Enhanced therapeutic effect of B cell-depleting anti-CD20 antibodies upon combination with in-situ dendritic cell vaccination in advanced lymphoma.

The present standard of care for B cell non-Hodgkin's lymphoma includes the anti-CD20 monoclonal antibody rituximab. Although combination treatments with chemotherapy and rituximab improved the duration of remissions and overall survival in indolent B cell lymphoma, the disease is essentially incurable. Thus, new therapeutic approaches are needed. One such approach is active immunization. Given that rituximab depletes both malignant and normal B cells, it is expected to impair humoral immune responses in vaccinated patients. Hence, optimal vaccination strategies for rituximab-treated patients require induction of effector T cells, which can be achieved by dendritic cell (DC) vaccines. We have demonstrated in a mouse model that chemotherapy combined with DC vaccines was therapeutically effective. However, efficacy was related to tumour size at the onset of treatment, decreasing in correlation with increasing tumour burdens. We therefore examined whether, in spite of its low efficacy in advanced disease, DC vaccination may synergize with anti-CD20 antibodies to enhance therapy. Lymphoma-bearing mice were treated with cyclophosphamide, anti-CD20 antibodies and an intratumoral DC vaccine. Results clearly demonstrated the enhanced therapeutic effect of this combination treatment. Thus, under conditions of disseminated disease, when either anti-CD20 antibody treatment or vaccination showed insufficient efficacy, their combination resulted in synergism that mediated long-term survival. We demonstrated further that the combination of antibody and vaccine induced T cell-mediated anti-tumour immune responses with long-term memory. Combination treatments including tumour cell-loaded DC vaccines may therefore provide a strategy for enhancing therapy in rituximab-treated patients.

The monoclonality of human B-cell lymphomas.

Human tissues involved with lymphoma have been examined in frozen sections for immunoglobulin-bearing cells by a technique involving double-label immunofluorescence with mixed anti-kappa and anti-lambda antibodies. F (ab')2 fragments of purified antibodies were employed to avoid any binding via Fc receptors. B cell lymphomas were shown to be composed of monoclonal populations of Ig bearing cells, whereas normal or reactive lymphoid follicles contained a mosaic of Ig-bearing cells derived from multiple clones. Nodules of lymphoma were often surrounded by normal polyclonal B cell populations. We anticipates that the approach described here will be useful in the diagnosis of lymphoma, differentiating it from reactive lymphoid hyperplasia by the demostration of monoclonality. In addition, it should provide a sensitive and reliable tool for investigating the immunobiology of human lymphoma.

In vitro induction of a primary response to the dinitrophenyl determinant.

Primary antibody response against the dinitrophenyl group has been elicited in vitro after the stimulation of normal mouse spleen explants with 2,4-dinitrophenyl (DNP)-hemocyanin or alpha-DNP-poly-L-lysine (PLL). Antibodies were detected in the culture medium by the inactivation of DNP-T4 phage. The specificity of the reaction was manifested by the lack of the capacity of the medium to inactivate the unmodified bacteriophage and by the inhibition of the inactivation of DNP-T4 with DNP-lysine.

Protein-bacteriophage conjugates: application in detection of antibodies and antigens.

Covalent attachment of proteins to bacteriophage yielded modified phage preparations with which it is possible to detect antibodies to proteins at concentrations as low as 0.5 to 2.0 nanograms per milliliter. Similarly, antibodies may be linked covalently to phage, and the resulting antibody-phage conjugate is useful in detecting proteins. An alternative method for quantitative determination of proteins is suggested, in which the inactivation of protein-phage by antibodies to protein is inhibited by the protein tested. With rabbit immunoglobulin G as the protein, as little as 0.3 nanogram per milliliter could he determind.

Characterization of productive and sterile transcripts from the immunoglobulin heavy-chain locus: processing of micron and muS mRNA.

