Immunotherapy of established murine B cell lymphoma. Combination of idiotype immunization and cyclophosphamide

A murine B cell lymphoma (38C13) was used to study the efficacy of idiotype immunotherapy against established tumors. Immunization of mice with 38C13 tumor-derived Ig, administered after a lethal tumor inoculation, significantly prolonged survival of animals compared to control groups. The efficacy of active immunotherapy was dramatically enhanced when combined with chemotherapy. Cyclophosphamide (100 mg/kg), administered in combination with idiotype immunization to mice bearing 10-day-old, 1 to 2 cm diameter s.c. tumors, resulted in a significant prolongation of survival as compared with either cyclophosphamide or immunization alone and yielded approximately 50% cures. Additional studies combining active immunotherapy with surgical excision of the primary s.c. tumor nodule were less effective than combination chemoimmunotherapy, indicating that reduction of tumor burden was necessary, but not sufficient for effective treatment of established 38C13 lymphoma.     

Heterogeneity of a murine B cell lymphoma. Isolation and characterization of idiotypic variants

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy. The variant tumor cells were fused to myeloma cells and hybrids were isolated which secreted large quantities of the altered idiotype proteins. Polyclonal antibodies and mAb prepared against the mutant proteins demonstrated cross-reactivity with the original 38C13 protein and its other variants. But the variants and wild type cells could be distinguished from each other by their patterns of reactivity with the panels of anti-idiotype antibodies. Differences in apparent m.w. were demonstrated in the L chains of each of the mutant proteins. Southern blot analysis of the H chain locus of these mutants established that they were all clonally related; however, the L chain loci were grossly different. Thus, rare cells with alteration in their Ig L chain genes and expressed proteins can give rise to idiotype variants in this B cell tumor.     

DNA vaccination against the idiotype of a murine B cell lymphoma: mechanism of tumor protection

Several studies have shown that immunization with DNA, which encodes the idiotypic determinants of a B cell lymphoma, generates tumor-specific immunity. Although induction of antiidiotypic Abs has correlated with tumor protection, the effector mechanisms that contribute to tumor protection have not been clearly identified. This study evaluated the tumor protective effects of humoral and cellular immune mechanisms recruited by idiotype-directed DNA vaccines in the 38C13 murine B cell lymphoma model. Antiidiotypic Abs induced by DNA vaccination supported in vitro complement-mediated cytotoxicity of tumor cells, and simultaneous transfer of tumor cells and hyperimmune sera protected naive animals against tumor growth. However, in vitro stimulation of immune splenocytes with tumor cells failed to induce idiotype-specific cytotoxicity, and following vaccination, depletion of CD4 or CD8 T cell subsets did not compromise protection. Furthermore, protection of naive recipients against tumor challenge could not be demonstrated either by a Winn assay approach or by adoptive transfer of spleen and lymph node cells. Thus, in this experimental model, current evidence suggests that the tumor-protective effects of DNA vaccination can be largely attributed to idiotype-specific humoral immunity.     

Molecular characterization of anti-idiotype antibody-resistant variants of a murine B cell lymphoma

We have previously identified Id- tumor vaiants that emerge after anti-Id mAb therapy of the murine B cell lymphoma 38C13. This report characterizes the molecular basis for these variants. By using a modification of the polymerase chain reaction (PCR), mu and kappa Ig loci were sequenced from nine Id- variants derived directly by anti-Id immunoselection in vivo. Ig kappa loci sequence analysis was also performed from 10 additional variants amplified directly from tumor cells in vitro without immunoselection. We demonstrate that the molecular mechanism underlying tumor cell escape in this model is the spontaneous emergence of variants that have undergone kappa L chain gene "re-rearrangement" before positive selection by the anti-Id antibody. A second round of re-rearrangement was also demonstrated to occur within primary tumor variants. Re-rearrangement of the 38C13 tumor cell Ig kappa locus is strongly biased toward use of variable kappa genes within the conserved V kappa-Ox1 gene family, although their use is not exclusive. With the use of RNA PCR re-rearrangement was documented to occur in vitro at a frequency of approximately 1.0 x 10(-5)/cell. These findings may have important implications for the application of anti-Id antibodies as a therapeutic approach for human lymphomas and for understanding of the Ig gene rearrangement process.     

Idiotype vaccination against murine B cell lymphoma. Inhibition of tumor immunity by free idiotype protein

A murine B cell lymphoma (38C13) was used as a model to study the induction of idiotype (Id)-specific tumor immunity. Immunization of syngeneic mice with Id protein derived from the tumor resulted in the production of anti-Id antibodies by the host and in the induction of a state of resistance to tumor growth. Tumor immunity could be established only if the Id protein was conjugated to a strongly immunogenic carrier protein such as keyhole limpet hemocyanin or thyroglobulin, and if the conjugate was administered at least 1 week prior to tumor challenge. Free Id protein, such as that present in tumor bearing animals, was found to inhibit tumor immunity in a dose-dependent manner. Although tumor immunity could be induced in animals with pre-existent serum Id protein, the expression of the immune state was inhibited by the presence of the soluble protein.     

