A prospective multicenter study of the efficacy and tolerability of cryopreserved allogenic human keratinocytes to treat venous leg ulcers.

Allogeneic human keratinocyte cultures have been used to treat burn wounds, donor sites, and chronic skin ulcers with some success. Cryopreservation of these cultures allows for the production of large standardized batches that are readily available for use. The aim of the study presented in this report was to study effects of cryopreserved cultured allogenic human keratinocytes (CryoCeal) on chronic lower extremity wounds. Parameters were measured to study efficacy, tolerability, pain associated with chronic wounds, and quality of life of patients. Twenty-seven patients with hard-to-heal venous leg ulcers received a maximum of 9 applications of CryoCeal in a prospective, uncontrolled multicenter study lasting 48 weeks. Eleven out of 27 patients (41%; 95% CI: 22%-61%) had complete wound closure within 24 weeks (1 week). The time required for complete wound closure in these 11 patients ranged from 4.1 to 24.9 weeks. Only 1 patient had recurrence of the ulcer at 48 weeks. Local (wound) pain scores decreased from a mean of 2.5 at baseline to 0.9 at week 24. Fifty percent of the patients attained a pain score of 0 after 12 weeks and remained stable at this score until the end of the study. Overall, the patient quality of life was better at week 24, compared to baseline values. The treatment was well tolerated, and wound infection was the most frequently occurring adverse event.

The genetic variability of the VH genes in follicular lymphoma: the impact of the hypermutation mechanism.

Follicular lymphoma (FL) cells have inherited an activated hypermutation mechanism from their origin of germinal centre B cells. Based on today's knowledge of the intrinsic properties related to this mechanism and the VH base composition, reconsideration of previous reports should be made on a broader range of samples. The present study examined the mutation pattern of the VH genes expressed by 55 cases of FL. FL VH genes showed evidence of antigenic selection in 30% of cases with 88% carrying a functional sIg and 78.2% showing intraclonal variation. VH family and gene segment utilization was found to be roughly similar to that of normal B lymphocytes. FL VH genes revealed extensive variations. 17% of the VH exons harboured a total of five deletions, three duplications and two insertions as compared to the most homologous germline counterpart. The VH genes of one tumour displayed three populations with varying CDR3 length at diagnosis. At relapse, emergence of a differently mutated gene, additional mutations reminiscent of ongoing mutations or no variation was prominent. From this study the heterogeneity of FLs is well established and ongoing mutations are seen in the scope of the activated status of the hypermutation mechanism rather than antigen-stimulated tumour growth.

Bispecific antibody treatment of murine B cell lymphoma.

This report summarizes our experimental data concerning the use of bispecific antibodies (bsAb) for the treatment of the murine BCL1 B cell lymphoma model. Initially we used a hybrid-hybridoma-derived bsAb with specificity for the TcR/CD3 complex on T cells and the idiotype of the membrane-bound IgM on the tumor cells. The bsAb used as a single agent could cure animals with a low tumor load (resembling minimal residual disease). However, in experiments aimed at increasing the therapeutic effect in animals with a higher tumor burden, we could demonstrate the importance of additional T-cell-costimulatory signals and the careful timing of the bsAb administration. Recently we have generated a bispecific single-chain Fv (bsscFv) fusion protein with the same dual specificity as the hybrid-hybridoma-derived bsAb. Immunotherapy with this smaller molecule also resulted in tumor elimination in BCL1-bearing mice. A second bsscFv (alpha-CD19 x alpha-CD3) with a broader applicability is now being characterized and tested in vivo.

Vaccine strategies in non-Hodgkin's lymphomas.

As described above, the most recent advances in anti-idiotype vaccination strategies have gone hand in hand with recent developments in molecular biology and other forms of cancer therapy. The techniques that are currently available in antibody engineering will greatly facilitate protein production and purification and will reduce the time and effort needed to produce the patient specific vaccines. Cytokine (gene) therapy has extensively been studied in cancer treatment and cancer vaccination and some therapeutic strategies are currently being evaluated in clinical trials (Bubenik, 1996). Combination therapy of idiotypic vaccination with cytokine therapy has recently been explored with promising results. The main focus so far has been on GM-CSF and IL-2, although other cytokines might prove to be efficient in stimulating different effector arms of the immune system. The nature of the immune response mounted by the host against the tumour and the mechanisms by which the tumour cells escape the effector functions of the immune system are not yet fully known. A better knowledge of the nature of B-cell lymphomas and the relation to the patient's immune system will therefore benefit the further development of the therapeutic strategies. Further research will provide us with a better view of how to break the immune tolerance and of which components of the immune system have to be targeted in order to obtain optimal therapeutic results.

Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28-induced costimulation.

The BCL1 lymphoma in Balb/c mice can be successfully treated with bispecific (anti-CD3 x anti-idiotype) antibodies (BSABs). In these experiments, animals were injected intraperitoneally (IP) with 5 x 10(3) tumor cells (day 0) and treated with one single intravenous (IV) injection of 5 micrograms BSAB (day 9). Because cross-linking of the CD3 complex is not in itself sufficient to activate resting T cells, the therapeutic success was mainly based on the progressive retargeting of the relatively small cytotoxic T-lymphocyte effector cell pool already in existence in vivo. For this reason, the therapy lost its effectiveness at higher tumor loads. Two possibilities were explored to treat mice with a higher tumor load (10(5) tumor cells). First multiple injections of BSABs were used, but a dose-related monovalent anti-CD3 immunosuppression was induced. This approach was only beneficial when the immune system was able to recover from the previous injection of BSAB. As a second approach, CD28 costimulation together with BSABs were used in an attempt to activate resting T cells, ultimately enlarging the effector T-cell pool. However, we were repeatedly unsuccessful in attempts to improve our in vivo results using young, naive animals in which the majority of T cells are of the naive phenotype. Only when animals were primed to induce the memory T-cell type was a significantly better outcome observed, and then only with multiple injections of BSABs and anti-CD28 monoclonal antibodies (MoAbs), rather than with BSAB or anti-CD28 MoAb alone. These results suggest that this combination was able to activate memory and effector T cells and to focus them on the tumor, but was unable to activate naive T cells fully. The in vivo potency of the BSAB and CD28 costimulation was shown by the fact that one-tenth of the quantity of BSAB was required to cure animals with 20 times the tumor load.

Specific killing of lymphoma cells by cytotoxic T-cells mediated by a bispecific diabody.

Antibody fragments produced by bacterial fermentation lack natural effector functions. Bispecific antibody fragments, however, can be endowed with effector functions, for example cell-mediated killing, by binding to and retargeting of cytotoxic cells. Diabodies are a class of engineered antibody fragments with two antigen binding sites, consisting of two associated chains; each chain consists of heavy and light chain variable domains linked by a short polypeptide linker. In contrast to IgG, or other antibody fragments in which the two binding sites can take up a range of orientations and spacings, the diabody structure is more rigid and compact, with the two binding sites separated by 65 lA (less than half the distance in IgG). To establish whether diabodies could also be used in cell-mediated killing, we have explored the use of a bispecific diabody binding to an idiotypic marker on mouse B-cell lymphoma (BCL-1) and to mouse CD3. The bispecific diabody activated naive T-cells and also mediated the specific killing of the lymphoma cells by cytotoxic T-cells. The diabody was less active in T-cell activation but 10-fold more active (w/v) in killing than an analogous bispecific IgG.

Production and characterization of bispecific single-chain antibody fragments.

We report the construction, expression and purification of a bispecific single-chain Fv antibody fragment produced in Escherichia coli. The protein possesses a dual specificity: the single-chain FvB1 portion is directed to the Idiotype of BCL1 lymphoma cells, the single-chain Fv2C11 moiety binds to the CD3 marker on T cells. The two domains are joined by a flexible peptide linker. Using Immobilized Metal Affinity Chromatography, the recombinant protein was purified from bacterial insoluble membrane fractions. After refolding of the bispecific protein, it was affinity-purified. As demonstrated by flow cytometry, both binding sites are retained in the refolded protein. Retargeted cytotoxicity and T cell proliferation assays further prove the biological activity and specificity of the bispecific single-chain Fv. Thus, these bispecific molecules show a potential anti-tumor activity.

In vivo studies using bispecific antibodies (anti-CD3 x anti-idiotype) and CD28-induced costimulation in the BCL1 lymphoma.

