DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma.

Vaccination with idiotypic protein protects against B-cell lymphoma, mainly through anti-idiotypic antibody. For use in patients, DNA vaccines containing single-chain Fv derived from tumor provide a convenient alternative vaccine delivery system. However, single-chain Fv sequence alone induces low anti-idiotypic response and poor protection against lymphoma. Fusion of the gene encoding fragment C of tetanus toxin to single-chain Fv substantially promotes the anti-idiotypic response and induces strong protection against B-cell lymphoma. The same fusion design also induces protective immunity against a surface Ig-negative myeloma. These findings indicate that fusion to a pathogen sequence allows a tumor antigen to engage diverse immune mechanisms that suppress growth. This fusion design has the added advantage of overcoming potential tolerance to tumor that may exist in patients.

A genetic approach to idiotypic vaccination for B cell lymphoma.

Idiotypic immunoglobulin expressed by a B cell tumor presents a clear tumor antigen which could be attacked by vaccination of the host. Vaccination with idiotypic protein has been shown to induce protective immunity against lymphoma, but application to patients is limited by the requirement of "personal" vaccines for each patient. A genetic approach enables V-region sequences encoding idiotypic antigen to be rescued from tumor biopsies, and to be assembled as scFv fragments. These can be expressed in bacteria to produce recombinant protein, or used directly as naked DNA vaccines. Intramuscular injection of idiotypic DNA from a mouse B cell lymphoma induces low levels of syngeneic anti-idiotypic antibody in serum. Response can be stimulated by co-injection of DNA plasmids encoding either IL-2 or GM-CSF, and T cells which proliferate in response to idiotypic IgM are generated. However, protection against tumor appears to be blocked by continuing secretion of idiotypic antigen from the persisting vaccine vector, which forms immune complexes with serum antibody. Methods for regulating the level of scFv to engage the immune system, but not to block the effector arm are being investigated. Similar control will be applicable to the cytokine vectors, which can deliver encoded cytokines designed to activate immune pathways for tumor destruction. Experience gained in lymphoma may be extended to other tumors with defined tumor antigens.

DNA fusion vaccines against B-cell tumors.

The ability of naked DNA to induce immune responses against encoded antigen has been clearly demonstrated for infectious diseases (1). In many cases, the induced immunity is able to protect against infection, and can approach the efficacy of exogenous antigen (2).

Differential rates of somatic hypermutation in V(H) genes among subsets of chronic lymphocytic leukemia defined by chromosomal abnormalities.

Chronic lymphocytic leukemia (CLL) is a B-cell tumor involving small lymphocytes that generally express the CD5 antigen and low levels of surface Ig. Within this definition, there is heterogeneity among cases in cell morphology, karyotypic abnormalities, and clinical course. Trisomy 12, the most frequent karyotypic abnormality, is commonly found in a subset of CLL with atypical morphology. It has also been associated with advanced disease, and possibly with a less favorable prognosis. A further subset of cases with abnormalities involving chromosome 13q14 have typical lymphocyte morphology. Occasionally, the two abnormalities are found together. To assess the clonal history of the cell of origin in disease subsets defined by these two chromosomal abnormalities, we investigated the usage of V(H) genes and the pattern of somatic mutation in 10 cases of trisomy 12 with atypical morphology and eight cases of 13q14 abnormality with typical morphology. In addition, four cases with both chromosomal abnormalities were analyzed. Results confirm a common usage of the V(H)1 family in all subsets. However, the patterns of somatic mutation were distinct, with cases of trisomy 12 showing a minimal level of mutation (mean +/- SD, 0.34% +/- 0.86%) and cases of 13q14 abnormality showing significant levels (6.5% +/- 1.67%). The four cases with both abnormalities showed a mixed pattern. All mutated cases had intraclonal homogeneity, and three of 10 had a pattern indicative of antigen selection. These results suggest that the clonal history of the two subsets of CLL may differ.

VH gene sequences from a novel tropical splenic lymphoma reveal a naive B cell as the cell of origin.

