Engineering DNA vaccines that include plant virus coat proteins.
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Animals↗
Biomedical subjects
Freda K Stevenson
Publications and source records attributed to Freda K Stevenson.
Vaccine therapy in NHL: future promises and current limitations.
Recent advances in genomics are revealing new molecular targets for vaccination strategies in cancer, and unraveling the immune pathways that must be activated. Initially, work focused on idiotypic antigen expressed by the clonal immunoglobulin (Ig) of B-cell tumors. This involved assembling the encoding V(H) and V(L) genes as single-chain Fv (scFv), which has been fused to Fragment C (FrC) of tetanus toxin to enhance recognition and response. The fusion gene induces strong anti-idiotypic protection against lymphoma in experimental models, and is now in clinical trials with encouraging results emerging. This principle of fusing a pathogen-derived sequence is widely applicable. A coat protein from a potato virus substituted for the FrC sequence has also been found to be capable of promoting anti-tumor immunity. The current design is effective in inducing anti-idiotypic (Id) antibody and CD4+ T-cell attack. For intracellular tumor antigens expressed as MHC Class I-associated peptides, a design consisting of a gene encoding a single domain of FrC linked to a candidate peptide sequence has been developed. This induces fast cytotoxic T-cell attack on peptide-expressing tumor cells. Injection of DNA appears safe and clinical trials will test the efficacy of this approach, and probably lead to improvements in these flexible vaccines.
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Immunotherapy of hematologic malignancy.
Over the past few years, improved understanding of the molecular basis of interactions between antigen presenting cells and effector cells and advances in informatics have both led to the identification of many candidate antigens that are targets for immunotherapy. However, while immunotherapy has successfully eradicated relapsed hematologic malignancy after allogeneic transplant as well as virally induced tumors, limitations have been identified in extending immunotherapy to a wider range of hematologic malignancies. This review provides an overview of three immunotherapy strategies and how they may be improved. In Section I, Dr. Stevenson reviews the clinical experience with genetic vaccines delivered through naked DNA alone or viral vectors, which are showing promise in clinical trials in lymphoma and myeloma patients. She describes efforts to manipulate constructs genetically to enhance immunogenicity and to add additional elements to generate a more sustained immune response. In Section II, Dr. Molldrem describes clinical experience with peptide vaccines, with a particular focus on myeloid tissue-restricted proteins as GVL target antigens in CML and AML. Proteinase 3 and other azurophil granule proteins may be particularly good targets for both autologous and allogeneic T-cell responses. The potency of peptide vaccines may potentially be increased by genetically modifying peptides to enhance T-cell receptor affinity. Finally, in Section III, Dr. Heslop reviews clinical experience with adoptive immunotherapy with T cells. Transferred T cells have clinical benefit in treating relapsed malignancy post transplant, and Epstein-Barr virus associated tumors. However, T cells have been less successful in treating other hematologic malignancies due to inadequate persistence or expansion of adoptively transferred cells and the presence of tumor evasion mechanisms. An improved understanding of the interactions of antigen presenting cells with T cells should optimize efforts to manufacture effector T cells, while manipulation of lymphocyte homeostasis in vivo and development of gene therapy approaches may enhance the persistence and function of adoptively transferred T cells.
Hematologic Neoplasms↗
Features of the overexpressed V1-69 genes in the unmutated subset of chronic lymphocytic leukemia are distinct from those in the healthy elderly repertoire.
Chronic lymphocytic leukemia (CLL) comprises 2 subsets, distinguished by expression of unmutated or mutated V(H) genes, with the former having a worse prognosis. Biased usage of the V1-69 gene is found in unmutated cases and is combined with selected D gene segments and J(H)6. It is controversial whether this is a CLL-associated feature or mirrors the normal B-cell pattern. Since CLL is a disease of the elderly, where changes in the B-cell repertoire may occur, we have analyzed V1-69 usage in the elderly (older than 75 years) population. Using monoclonal antibody (MoAb) G6, specific for 51p1-related V1-69 alleles, we found no increased expression with age. In 51p1-encoded immunoglobulin M (IgM), complementarity-determining region 3 (CDR3) length and frequency of D and J(H) genes were similar to those in the healthy young and distinct from those in CLL. These findings support the concept that CLL arises from B cells driven by antigen/superantigen and is not a stochastic event in the elderly B-cell population.
Adult↗
V(H) gene analysis of splenic marginal zone lymphomas reveals diversity in mutational status and initiation of somatic mutation in vivo.