Treatment of a murine B cell lymphoma with monoclonal antibodies and IL 2

A transplantable murine B cell lymphoma was used to study combination therapy with anti-idiotype antibody and interleukin 2 (IL 2). Class-switched IgG2a and IgG2b antibodies were compared. A marked additive and sometimes synergistic effect was seen when IL 2 was combined with either IgG2a or IgG2b anti-idiotype antibodies. A synergistic effect was also seen when similar experiments were performed in nude mice. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays showed that IL 2 enhanced antibody-mediated lysis by peritoneal cells exposed to IL 2 in vitro in a dose-related manner. Peritoneal cells harvested from mice treated in vivo with IL 2 contained an increased number of T cells and asialo GM+ natural killer cells, and also mediated enhanced ADCC. Depletion of natural killer cells with anti-asialo GM and complement resulted in a marked decrease in the antibody-dependent cytotoxicity mediated by these peritoneal cells. The mechanism of synergy between monoclonal antibody and IL 2 may be due to the direct or indirect activation of natural killer cells mediating ADCC.     

Idiotype variants emerging after anti-idiotype monoclonal antibody therapy of a murine B cell lymphoma

One of the difficulties encountered with the treatment of human B cell malignancies with anti-Id antibodies is the emergence of Id variants. The current study was designed to investigate this phenomenon further by using the murine B cell lymphoma model 38C13. Tumors were harvested that developed despite treatment with the anti-Id antibody S1C5 in mice inoculated with 38C13 cells and evaluated by immunofluorescence. Various phenotypes were found among escaping tumor cells. Some cells continued to react with S1C5 whereas others lost S1C5 reactivity. Among these latter cells, some continued to express surface IgM kappa, whereas others no longer expressed surface mu or kappa. After Id variant cell lines were established, immunofluorescence and ELISA of cell lysates from the surface IgM kappa- lines revealed persistent intracellular mu H chain but no detectable kappa. Surface IgM kappa+ lines were fused with myeloma cells and the Ig proteins secreted by the resultant hybridomas analyzed. The apparent m.w. of the mu-chains of these rescued Ig was the same as wild-type 38C13, whereas the kappa-chains were either the same or different in m.w. from the wild type. The IgM kappa of the variant line, T3C, weakly reacted with S1C5 and did not react with other anti-Id antibodies. The IgM kappa of the other variants were nonreactive with all the antibodies. Immunofluorescence of these surface Ig+ variants confirmed this finding. Some of the surface Ig+ and Ig- variant lines grew identically to wild-type tumor in vivo, but only the weakly S1C5-reactive variant T3C was inhibited in its growth by S1C5. Moreover, T3C was the only one of these lines capable of being lysed in vitro with S1C5 by antibody-dependent cellular cytotoxicity. Further studies revealed that surface Ig+ and Ig- variants emerge in escaping tumors with similar frequency and that these variants represent a major mode of tumor escape from anti-Id treatment in this model.     

Anti-idiotypic mechanisms involved in suppression of a mouse B cell lymphoma, BCL1

Immunization of BALB/c mice with idiotypic IgM rescued by hybridization from the syngeneic BCL1 lymphoma protects specifically against challenge with tumor cells, with 83% surviving greater than 100 days compared with controls (38 +/- 10 days). Spleens from long-term survivors (greater than 6 mo) with no macroscopically visible tumor, when examined with anti-idiotypic antibody, showed a range of apparently dormant tumor with BCL1 cells present at 2 to 50% of total. A spectrum of protection against tumor resulted from immunization, and tumor emerging in the period 53 to 173 days postpassage was investigated for expression of idiotype. It was found that cells from individual mice expressed variable amounts of idiotypic IgM at the cell surface, although it was always detectable in the intracellular compartment. Unlike typical BCL1 cells, tumor cells developing in immune spleens often secreted little idiotypic IgM either in vitro or in vivo. This modulation of expression and secretion of idiotype was detected even in the apparent absence of serum anti-idiotypic antibody. On passage of spleen cells from the long-term survivors into naive animals, BCL1 tumor developed and killed the recipients in a way indistinguishable from routine tumor passage. These tumor cells, however, both expressed and secreted IgM of the same idiotype as the original tumor. It appears therefore that tumor development in immunized mice is suppressed by a process that includes modulation but not selection of the tumor cell idiotypic determinants. Analysis of possible mechanisms of suppression revealed the presence of cytotoxic anti-idiotypic antibody at variable levels in sera of immunized mice, and splenic T cells that proliferated specifically in response to idiotypic IgM. Only low levels of cytotoxic T cells were found. Passive transfer studies demonstrated a major role for antibody in protection against tumor, with no significant enhancement by immune lymphocytes.     

