Joseph Haimovich

Synergistic Effect of Dendritic Cell Vaccination and Anti-CD20 Antibody Treatment in the Therapy of Murine Lymphoma

Indolent B-cell lymphomas are characterized by repeated remissions and relapses with most patients eventually dying of the disease. Although combination treatments with chemotherapy and the anti-CD20 antibody rituximab improved duration of remissions and overall survival, the disease is essentially incurable. Thus, novel therapeutic approaches are needed. One such approach is active immunization with dendritic cells (DCs). Given that rituximab depletes patients of normal B cells, optimal vaccination strategies for rituximab-treated patients require induction of effector T cells. We have previously demonstrated in a murine model that idiotype (Id)-keyhole limpet hemocyanin-pulsed DCs induced Id-reactive CD8+ T cells and protection against tumor challenge in the absence of anti-Id antibodies. On the basis of these results, we investigated vaccination in a therapeutic model, in which mice carrying advanced tumors of the highly aggressive 38C-13 lymphoma were treated with chemotherapy and anti-CD20 antibodies combined with a DC-based vaccine. As a rule, cytoreduction by cyclophosphamide was required in each regimen of combination treatment, and vaccination with tumor cell-loaded DCs was more effective than vaccination with Id-keyhole limpet hemocyanin-loaded DCs. We demonstrated that under conditions of large primary tumors that had already spread to lymph nodes, when anti-CD20 antibody treatment showed minimal effect and DC vaccination had no effect, synergism between anti-CD20 antibodies and DC vaccines resulted in significant long-term survival that did not involve active antitumor antibody production. Combination treatments including tumor cell-loaded DC vaccines may therefore provide a strategy for enhancing the potency of therapy in rituximab-treated patients.

Dendritic cell-based therapeutic vaccination against myeloma: vaccine formulation determines efficacy against light chain myeloma.

Multiple myeloma is an incurable plasma cell malignancy. Immunotherapy in myeloma patients had limited success to date. We have previously demonstrated that dendritic cells (DCs) pulsed with autologous Ig Id induced Id-reactive CD8+ T cells and protection against a myeloma tumor challenge. In this work, we studied the therapeutic efficacy of chemotherapy combined with different formulations of DC-based vaccines in mice bearing large plasma cell tumors. The comparative study demonstrated that s.c. injection of DCs loaded with Id coupled to keyhole limpet hemocyanin, s.c. injection of DCs loaded with irradiated tumor cells, and intratumoral injection of naive DCs were similarly effective in mediating tumor regression and long-term survival. However, whereas the Id-keyhole limpet hemocyanin-DC vaccine was inefficient against myeloma cells that lost expression of the Ig H chain, intratumoral injection of naive DCs and s.c. injection of DCs loaded with irradiated tumor cells were highly effective against cells producing L chains only. This may be of particular importance for patients with L chain myeloma. Given that T cells respond primarily to peptides derived from H chain CDRs, attempts to treat L chain disease with myeloma protein-pulsed DCs may be futile. Vaccination with tumor cell-loaded DCs may, however, induce an effective antitumor response.

Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma.

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id × anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id × anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.

Selective cytotoxicity against tumor cells by cisplatin complexed to antitumor antibodies via carboxymethyl dextran

SummaryCis-diamminedichloroplatinum (II) (cis-DDP) and its structural analogue cis-diamminediaquoplatinum(II) nitrate (cis-aq) were complexed via an intermediate dextran carrier to antibodies specifically reactive with B lymphoma cells (38C-13). The potential use of these drugs in site-directed immunotargeting was evaluated. The two platinum(II) compounds were previously shown to form pharmacologically active complexes with carboxymethyl dextran (CM-dex). For the purpose of preparing drug-antibody complexes, CM-dex was first conjugated to idiotypic antibodies that recognize a specific membrane IgM on the B lymphoma cells. The conjugates were prepared by a modified water-soluble carbodiimide method in which N-hydroxysuccinimide was used to enhance the coupling reaction. The conjugation was followed by separation of the CM-dex-IgG conjugates from unconjugated CM-dex or IgG. The platinum(II) compounds were then complexed to the CM-dex-IgG resulting in complexes carrying up to 50 mole drug/mole IgG. Both cis-DDP and cis-aq complexes of CM-dex-antibody conjugates maintained most of the original cell-binding activity of the antibodies. An in vitro assay was used to demonstrate selective binding to tumor cells in which the target cells were treated with specific immune complexes and washed before culture. In this assay the specific complexes showed preferential cytotoxicity for the B lymphoma cells in comparison to the free drugs, drug CM-dex, or nonspecific immune complexes.

Rejection of tumors of the B cell lineage by idiotype-vaccinated mice.