We previously reported the successful treatment of the BCL1 lymphoma in Balb/c mice using bispecific (anti-CD3 x anti-idiotype) antibodies (BsAb). The in vivo effect was dependent on the bridging of tumor cells and CD3-positive cells. This direct CD3/TCR cross-linking induced targeted cytolytic and cytotoxic activity toward the tumor cells. Definitive proof of an underlying T cell-mediated mechanism was obtained, as the therapeutic effect was completely lost when animals were T cell depleted before treatment. In all these experiments, animals were injected intraperitoneally (i.p.) with 5000 tumor cells (day 0) and treated with one single intravenous (i.v.) injection of 5 micrograms BsAb (day 9). At a higher tumor load, the therapy lost its effectiveness. We evaluated repeated injections of bispecific antibodies (BsAb) to treat mice with a higher tumor burden (10(5) tumor cells). However, a dose-related immunosuppression (even with 5 micrograms BsAb) was induced. Therefore, this approach was only beneficial if the immune system could recover from the previous injection of BsAb. To prevent this induction of anergy, costimulation with bivalent anti-CD28 has been proposed, but despite the high in vitro T cell proliferation using BsAb + anti-CD28 versus BsAb alone, we were repeatedly unsuccessful in improving our in vivo results using immunologically naive animals. Only when T cell preactivation was induced was a significantly better outcome observed when the animals were treated with a mixture of BsAb + anti-CD28 compared with BsAb or anti-CD28 alone. The potency of the BsAb + anti-CD28 combination was demonstrated by the fact that 10 times less BsAb was necessary to cure animals with a 20 times higher tumor burden.

Role of T-cell subsets in the bispecific antibody (anti-idiotype x anti-CD3) treatment of the BCL1 lymphoma.

We reported previously on the successful use of bispecific antibodies in two well characterized B-cell lymphoma models. These bispecific antibodies were hybrid-hybridoma antibodies with dual specificity for the TcR/CD3 complex and for the tumor-specific idiotype of the surface IgM expressed by the lymphoma cells. Class-matched control antibodies, either monovalent for CD3, monovalent for idiotype, or bivalent for these surface markers, were always used in parallel with the bispecific antibodies. We extended our studies to determine the relative contribution of antibody-dependent cellular cytotoxicity and a T-cell-mediated therapeutic effect in the BCL1 lymphoma model. In tumor-bearing mice depleted of CD4+, CD8+ or both T-cell subsets and treated with bispecific antibodies, we could show that both T-cell populations contribute to the therapeutic outcome and have an additive role. In vitro studies demonstrate that bridging BCL1 tumor cells to T-cells by bispecific antibodies induces T-cell activation and secretion of tumor growth inhibiting lymphokines by both CD4+ and CD8+ T-cell populations. Particularly gamma-interferon seems to be the major tumor-inhibiting substance for BCL1 tumor cells. However, in vivo experiments using anti-cytokine antibodies showed that both gamma-interferon and tumor necrosis factor alpha have an effect on the tumor growth. The former acts directly by inhibiting tumor growth, the latter via an indirect mechanism, possibly by activating macrophages. In conclusion, our results show that induction of targeted cytolytic activity by the direct CD3/TcR cross-linking and development of targeted cytotoxic activity, mediated by gamma-interferon, by both T-cell subsets, contribute to the therapeutic success of bispecific antibody therapy.

Bispecific antibody therapy of two murine B-cell lymphomas.

Numerous in vitro studies have shown that T lymphocytes can be targeted towards any target cell by using bispecific antibodies (bsAbs) with specificity of the CD3/TCR complex and a target cell antigen. We have produced bsAbs directed against the membrane expressed idiotype of the murine B cell lymphomas BCLI and 38C13, and murine CD3 complex. The dual specificity of the hybrid-hybridoma produced monoclonal antibodies (MAbs) could be demonstrated by flow cytometry, the induction of T cell proliferation, the induction of IL2 secretion by polyclonal T cells, and redirected lysis of the relevant target cells. Immunotherapy of tumor bearing animals demonstrated that bsAbs could efficiently target T cells towards the tumor cells, that tumor cell--T cell bridging is established in vivo, and that both T cell subsets contribute to tumor regression resulting in long-term survival and cure of the lymphomas.