Tumors of the splenic marginal zone can present in spleen or blood. The maturational status of the neoplastic B cells from each site appears heterogeneous, with either unmutated or mutated variable-region heavy chain (V(H)) genes. To determine an influence of tissue location, we assessed matched blood and splenic tumor cells from 4 patients and found them identical. However, one patient with unmutated V(H) genes in blood and spleen developed a clonally related diffuse large B-cell lymphoma in the chest wall. Strikingly, this subclone had undergone significant somatic mutation, with clear intraclonal heterogeneity. To our knowledge, this is the first case of a B-cell tumor showing initiation of somatic mutation in vivo. The finding emphasizes that the tissue microenvironment can influence tumor cell behavior and possibly affect disease progression. Importantly, because several replacement mutations were located within or close to the complementarity-determining regions (CDRs), it raises the question of a role for antigen in driving tumor growth.
Amino Acid Sequence↗
Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen.
The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags.
Agglutinins↗
Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen.
DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.
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Typical Waldenstrom macroglobulinemia is derived from a B-cell arrested after cessation of somatic mutation but prior to isotype switch events.
There exists a wide spectrum of IgM-secreting B-cell tumors with different clinical behavior. Knowledge of the V(H) gene status can reveal their origin and clonal history. For Waldenstrom macroglobulinemia (WM), a distinct subtype of lymphoplasmacytic lymphoma, early data on limited sequences showed evidence for somatic mutation. A recent report of one case demonstrated intraclonal mutational activity occurring after transformation, a characteristic of germinal center lymphomas. To extend the investigation, we have analyzed 7 cases of WM. V(H) genes were somatically mutated with no evidence of intraclonal variation in all cases. In contrast to IgM-secreting multiple myeloma, there was no evidence for isotype switch transcripts in any of the cases. These data support the concept that typical WM is derived from a B cell that has undergone somatic mutation prior to transformation, at a point where isotype switch events have not been initiated. (Blood. 2002;100:1505-1507)
Amino Acid Sequence↗
Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma.
Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site. Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional V(H) genes as a consequence of somatic mutation. FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation. Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P <.001). Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 [41%] patients). Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively. In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv in Pichia pastoris, and glycosylation was demonstrated. These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL. Most novel sites are located in the complementarity-determining regions. V(H) sequences of nonfunctional V(H) genes contained few sites, arguing for positive selection in FL. One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment. This common feature of FL may be critical for tumor behavior.
Amino Acid Sequence↗
Vaccination with DNA encoding a single-chain TCR fusion protein induces anticlonotypic immunity and protects against T-cell lymphoma.
The clonotypic T-cell antigen receptor (TCR) provides unique Valpha and Vbeta sequences with potential as idiotypic targets for immunoregulation. For T-cell malignancies, vaccination with the TCR could induce therapeutic anti-idiotypic responses. To facilitate this approach, we have developed DNA vaccines that include the genes encoding TCR sequences from a T-cell lymphoma (TCL). To combine requirements for stable folding with a simple minimized single-chain construction, we used a three-domain ValphaVbetaCbeta sequence. To promote anti-TCR immunity, we fused a pathogen-derived sequence from tetanus toxin to the 3'-end of the single-chain TCR. The fusion gene vaccine induced anti-idiotypic antibodies and generated protection against the TCL. The critical requirement for the conformational integrity of the delivered TCR antigen was highlighted by the observation that DNA fusion vaccines containing either ValphaVbeta or VbetaCbeta sequences failed to generate antibodies reactive with the native TCR or provide protection. This is the first report of a DNA vaccine able to induce anti-idiotypic immunity against TCL, and it presents a simple strategy for selectively eliminating T-cell clones in vivo.
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CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease.
Although the presence or absence of somatic mutations in the immunoglobulin variable region (IgV(H)) genes in chronic lymphocytic leukemia (B-CLL) identifies subtypes with very different prognoses, the assay is technically complex and unavailable to most laboratories. CD38 expression has been suggested as a surrogate marker for the 2 subtypes. IgV(H) mutations and CD38 expression in 145 patients with B-CLL with a long follow-up were compared. The 2 assays gave discordant results in 41 patients (28.3%). Multivariate analysis demonstrated that Binet stage, IgV(H) mutations and CD38 were independent prognostic indicators. Median survival time in patients whose cells had unmutated IgV(H) genes and expressed CD38 was 8 years; in those with mutated IgV(H) genes not expressing CD38, it was 26 years. For those with discordant results, median survival time was 15 years. Thus, although CD38 expression does not identify the same 2 subsets as IgV(H) mutations in CLL, it is an independent risk factor that can be used with IgV(H) mutations and clinical stage to select patients with B-CLL with the worst prognoses. Using cryopreserved cells taken at intervals during the course of the disease, however, changes of CD38 expression over time were demonstrated in 10 of 41 patients. Causes of the variation of CD38 expression require further study. Additional prospective studies are required for comparing CD38 expression with other prognostic factors and for taking sequential measurements during the course of the disease.
ADP-ribosyl Cyclase↗
A gamma-herpesvirus immune evasion gene allows tumor cells in vivo to escape attack by cytotoxic T cells specific for a tumor epitope.
