Immunomodulation in the treatment of haematological malignancies.

Despite the continuous advances in immunology and cancer biology, haematological malignancies are often incurable. Conventional chemotherapy and radiation are efficacious for some lymphoma and leukaemia, however relapse and progressive disease often occurs. The evidence that the immune system can play an essential role in controlling cancer progression provide a basis for the development of active therapies, such as immunization, aimed to evoke or amplify a tumour-specific immune response. However, the inability of the patient's own immune system to mount effective responses against tumour antigens is a major limit of vaccination approaches. The adoptive transfer of effectors of the adaptive immune system is an attractive strategy to circumvent the limitations of autologous immune responses. Donor lymphocyte infusion and the transfer of monoclonal antibodies (MoAbs) have been the first forms of adoptive therapy approved for clinical use and are still fundamental components of immunotherapy of haematological malignancies. Due to the continuous characterization of tumour-specific antigen, the development of tumour-tailored therapies that exploit the specificity of antibodies and T cell receptors (TCRs) is progressing rapidly. This review highlights the current advances in the field of adoptive immunotherapy of haematological malignancies, starting by elucidating the ongoing progress in passive transfer of MoAbs. We will also discuss recent advances in the adoptive transfer with tumour-specific high avidity T cells, which can be generated ex vivo by the transfer of gene constructs encoding single chain antibodies or TCRs, thus redirecting T cell specificity to selected tumour antigens. The ability to produce gene-modified T cells of desired specificity and defined functional activity may improve in the future T cell based immunotherapy of cancer.

Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner.

P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.

Efficient folding of the FcepsilonRI alpha-chain membrane-proximal domain D2 depends on the presence of the N-terminal domain D1.

Human high affinity receptor for IgE is a membrane glycoprotein multichain complex presenting two extracellular Ig modules in its alpha-chain (D1D2). The receptor IgE binding region is located within the membrane-proximal module D2, while the N-terminal module D1 appears to promote an optimal receptor conformation for IgE binding. To understand the structural relationship between the two modules, we dissected FcepsilonRI alpha-chain into its discrete Ig units and expressed them in mammalian cells. Unexpectedly, D2 was secreted as a disulphide-linked dimer, while D1 was monomeric. Active secretion and full glycosylation of dimeric D2 suggest a native-like conformation of the protein, justifying the escape from the endoplasmic reticulum/Golgi quality control systems. We then propose a domain-swapping model for D2, in which two interdigitated polypeptide chains assume the overall conformation of two Ig modules, as observed for rat CD2 N-terminal domain. Fusion of an unrelated Ig fold moiety at the N terminus of D2 did not interfere with its dimerisation. While D1D2 assumes a correct fold, co-expression of both isolated domains in the same cell did not restore monomeric folding of D2. Thus, D1 appears to assist the appropriate folding of FcepsilonRI alpha-chain, acting as an uncleavable intramolecular chaperone-like block towards D2.

Anti-idiotypic DNA vaccines for B-cell lymphoma therapy.

The idiotypic determinants expressed by immunoglobulin at the surface of malignant B-cells provide specific targets for vaccination strategies. However, as self-antigens they are poorly immunogenic and vaccines must include carriers to improve immune responses. Chemical cross-linking of purified idiotypic protein is so far the only method which has been employed in clinical trials while a number of second-generation vaccines have been developed in mouse models. These strategies are based on the use of recombinant DNA technology to create fusion proteins that contain the idiotype genetically linked to molecules that act as immunological adjuvants. Fusion proteins can be made available to the immune system by direct delivery of naked DNA with great advantages in terms of time and costs. The most relevant approaches are listed and discussed with particular emphasis to the mechanism by which different molecules exert their adjuvant effect. The role of cellular versus antibody mediated suppression of tumor growth following Id vaccination is examined by comparing the most recent data on the mechanism of tumor protection. In addition, an analysis of the specificity of anti-idiotypic antibodies induced by scFv-DNA vaccination as compared to protein immunization is provided.

Idiotypes as immunogens: facing the challenge of inducing strong therapeutic immune responses against the variable region of immunoglobulins.

Idiotype (Id)-based immunotherapy has been exploited as cancer treatment option. Conceived as therapy for malignancies bearing idiotypic antigens, it has been also extended to solid tumors because of the capacity of anti-idiotypic antibodies to mimic Id-unrelated antigens. In both these two settings, efforts are being made to overcome the poor immune responsiveness often experienced when using self immunoglobulins as immunogens. Despite bearing a unique gene combination, and thus particular epitopes, it is normally difficult to stimulate the immune response against antibody variable regions. Different strategies are currently used to strengthen Id immunogenicity, such as concomitant use of immune-stimulating molecules, design of Id-containing immunogenic recombinant proteins, specific targeting of relevant immune cells, and genetic immunization. This review focuses on the role of anti-Id vaccination in cancer management and on the current developments used to foster anti-idiotypic B and T cell responses.

WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

T-cell receptor gene therapy for cancer: the progress to date and future objectives.

In the last decade research has begun into the use of T-cell receptor (TCR) gene therapy as a means to control and eradicate malignancies. There is now a large body of evidence to demonstrate that through the use of this technology one can redirect T-cell antigen specificity to produce both cytotoxic and helper T cells, which are functionally competent both in vitro and in vivo and show promising antitumour effects in humans. This review focuses on the means by which TCR gene transfer is achieved and the recent advances to modify the TCRs and vector delivery systems which aim to enhance the efficiency and safety of TCR gene transfer protocols.

Monoclonal T-cell receptors: new reagents for cancer therapy.

Adoptive transfer of antigen-specific T lymphocytes is an effective form of immunotherapy for persistent virus infections and cancer. A major limitation of adoptive therapy is the inability to isolate antigen-specific T lymphocytes reproducibly. The demonstration that cloned T-cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. TCR gene-modified lymphocytes display antigen-specific function in vitro, and were shown to protect against virus infection and tumor growth in animal models. A recent trial in humans demonstrated that TCR gene-modified T cells persisted in all and reduced melanoma burden in 2/15 patients. In future trials, it may be possible to use TCR gene transfer to equip helper and cytotoxic T cells with new antigen-specificity, allowing both T-cell subsets to cooperate in achieving improved clinical responses. Sequence modifications of TCR genes are being explored to enhance TCR surface expression, while minimizing the risk of pairing between introduced and endogenous TCR chains. Current T-cell transduction protocols that trigger T-cell differentiation need to be modified to generate "undifferentiated" T cells, which, upon adoptive transfer, display improved in vivo expansion and survival. Both, expression of only the introduced TCR chains and the production of naïve T cells may be possible in the future by TCR gene transfer into stem cells.

Targeting the Wilms tumor antigen 1 by TCR gene transfer: TCR variants improve tetramer binding but not the function of gene modified human T cells.

We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the alpha- and beta-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated approximately 90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-gamma production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.

BCL1 lymphoma protection induced by idiotype DNA vaccination is entirely dependent on anti-idiotypic antibodies.

DNA vaccination with the idiotype (Id) of tumour B-cell membrane immunoglobulins (Ig) is a validated strategy to induce tumour protection to several mouse lymphomas. The relative contribution of anti-Id antibodies and T lymphocytes to tumour rejection is still debated. Previous studies in the BCL1 lymphoma model showed that scFv DNA immunisation induces a polyclonal antibody response restricted to conformational epitopes formed by the parental V(L)/V(H) association. We implemented a system based on this specificity to investigate the mechanism of BCL1 lymphoma protection induced by DNA immunisation. Antibody response and survival of mice immunised with the tumour Id scFv were compared with those of mice immunised simultaneously with two chimeric scFvs, containing either the tumour-derived V(L) or V(H) paired to an irrelevant V(H) or V(L) domain, respectively. Animals vaccinated with one or both chimeric constructs were not protected, despite the exposure to all putative tumour Id-derived MHC class I and class II T-cell epitopes. In addition, conformational antibodies induced by DNA vaccination caused tumour cells apoptosis and cell cycle arrest in vitro and transferred protection in vivo. Therefore, lymphoma rejection appears to be completely dependent on the induction of anti-Id antibodies.

Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner.

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.

A minimal receptor-Ig chimera of human FcepsilonRI alpha-chain efficiently binds secretory and membrane IgE.

We constructed a soluble minimal receptor-Ig chimera in which the two extracellular domains of human Fcepsilonhain (D1 and D2) were fused to the dimerizing C-terminal domain of human IgG1 heavy chain (gamma1-CH3). The protein was expressed and actively secreted by Chinese hamster ovary (CHO) cells as a fully glycosylated soluble dimeric protein. It showed efficient binding both to human membrane-bound IgE isoforms and to the two secretory IgE isoforms. Moreover, the dimeric receptor binds IgE with the expected 1:2 stoichiometry. The receptor-Ig chimera, in 2-fold molar excess, inhibited engagement of secretory IgE to rat basophilic leukemia cells expressing the human alphabetagamma receptor. Full self-nature and inability to bind Fcgamma receptors make this protein an attractive candidate for clinical applications and a novel biotechnological tool for atopic allergy research.