Treatment of mice bearing BCL1 lymphoma with bispecific antibodies.

Bispecific antibodies with specificity for the CD3/TCR complex of CTL and a target cell Ag can bridge both cell types and trigger cellular cytoxicity. We have produced bispecific antibodies, directed against the surface-expressed Id of the mouse BCL1 lymphoma and the mouse CD3 complex, by hybrid-hybridoma fusion. Two recombination Ig were purified to homogeneity: B1 X 7D6F, which is univalent for Id and CD3 binding and B1 X 7D6M, which is univalent for Id binding but has lost the CD3 binding because of association of the anti-CD3 H chain with the inappropriate L chain. In vitro studies indicate that bridging the TCR/CD3 complex of resting T cells with tumor IgM Id and the appropriate bispecific antibody induced proliferation and secretion of IL-2. Furthermore, in cytotoxicity assays using 51Cr-labeled tumor cells, preactivated T cells could be targeted with the bispecific antibody to give complete lysis of the Ag+ tumor. Finally, the activity of the bispecific antibody was confirmed in vivo. Animals treated i.v. with 5 micrograms of bispecific antibody 9 days after receiving BCL1 cells were cured. Furthermore, when these animals were checked at 150 days for dormant or variant tumors, as have been reported after other forms of immunotherapy in this model, none could be found. Immunotherapy experiments comparing a mixture of control antibodies with the bispecific antibody demonstrate that tumor cell-T cell bridging is established in vivo and is required for therapeutic success. These results indicate the importance of bispecific antibodies as a novel form of treatment for cancer.

Treatment of murine B cell lymphoma with bispecific monoclonal antibodies (anti-idiotype x anti-CD3).

It has previously been reported that T lymphocytes can be targeted by using bispecific antibodies consisting of anti-target antibody and anti-CD3. In the present study, a bispecific mAb was developed by somatic hybridization of mouse hybridomas, one producing a mAb against the Id determinant of the mouse B cell lymphoma 38C13 and the other a mAb against a polymorphic determinant on murine CD3. The bispecific antibody, anti-38C13 x anti-CD3, is bi-isotypic (IgG1 x IgG2a) and was purified by ion exchange and affinity chromatography. The dual specificity of the hybrid hybridoma-produced mAb could be demonstrated by flow cytometry, the induction of T cell proliferation, the induction of IL-2 secretion by polyclonal T cells, and redirected lysis of the relevant target cells. The hybrid (bi-isotypic) Fc part of the bispecific antibodies was nonfunctional in FcR-dependent redirected lysis. In vivo studies demonstrate that this bispecific mAb could efficiently target T cells towards the tumor cells, resulting in long term survival and cure of the lymphoma.

Interaction of B-cell hybridomas with fibroblast or hepatocyte monolayers in vitro and their metastatic behaviour in vivo.

Using an in vitro monolayer assay (MIA) we analyzed the invasive behaviour of a panel of B-cell hybridomas prepared by the fusion of non-invasive, non-metastatic NSO plasmacytoma cells and normal murine B-cells. Interaction of these hybridomas with fibroblast-like monolayers consisted mostly of adhesion on top of the monolayers, whereas only a fraction of these cells penetrated through the monolayer. This is in sharp contrast with the highly invasive properties displayed by T-cell hybridomas. Whereas T-cell hybridomas highly infiltrated monolayers of rat hepatocyte in vitro, B-cell hybridomas neither adhered to nor infiltrated hepatocyte monolayers. We found a good correlation between the degree of adhesion of B-cell hybridomas to fibroblast-like monolayers and their metastatic capabilities upon i.v. injection into syngeneic animals. Unlike T-cell hybridomas which formed diffuse metastasis in liver and spleen, B-cell hybridomas generated nodular metastatic lesions. . When normal LPS-stimulated B-lymphocytes were tested in the fibroblast-MIA, only part of the population infiltrated the monolayers. This again contrasts with T-lymphocytes where a majority of the cells penetrated through the monolayers. These results suggest that (i) B-lymphocytes express invasive properties, albeit to a lesser extent than T-lymphocytes, (ii) non-invasive B-lymphoma cells can acquire invasiveness following cell fusion with a normal B-cell, (iii) these invasive properties contribute to the malignancy of the hybridomas when tested in recipient animals.