DNA vaccines induce CTL attack on target tumor epitopes, but tumor elimination in vivo also requires sufficient effector CTL to enter the site, guided by inflammatory chemokines. Many herpesviruses contain genes for chemokine and chemokine receptor-like proteins to protect infected cells from immune attack. To assess if this evasion strategy could protect tumor cells, we used a model where CTL specific for a single epitope were the only effectors. Following DNA vaccination, CTL eliminated tumor cells from a subcutaneous site. However, introducing a viral gene encoding a secreted broad-spectrum chemokine-binding protein (M3) into tumor cells completely blocked CTL attack. Transduced tumor cells also protected neighboring non-transduced tumor. These findings confirm the importance of chemokines for migration of CTL to a non-lymphoid site. They may have relevance for escape of human virus-associated malignancies, and raise the question of whether analogous molecules might contribute to the failure of CTL to eliminate tumors.
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Insight into the potential for DNA idiotypic fusion vaccines designed for patients by analysing xenogeneic anti-idiotypic antibody responses.
DNA vaccines induce immune responses against encoded proteins, and have clear potential for cancer vaccines. For B-cell tumours, idiotypic (Id) immunoglobulin encoded by the variable region genes provides a target antigen. When assembled as single chain Fv (scFv), and fused to an immunoenhancing sequence from tetanus toxin (TT), DNA fusion vaccines induce anti-Id antibodies. In lymphoma models, these antibodies have a critical role in mediating protection. For application to patients with lymphoma, two questions arise: first, whether pre-existing antibody against TT affects induction of anti-scFv antibodies; second, whether individual human scFv fusion sequences are able to fold consistently to generate antibodies able to recognize private conformational Id determinants expressed by tumour cells. Using xenogeneic vaccination with scFv sequences from four patients, we have shown that pre-existing anti-TT immunity slows, but does not prevent, anti-Id antibody responses. To determine folding, we have monitored the ability of nine DNAscFv-FrC patients' vaccines to induce xenogeneic anti-Id antibodies. Antibodies were induced in all cases, and were strikingly specific for each patient's immunoglobulin with little cross-reactivity between patients, even when similar VH or VL genes were involved. Blocking experiments with human serum confirmed reactivity against private determinants in 26-97% of total antibody. Both immunoglobulin G1 (IgG1) and IgG2a subclasses were present at 1.3 : 1-15 : 1 consistent with a T helper 2-dominated response. Xenogeneic vaccination provides a simple route for testing individual patients' DNAscFv-FrC fusion vaccines, and offers a strategy for production of anti-Id antibodies. The findings underpin the approach of DNA idiotypic fusion vaccination for patients with B-cell tumours.
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DNA gene fusion vaccines against cancer.
The ability of DNA vaccination to induce effective immune responses has been shown in a range of preclinical disease models. However, the potency of DNA vaccines must be further improved for their use in patients. DNA fusion vaccine strategies, whereby target antigens are genetically linked to immuno-enhancing molecules, are currently being explored. The ease of DNA manipulation has allowed incorporation of a wide variety of molecules able to promote antigen uptake, processing and presentation by professional antigen-presenting cells, to provide critical CD4 T-cell help and to activate more effective immune effector pathways. These strategies are particularly important for cancer vaccines to increase their immunogenicity and to overcome tolerance.
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Failure of vaccination with idiotypic protein or DNA, (+/-IL-2), the depletion of regulatory T cells, or the blockade of CTLA-4 to prolong dormancy in mice with BCL1 lymphoma.
Immunization of mice with the idiotype (Id) immunoglobulin from the murine B cell lymphoma, BCL1, before inoculating tumor cells can induce tumor dormancy. In this model, the tumor cells grow for a short period of time and then regress. The mice live for months or years with approximately 1 million tumor cells in their spleens. Some mice relapse due to decreases in the anti-Id antibody titers or the development of mutations in the residual tumor cells which render them refractory to negative signaling by the anti-Id antibody. In this study we determined whether we could eliminate the residual dormant cells by using a DNA vaccine against the Id or by immunomodulation of T-cell subsets in vivo. Our results demonstrate that dormancy can be maintained by further immunizations with either the BCL1 Id protein or DNA vaccine encoding its single-chain Fv fragment. We also found that a cytotoxic T-cell response was not induced by either in vivo administration of vaccine alone or by the vaccine plus interleukin-2. In addition the injection of anti-cytotoxic T-lymphocyte-associate antigen did not prolong dormancy. Finally, the in vivo administration of anti-CD25 to deplete regulatory T cells did not prolong dormancy. Dormancy in this model is dependent primarily upon anti-Id antibodies, our results suggest that other strategies to target residual dormant BCL1 cells are warranted. They also suggest that the elimination of dormant tumor may represent a greater challenge than the elimination of primary tumors.
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