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.     

The "lamin B-fold". Anti-idiotypic antibodies reveal a structural complementarity between nuclear lamin B and cytoplasmic intermediate filament epitopes.

Previous studies have shown that nuclear lamin B binds specifically to the C-terminal domains of type III intermediate filament (IF) proteins under in vitro conditions. To further explore such site-specific interactions, we have used a two-step anti-idiotypic antibody approach. First, a monoclonal antibody disrupting the cytoplasmic IF network organization of living cells (mAb7A3) (Matteoni, R., and Kreis, T. E. (1987) J. Cell Biol. 105, 1253-1265) was characterized. Epitope mapping demonstrated that this antibody recognized a site located in the C-terminal domains of vimentin and peripherin (type III IF proteins). mAb7A3 was able to inhibit more than 80% of the in vitro binding of nuclear lamin B to PI, a synthetic peptide modeled after the C-terminal domain of peripherin that comprises a lamin B-binding site (Djabali, K., Portier, M. M., Gros, F., Blobel, G., and Georgatos, S. D. (1991) Cell 64, 109-121). In a second step, animals were immunized with mAb7A3 and the resulting anti-idiotypic sera were screened. Two of these antisera reacted specifically with nuclear lamin B but not with type A lamins or cytoplasmic IF proteins. The anti-lamin B activity of one of the antisera was isolated by affinity chromatography using a lamin B-agarose matrix. The reaction of these affinity-purified antibodies with lamin B was inhibited by mAb7A3. Furthermore, the anti-lamin B antibodies reacted with Fab fragments of mAb7A3 and abolished binding of lamin B to PI. From these data we conclude that anti-idiotypic antibodies against the paratope of mAb7A3 recognize specific epitopes of the lamin B molecule that have shapes complementary to the one of the C-terminal domain of type III IF proteins. We speculate that these (regional) conformations, which we term the "lamin B-fold," may also occur in non-lamin proteins that mediate the anchorage of IFs to various membranous organelles.     

Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B cell lymphoma. A study of hybridoma class switch variants

An initial panel of four syngeneic monoclonal antibodies directed against the idiotype of a murine B cell lymphoma was used to treat this tumor in vivo. The antibody in the panel of the IgG2a isotype was more effective in treatment than the other antibodies, which were of the IgG1 and IgG2b isotypes. To independently assess the role of antibody isotype in mediating antitumor effects, switch variant hybridoma families were isolated from the hybridomas secreting the less effective IgG1 and IgG2b antibodies. A family isolated from an IgG1-secreting parent consisted of IgG1-, IgG2b-, and IgG2a-secreting members, and an IgG2a variant was isolated from an IgG2b-secreting parent for another family. Antibody members of each family differed only in heavy chain composition and were the same with respect to their light chains and their affinity and specificity for idiotype. The IgG2a members of both families were superior to the other members in inhibiting tumor growth with an order of effectiveness of IgG2a greater than IgG1 greater than IgG2b. These in vivo results paralleled the abilities of these different isotype antibodies to mediate antibody-dependent cellular cytolysis in vitro. For the IgG2b----IgG2a family, in vivo treatment with the IgG2a member given i.p. after i.p. tumor challenge at one-tenth the dose of the IgG2b member was still superior to the latter. At one-hundredth the dose of the IgG2b, the IgG2a was still superior to the latter when the antibodies were given i.p. and tumors subcutaneously. These data and those showing that the clearance of these antibodies from the serum differed in only a relatively minor way indicate that the IgG2a antibodies in this system had greater antitumor effects primarily by virtue of their greater capacity for host effector interaction.     

Antigen-induced murine B cell lymphomas. II. Exploitation of the surface idiotype as tumor specific antigen.

In the accompanying report, we have described the characterization of two unusual murine B cell lymphomas, CH1 and CH2. A heterologous antiserum, which we refer to as "anti-idiotype" serum, has been raised to the detergent-solubilized surface immunoglobulin of CH1. The following criteria have established that this antiserum is specific for the CH1 tumor and that it reacts with V region determinants of the tumor surface IgM: 1) the antiserum reacts with CH1 tumor cells, but not normal mouse lymphoid cells or CH2 tumor cells, in indirect immunofluorescence and C-dependent cytotoxicity testing, 2) capping with the anti-idiotype serum removes all or most of the tumor surface Ig, 3) the antiserum forms a single band of precipitation against serum from CH1 tumor-bearing mice, when tested by double diffusion precipitin analysis, and 4) a single band of precipitation is formed in the electrophoretic migration position of IgM when the anti-idiotype antiserum is tested against serum from CH1 tumor-bearing mice in immunoelectrophoresis. Furthermore, we have demonstrated that this antiserum is useful in monitoring tumor growth and is a potent immunotherapeutic agent. Specifically, 50% of mice injected with a lethal tumor inoculum and given a small dose of anti-idiotype serum 2 days later remain tumor free, whereas all tumor-challenged control mice died within 30 days.     