An analysis of the sizes and sequence content of nuclear RNA transcripts of the heavy-chain locus in two B-cell lymphomas, 70Z/3 and 38C-13, and in selected hybridoma derivatives of 38C has led to the identification of two distinct precursors of the mRNAs encoding the membrane and secretory forms of mu chain. These precursors, termed Pm1 and Ps1, extend from a common 5' terminus (presumably the cap site) to alternative polyadenylation sites located 3' of the membrane and secretory tailpieces, Pm1 and Ps1 are present in similar amounts in lymphomas, indicating roughly equivalent usage of the two polyadenylation sites, whereas Ps1 much greater than Pm1 in hybridomas, indicating that mature plasma cells produce a trans-acting factor which enhances cleavage at the proximal (muS) site. The lymphomas also synthesize several nonproductive or sterile mu (Smu) transcripts from the second H allele. One class of sterile mu transcripts appears to be initiated about 1 kilobase downstream from the JH4 element. In 70Z, in which the nonproductive H allele has undergone a D1J2 fusion, another initiation site was located about 0.3 kilobase upstream of the D1 element. The sterile mu transcripts exhibit the same regulated termination at alternative polyadenylation sites as the mu mRNA precursors, although their rate of production is not necessarily coupled to that of the productive allele. This analysis has also defined probable processing pathways for productive and sterile components in which there is a 5' leads to 3' order for the excision of the large introns.

Purification and characterization of tumor inhibitory factor-2: its identity to interleukin 1.

The tumor inhibitory factor-2 from the conditioned medium of the human rhabdomyosarcoma cell line A673 was purified and sequenced. The 19 N-terminal amino acid residues were identical to those of human interleukin 1 (IL-1), corresponding to the residues 119-137 of the IL-1 alpha precursor. The purified material had an apparent molecular weight similar to that of the mature secreted form of IL-1 alpha (Mr 17,400). In addition, similarly to IL-1, it induced the production of IL-2 by T-cells. The purified protein inhibited the growth of the A673 cells from which it was derived, suggesting that it may act as an autocrine growth inhibitor. It also inhibited the growth of a human adenocarcinoma of the lung and three human mammary carcinomas, but not of two human melanoma cell lines. In contrast, it stimulated the proliferation of normal human fibroblasts. These biological activities, previously assigned to a putative tumor inhibitory factor molecule, are apparently due to the production by the tumor cells of IL-1 alpha.

Light chain loss and reexpression leads to idiotype switch. Surrogate light chains are probably responsible for this process.

The murine B-lymphocyte cell line 38C-13 is characterized by several cell surface markers typical for an early stage of B-cell differentiation. Cells of this cell line possess cell surface membrane IgM molecules composed of mu and kappa polypeptide chains. They also produce "surrogate" or "pseudo" light chains (psi L) coded by the lambda 5 and VpreB genes. Variants of the 38C-13 cell line which do not synthesize kappa chains can be isolated from the 38C-13 population by the use of anti-idiotype antibodies in vivo and in vitro. In some kappa chain-deficient variant cell lines, cells which have regained surface IgM expression but have lost the original idiotype specificity, can be isolated. This idiotype switch is probably due to a secondary rearrangement of the kappa gene. In the kappa chain-deficient variant cells, the mu chains assemble with the surrogate light chains but the assembled IgM-like molecules are not expressed on the cell surface. It is suggested that surrogate light chains play an important role in the induction of kappa gene rearrangement but that surface expression of mu-psi L complexes is not required for this process.

The effects of passive anti-viral immunotherapy in AKR mice: II. Susceptibility to B cell lymphomagenesis.

Prevention of high frequency spontaneous T cell lymphoma development in AKR mice by mAb 18-5 treatment was shown to involve inhibition of the recombinant Class I MCF virus formation and elimination of the early occurring potential lymphoma cells (PLCs). A low B cell lymphoma incidence (16% at a mean latency of 540 days) and a low level of PLCs (yielding 12% B cell lymphoma development following lymphoid cell transfer) was observed in mAb 18-5 treated mice (in contrast to a high PLC level in thymectomized AKR mice that could be experimentally triggered to progress to overt CD5+ B cell lymphomas). Administration of anti CD8 mAb or IL-4 to 12-month-old mAb 18-5 pre-treated mice only slightly increased B cell lymphoma incidence (up to 30-40%). Exposure to split-dose irradiation resulted in 26% B cell lymphomas at a 250 day mean latency. The phenotypes of the B lymphomas developing in mAb 18-5 treated mice were: B220+ (14.8+, 6B2+), 6C3+, Mac2+, CD5-. Most lymphomas expressed l-a and surface IgM, pointing to their mature B cell characteristics. Moreover, in some of the lymphomas, high levels of IgM production and secretion were determined. A comparison of the morphological characteristics (based on light and ultrastructure microscopy) of CD5+ and CD5- B cell lymphomas developing in AKR mice indicated marked differences. Analysis of the IgH locus of representative CD5- B lymphomas showed an identical pattern of IgH rearrangement in some tumors (similar to previous findings among CD5+ lymphomas). The virological analysis of the CD5- B cell lymphomas (similar to those observed in the CD5+ B cell lymphomas of AKR origin) showed that their development did not require formation of the pathogenic MCF recombinant viruses. The differences observed between the CD5+ and CD5- B cell lymphomas developing in AKR mice (following prevention of spontaneous T cell lymphomagenesis) may be due to their origin of different B cell precursors or from B cells at different levels of differentiation.