The prevalence of malaria and other infections in tropical Africa provides a setting for the emergence of B-cell tumours distinct from that in Western countries. Attempts to draw comparisons with Western lymphomas have led to difficulties, with so-called African chronic lymphocytic leukaemia (CLL) having a different pattern of incidence from Western CLL. Splenomegaly is common in African CLL, and this has posed diagnostic problems in differentiating the tumour from malaria-associated hyper-reactive malarial splenomegaly (HMS). One feature of the splenomegalic form of African CLL is that the tumour cells often possess short but fine cytoplasmic projections reminiscent of those observed in Western splenic lymphoma with villous lymphocytes (SLVL). Analysis of Ig VH genes both facilitates discrimination between clonal B-cell tumours and HMS, and reveals the differentiation status of the cell of origin. This study indicated that VH genes of nine cases of clonal splenic B-cell tumours with villous lymphocytes from Ghana were relatively unmutated, consistent with an origin from a naive B cell. These features differ from SLVL which arises from a post-follicular antigen-selected B cell. One possibility is that these splenic B-cell tumours derive from a splenic T-independent B cell, with malaria infection as a potential influence.

V(H) gene sequences from primary central nervous system lymphomas indicate derivation from highly mutated germinal center B cells with ongoing mutational activity.

Primary central nervous system lymphoma (PCNSL) represents 1% to 3% intracranial tumors. Most PCNSL are located in the brain, and 75% are large B-cell lymphomas. The largest subgroup of these tumors contains cells that resemble centroblasts and has been labelled diffuse centroblastic (polymorphous) lymphoma. To investigate the cell of origin and the clonal history of these tumors, we have analyzed V(H) gene of 5 cases of PCNSL, all confirmed by histological studies to be Epstein-Barr virus (EBV)-negative, high-grade diffuse B-cell lymphomas. The V4-34 gene of the V(H)4 family was used in 4 of 5 cases. All V(H) genes were found to have accumulated very high levels of somatic mutation (14% to 25%). In 3 of 5 cases, intraclonal nucleotide heterogeneity, including codon deletion in some clones in 1 case, was observed, indicating that the V(H) genes were still under the influence of the somatic hypermutation mechanism. Analysis of the distribution of silent and replacement mutations showed evidence for preservation of immunoglobulin structure in all cases. These results suggest that, although there is no evidence for germinal center formation in the brain tissue, PCNSL is derived from a B cell with features associated with location in a germinal center environment.

Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression.

Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VH genes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 "clonal signature" was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.

DNA vaccines against lymphoma: promotion of anti-idiotypic antibody responses induced by single chain Fv genes by fusion to tetanus toxin fragment C.

Idiotypic determinants can act as tumor-associated Ags for B cell lymphoma. Vaccination with idiotypic protein and adjuvant is known to induce specific protection against lymphoma challenge in mice, largely mediated by anti-idiotypic Ab. For facilitating the approach for patients, the V(H) and V(L) genes used to encode the individual idiotypic determinants of each tumor can be obtained by PCR and assembled as single chain Fv (scFv). DNA vaccines containing scFv sequences alone induce low and poorly reproducible levels of anti-idiotypic Ab, likely to be insufficient to suppress tumor in patients. In addition, it may be necessary to break tolerance to Id in tumor bearers. By fusing the gene for fragment C of tetanus toxin to the C terminus of human scFv, we have promoted the anti-scFv Ab response in mice by >50-fold in three of three cases. The induced Abs are mainly against idiotypic determinants, and react specifically with patients' tumor cells, indicating optimal folding of the scFv molecule in the fusion protein. For both antigenic components of the DNA vaccine, the IgG subclass distribution showed a relative increase in IgG2a as compared with vaccination with IgM protein in adjuvant. In patients, the fusion gene should both promote anti-idiotypic Ab and induce Abs against fragment C of tetanus toxin. The latter response would provide a potentially useful comparative measure of the ability of patients to respond to conventional Ag delivered via DNA.

DNA vaccines against haematological malignancies.

DNA vaccines against cancer have to activate an inadequate or damaged immune system in order to attack residual cancer cells. Although the potential problem of tolerance may be overcome by transplantation, provision of high levels of T-cell help is likely to be an important factor in stimulating effective immune pathways. The fusion gene approach appears to provide the required help, and offers a rational design for raising both antibody and T-cell mediated attack against lymphoma and myeloma, which express idiotypic antigen at the cell surface or as a secreted protein respectively. Intriguingly, preliminary data indicate that the fusion gene approach promotes antibody responses against a different cell surface tumour antigen, CEA. Strategies for using DNA vaccines to induce attack on processed peptides bound to MHC class I molecules are also being developed. We hope and anticipate that all categories of tumour antigen may be susceptible to this powerful new technology. The critical clinical requirement, however, will be to treat the presenting tumour with maintenance or restoration of immune capacity. We await results of the preliminary clinical trials with great interest.