A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.     

The control of IgM expression in a murine B cell lymphoma

The regulation of IgM expression was studied in clones derived from a murine B lymphocyte cell line, WEHI279.1. During normal B cell development IgM heavy chain synthesis increases concomitantly with heightened IgM secretion and reduced cell-surface IgM. However, in these subclones, the levels of membrane-bound and secreted IgM were regulated independently of one another. The amount of IgM secreted by the cells was tightly coupled to the amount of heavy chain synthesis, suggesting that the major control of secretion is pretranslational. Surface IgM exhibited a more complex regulation, with both pre- and posttranslational components. Variation in the expression of both forms of IgM occurred at high frequency. Although IgM expression follows a unidirectional pathway in nontransformed cells, the variability in these tumor cells was reversible and cellautonomous. High levels of phenotypic variability may be important in the ability of transformed cells to escape the immune response.     

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 (α-CDl9×α-CD3) with a broader applicability is now being characterized and tested in vivo. Accepted: 14 October 1997     

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.     

Mycobacterial HSP70 as an adjuvant in the design of an idiotype vaccine against a murine lymphoma

Mycobacterial HSP70 protein coupled with ovalbumin is known to elicit antigen-specific CD8⁺ T-cell response. We investigated whether anti-idiotype immunity can likewise be enhanced using a conjugate of recombinant mycHSP70 and the Ig Id in a murine lymphoma model, A20. Plasmids were constructed of A20 tumor to generate A20Id-HSP70 (scFv-H), unconjugated A20Id (scFv), and mycHSP70 (H) recombinant proteins. We evaluated their relative efficacy in activating anti-tumor immunity that can reduce the mortality of tumor-challenged BALB/c mice; significantly, a longer term protection (>50% of the population) was observed in mice vaccinated with scFv-H compared to those receiving the scFv or H proteins. Concomitantly a much higher-level activation in anti-A20 cytotoxic T-cell activity, IFN-γ secretion and predominantly anti-A20 IgG2a response was also observed with the scFv-H group. Thus, conjugating HSP70 with the A20Id renders the latter significantly immunogenic and affords longer protection against A20 tumor progression.     

Bispecific anti-idiotype/anti-CD3 antibody therapy of murine B cell lymphoma.

Retargeting of T cells by bispecific IgG which binds to both CD3 and a tumor-associated Ag can induce T cell lysis of target cells irrespective of TCR specificity. The current studies were designed to further explore the efficacy and specificity of bispecific IgG-directed therapy in an immunocompetent animal model, and to evaluate the mechanisms responsible for bispecific IgG-directed inhibition of tumor cell growth by using the 38C13 murine lymphoma system. In vitro, proliferation of activated T cells in the presence of bispecific IgG was increased when the relevant, but not the irrelevant target cells were present. Bispecific IgG specifically induced activated T cell mediated lysis of cells expressing the target Ag, but not of cells expressing an irrelevant Ag, even when the irrelevant cells were in the same cell mixture, indicating contact between target cells and T cells plays a major role in bispecific IgG-mediated lysis. Bispecific IgG was less effective than anti-Id at inducing target cell lysis when peritoneal macrophages were used as effectors, suggesting bispecific IgG Fc is not responsible for cytotoxicity in this system. In vivo, bispecific IgG was significantly superior to anti-Id, anti-CD3, or a combination of anti-Id and anti-CD3 in preventing tumor growth in immunocompetent mice inoculated with syngeneic lymphoma. Phenotypic evaluation of tumors that emerged despite therapy indicated bispecific IgG selects for the emergence of Id variant lymphoma cells. In separate studies, 38C13 tumor inocula containing cells recognized by the therapeutic antibody were supplemented with a small number of 38C13 cells which expressed a distinct Id not recognized by the therapeutic antibody. Untreated mice inoculated with this mixture developed tumors containing cells of both phenotypes, whereas tumors emerging from mice treated with bispecific IgG contained only cells expressing the nonreactive Id. These studies demonstrate bispecific IgG-directed lysis is therapeutically superior to monospecific anti-Id therapy in the 38C13 tumor model, and that tumor lysis is mediated largely by cell-cell contact. As with other forms of anti-Id based therapy, Id variants can emerge as resistant cell populations after bispecific IgG therapy.