Abstract Idiotypic determinants of immunoglobulins of malignant B lymphocytes and plasma cells are tumor-specific antigens and have been used extensively in immunotherapy studies. The mechanisms involved in resistance to tumor challenge following idiotype vaccination are poorly understood. Although a predominant role has been attributed to anti-idiotype antibodies, both humoral and cellular immune responses are probably involved. Cell-mediated responses may be particularly effective against tumor cell variants that do not express the idiotype on the cell surface and are therefore resistant to anti-idiotype antibodies but continue to produce one of the original immunoglobulin polypeptides that may be processed and presented to T cells. In this report we describe two experimental models of idiotype vaccination in which antibodies are unlikely to play a role, and hence tumor immunity is attributed to cell-mediated responses. One model consists of the murine B lymphocyte tumor 38C-13 and its idiotype-negative variant DB2, which has lost the idiotypic specificity of the parental 38C-13 cell line through the production of a different light chain but expresses the original heavy chain. Vaccination of mice with the purified IgM of 38C-13 induced resistance to 38C-13 tumor cells as well as to the variant cells. Although immunized mice produced high levels of anti-idiotype antibodies that bound to 38C-13 cells, no binding of antibodies to DB2 cells occurred. The finding that idiotype-vaccinated mice were resistant to idiotype-negative DB2 cells suggested that cellular mechanisms are involved in mediating resistance. The second model consists of the two plasma cell line JLμs and JLμm, which produce IgM with an identical specificity. Whereas one of them (JLμs) secretes the IgM, the other one(JLμm) can neither secrete nor deposit it on the cell surface. Immunization against JLμs IgM followed by tumor challenge resulted in prolonged survival of both JLμs- and JLμm-challenged mice. Although sera of immunized mice contained high levels of anti-idiotype antibodies, they did not react with the plasmacytoma cells. Similarly to the results obtained in the 38C-13 experimental model, these results suggest that a non-antibody-mediated mechanism was involved in the resistance of mice to tumor growth.

Anti-idiotype × Anti-LFA-1 Bispecific Antibodies Inhibit Metastasis of B Cell Lymphoma

Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id × anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.

B-cell lymphoma and myeloma protection induced by idiotype vaccination with dendritic cells is mediated entirely by T cells in mice.

Immunoglobulin idiotypes (Id) of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Id-directed immunotherapy by immunization with autologous Id has been initiated in clinical trials to control residual disease in B-cell lymphoma and multiple myeloma. The effector mechanisms responsible for destruction of B-cell tumors are a controversial issue. The authors show that vaccination with Id-pulsed dendritic cells (DCs) or with soluble Id-KLH in adjuvant induced immune responses that eliminated both B-cell lymphoma and myeloma in tumor-bearing mice; however, the two vaccination regimens resulted in distinct immune responses. Whereas soluble Id plus adjuvant induced high levels of anti-Id antibodies, the Id-pulsed DCs did not induce anti-Id or any antitumor antibodies. Immunization with Id-pulsed DCs induced a significant increase in the frequency of Id-reactive T cells. Depletion studies in DC-vaccinated mice showed that the predominant effector cells responsible for tumor rejection were of the CD8+ subset. The finding that DC-based Id vaccines elicit tumor protection, which is entirely based on cell-mediated effector mechanisms, is of particular importance for plasma cell tumors because these tumors do not express Id on the surface and hence do not bind anti-Id antibodies.

Somatic cell hybridization between bovine leukemia virus-infected lymphocytes and murine plasma cell tumors: Cell fusion studies with bovine cells☆

Hybridization of peripheral blood lymphocytes from bovine leukemia virus-infected cows with murine myeloma cells resulted in the generation of hybrid cells secreting immunoglobulins composed of various combinations of heavy and light chains of both bovine and murine Ig origin. Some hybrid cells derived from the light-chain producer, but non-secretor murine myeloma NSI cell line, secreted IgM molecules composed of bovine mu-chain linked to bovine and/or murine light chains. Other hybridomas secreted mouse and bovine light-chain dimers and/or monomers, or failed to secrete any Ig polypeptide chain whatsoever. Immunoglobulins secreted by hybridomas obtained upon hybridization of bovine cells with the IgG-secreting murine myeloma P2X63 cell line, contained bovine mu-chain in one of the seven hybridomas obtained, and bovine light chain in two of them. All the cell lines secreted murine light- and gamma-chains.

Immunotherapy of B lymphoma by anti-idiotype antibodies: Characterization of variant tumour cells appearing a long time after the initial tumour inoculation

SummaryImmunotherapy of a murine B-cell lymphoma by antibodies to idiotypic determinants of its IgM, resulted in surviving tumour-free mice. Several of these mice, however, did develop tumours a long time after the initial inoculation of the tumour cells, or upon rechallange of the survivors with B-lymphoma cells. The presence of tumour cells during this long latent period may have been due to the development in the host of an anti-idiotype immune response. These late-developing tumours were detected by a radioimmunoassay and characterized by immunofluorescent staining and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Cells of some late-developing tumours lost the idiotype recognized by the antibodies used for the immunotherapy. Several of these tumours expressed IgM on the cell surface, while others did not, because of the absence of light chains. They were identical in the structure of their rearranged µ chain genes proving their derivation from the original inoculation. Cell lines obtained from the late-developing tumours differed in their tumorigenicity. The appearance of idiotype-negative tumour cells as a result of anti-idiotype immunotherapy has a great impact in our efforts to cure lymphoma by this modality.

The MAPK/ERK and PI(3)K Pathways Additively Coordinate the Transcription of Recombination-Activating Genes in B Lineage Cells

Rag-1 and Rag-2 are essential for the construction of the BCR repertoire. Regulation of Rag gene expression is tightly linked with BCR expression and signaling during B cell development. Earlier studies have shown a major role of the PI(3)K/Akt pathway in regulating the transcription of Rag genes. In this study, by using the 38c13 murine B cell lymphoma we show that transcription of Rag genes is also regulated by the MEK/ERK pathways, and that both pathways additively coordinate in this regulation. The additive effect is observed for both ligand-dependent (upon BCR ligation) and ligand independent (tonic) signals. However, whereas the PI(3)K/Akt regulation of Rag transcription is mediated by Foxo1, we show in this study that the MEK/ERK pathway coordinates with the regulation of Rag by controlling the phosphorylation and turnover of E47 and its consequential binding to the Rag enhancer regions. Our results suggest that the PI(3)K and MEK/ERK pathways additively coordinate in the regulation of Rag transcription in an independent manner.