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.

Human anti-mouse immunoglobulins in sera of patients treated chronically with monoclonal antibody-purified factor VIII.

In the process of isolation of factor VIII from human plasma making use of immunoadsorbents prepared by coupling monoclonal murine antibodies to resins, trace amounts of murine immunoglobulin G (IgG) antibodies are released from the resin into the final Monoclate product. This trace contamination, amounting to not more than 50 ng/100 units of Monoclate, was assumed to be below the threshold amounts necessary for inducing an immune response. Nevertheless, we have developed a series of highly sensitive and specific radioimmunoassays for the determination of human antibodies of the IgG, IgM, and IgE classes against the murine monoclonal IgG used for purification of Monoclate. Screening of sera from adults and children treated with Monoclate showed that in no case were any antibodies produced in response to injection of Monoclate. Surprisingly, sera from several patients had a high activity against murine IgG both before and after treatment with Monoclate.

Determination of anti-idiotype antibodies by surface plasmon resonance.

A method based on surface plasmon resonance has been used to detect and to quantitate antibodies against other antibodies. This method has been applied to the detection of syngeneic antibodies. Concentrations as low as 0.3 microg/ml could be detected even when the antibodies were present in undiluted serum. Quantitation of both monoclonal and polyclonal antibodies could be performed. Anti-idiotype antibodies could be detected equally well against an IgM molecule derived directly from the B cell tumour or against a chimeric protein composed of the same idiotypic determinants embedded in a human IgG molecule. Surface plasmon resonance will be the method of choice for the detection of antibody responses in humans vaccinated against idiotypic determinants of their own tumour.

Lack of immune response to mouse IgG in hemophilia A patients treated chronically with Monoclate, a monoclonal antibody affinity purified factor VIII preparation.

Hemophilia A is caused by factor VIII deficiency that historically has been treated with either a cryoprecipitate fraction of serum or factor VIII concentrate. Recently, the availability of affinity isolated factor VIII (Monoclate) has allowed for a highly purified preparation for the chronic therapy of hemophilia A. This factor VIII preparation contains a trace quantity (less than 50 ng/100 I. U.) of mouse IgG. Immunoassays for the measurement of human IgG, IgM and IgE anti-mouse IgG antibody (HAMA) were developed and used to measure HAMA levels in hemophilia A patients undergoing chronic therapy with Monoclate in three different clinical studies. Natural antibodies to mouse IgG were observed in patient sera prior to Monoclate infusion. Data is presented demonstrating that induction of HAMA upon Monoclate treatment does not occur. The low level of mouse IgG contained in Monoclate appears to be below the threshold of immunogenicity. Most importantly, clinical symptoms related to hypersensitivity or anaphylaxis were never observed in any patient undergoing chronic therapy with Monoclate in these clinical studies.

Idiotype-specific inhibition of LFA-1-mediated cell adhesion by anti-idiotype x anti-LFA-1 bispecific antibodies.

