ch.MK1 displays antitumor activity in SCID mice but does not activate complement-dependent cytolysis (CDC).

A monoclonal chimeric antibody ch.MK1 was generated by immunizing F004 mice expressing human instead of murine IgG1/κ immunoglobulin constant regions. The novel antibody specifically binds cell surface-expressed human neural cell adhesion molecule (NCAM) as shown by immunoprecipitation, flow cytometry and cytospins. Functional analysis revealed nearly complete absence of complement-dependent cytolysis in ch.MK1 and in all other anti-NCAM antibodies tested for reference (UJ13a, ERIC1, 123C3, ch.5A2, B159), indicating an unexpected and group-specific property of anti-NCAM antibodies. As a most plausible mechanism, posttranslational modification of NCAM by complement-inhibiting polysialic acid is discussed. The antibody ch.MK1 demonstrated significant in vivo activity against NCAM-positive neuroblastoma in SCID mice in presence of human peripheral blood mononuclear cell. In absence of human peripheral blood mononuclear cell no distinct antitumor activity of the antibody alone was observed. In ch.MK1 the cellular component of the immune system seems to be the dominant effector mechanism, whereas complement-dependent cytolysis seems not to be necessarily required for antitumor activity. These observations help us to understand immunotherapeutic mechanisms of native anti-NCAM antibodies and may additionally contribute to the understanding of results of currently ongoing clinical studies with conjugated anti-NCAM antibodies.

Implementation and Evaluation of a Central Coordination Office for Clinical Trials in a Tertiary Care Hospital

Background: Controlled clinical trials are essential tools for establishing new standards in patient care. Nevertheless, the majority of cancer patients are not treated within clinical trials. We report about a project now running for 7 years that was started in order to enhance the recruitment of patients into clinical trials, to improve trialrelated quality, and to comply with the regulatory issues related to these studies. Material and Methods: We established a Central Coordination Office (CCO) for clinical trials, an associated internal clinical trials review board, a register of active clinical trials, and a computer-based medical information system at our department. Results: Inpatient recruitment into clinical trials at our department improved over the last 7 years from 40% in 1997 to 70% in 2003. The internal review board approved 276 trial projects since its establishment. A clinical trials register is now in its 9th edition. Currently, 50 to 60 clinical trials in oncology/hematology are active while 10 to 20 new trials are being implemented per year. All clinical trials comply with the regulatory requirements, and trial documentation is provided in a timely manner. Conclusions: The establishment of a CCO for clinical trials substantially improves and maintains patient recruitment into clinical trials and improves the quality of clinical research.

Prolonged Remission in a Patient with Relapsed Follicular Lymphoma after a Single Course of Rituximab

Background: Rituximab monotherapy can achieve remissions of up to 80% in follicular lymphoma. As the antibody has only been on the market since 1997, long-term observations are still rare. Case Report: We report about a patient with follicular lymphoma who received a single treatment of 4 × 375 mg/m2 of the monoclonal antibody rituximab without chemotherapy and has been in complete remission (CR) for 8 years. Discussion: This remarkable response duration highlights the efficacy of rituximab monotherapy. A number of recently published studies have indicated that CR of more than 4 years after rituximab monotherapy may be a regularly occurring event. Conclusions: These patients are likely to represent a yet poorly characterized patient group that profits considerably from rituximab monotherapy.

Modulation of tumour directed B cell responses by NK cells

Background and aim Spontaneous natural killer cell (NK cell) activity against malignant cells is important for development of tumour directed Th1 biased and cytotoxic T cell (CTL) responses (Kelly et al., Nature 2002; Geldhof et al, Blood 2002) resulting in protection from tumour growth in mouse models. Aim of our investigations was to study the significance of NK cell activity on tumour directed B cell immunity.