Adhesion molecules are involved in lymphoma dissemination. Antibodies to adhesion molecules may block tumor metastasis. However, such antibody treatment may block as well normal functions of the immune system. We tested the hypothesis that a bispecific antibody with specificity for an adhesion molecule and for a tumor specific antigen binds preferentially to tumor cells which coexpress both antigens and hence selectively blocks adhesion. A bispecific antibody was developed by somatic cell hybridization of two hybridomas, one producing a monoclonal antibody against the immunoglobulin idiotypic determinant of the murine B cell lymphoma 38C-13 and the other producing an antibody against the alpha subunit (CD11a) of the adhesion molecule LFA-1. The bispecific antibody, anti-idiotype x anti-LFA-1, was purified by affinity chromatography. The dual specificity of the hybrid hybridoma product was demonstrated by a radioimmunoassay devised for detection of bifunctional activity. The bispecific antibody was shown by flow cytometry to bind efficiently to 38C-13 cells that coexpress idiotype and LFA-1. It bound only weakly to idiotype-negative variants of 38C-13 that express only LFA-1. In binding assays to immobilized ICAM-1, the anti-idiotype x anti-LFA-1 was highly active in blocking 38C-13 cell adhesion. However, it did not effect adhesion of idiotype-negative tumor cells or of normal T lymphocytes. In summary, the bispecific antibody preferentially blocks adhesion of cells that coexpress the tumor specific antigen and the adhesion receptor. The present approach may provide a general way for the selective adhesion blockade of a specific cell population.

Characterization of IgM molecules in light-chain deficient variants of a B-cell tumor.

Characterization of a membrane-IgM-negative variant cell line derived from the murine B-cell line 38C-13 revealed the absence of light chains and the presence of polypeptides with an apparent molecular size of 18 kDa and 14 kDa, previously denoted omega and iota and characteristic of pre-B cells. These polypeptides assemble with the mu chains into complexes with apparent molecular sizes of about 100 kDa and 200 kDa. It has been previously shown that light-chain-deficient variants of the 38C cell line undergo 'secondary' light chain rearrangements. It is suggested, therefore, that complexes of mu and the 'surrogate' light chains omega and iota play a role in this process. As these complexes do not reach the cell surface we would like to propose that the mechanism of secondary rearrangement is intracellularly controlled.

Membrane IgM of bovine lymphocytes.

The membrane immunoglobulins of peripheral blood lymphocytes (PBL) obtained from four bovine-leukemia-virus infected calves and from a normal cow were isolated and characterized. They were found to consist of an IgM exhibiting a mu-chain of an apparent molecular weight (AMW) of 95,000 daltons, which is 10,000 daltons more than found for the mu-chain of serum IgM. It thus seems that this is a property of membrane-bound IgM of bovine origin.

Monoclonal anti-idiotype antibodies against the murine B cell lymphoma 38C13: characterization and use as probes for the biology of the tumor in vivo and in vitro.

To establish a murine model for the monoclonal anti-idiotype immunotherapy of B cell lymphoma, a panel of rat and murine monoclonal anti-idiotype antibodies of several different isotypes was generated against the surface immunoglobulin of the murine B cell tumor 38C13 (38C). Xenogeneic antibodies were made from fusions of rat spleen cells immunized with the 38C idiotype. Syngeneic monoclonal anti-idiotypes were generated from mice immunized with the idiotype conjugated to the protein carrier KLH. Small differences were noted in the ability of the antibodies to cross-block one another, but all appeared to be directed against the same or closely spaced idiotopes on the immunoglobulin molecule. The antibodies selectively precipitated surface Ig from 38C tumor cells and not from normal mouse spleen cells. They were used to selectively stain 38C tumor cells in cell suspensions for FACS analysis or immunohistochemical staining of tissue sections from mice bearing the tumor. As the malignancy progressed, the number of tumor cells found in all tissues examined increased. Thus, the anti-Id antibodies provided a specific probe for tumor cell detection. The antibodies had no detectable effect on cell growth in vitro; however, they did cause the rapid transient loss of the expression of cell surface Ig. This modulation was concentration and time dependent but not 100% complete. Re-expression of the Id occurred by 24 h following removal of the anti-Id antibodies. When these antibodies were used in sensitive radioisotope and enzyme linked immunoassays, the tumor cells were found to secrete small amounts of idiotype in vitro and in vivo. The level of idiotype detected in vivo correlated with tumor growth and inversely with survival. This work is an attempt to develop further an animal model system in which to test the diagnostic and therapeutic effects of monoclonal anti-idiotype antibodies.