Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
2001-08-06
2003-11-04
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S205100, C424S231100, C424S093200, C424S093210, C435S235100, C435S236000, C435S320100, C435S325000, C435S372000
Reexamination Certificate
active
06641817
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to attenuated herpes simplex viruses capable of efficiently infecting dendritic cells. It also relates to the use of such viruses in immunotherapy approaches to the treatment of disease.
BACKGROUND TO THE INVENTION
Dendritic cells (DCs) are the most potent antigen presenting cells and are efficient at inducing responses even to antigens to which the immune system has become tolerant. Thus for tumour immunotherapy, in which an immune response is raised against a tumour, the use of DCs may be ideal if they were made to present tumour specific antigens. DCs might also be used to present antigens derived from infectious agents such as bacteria, viruses or parasites, providing protective or therapeutic vaccines for such diseases. However effective transfer of antigens into DCs for any of these targets has proved the greatest problem with this approach.
To provide a realistic chance of generating a therapeutic immune response against a tumour antigen or other disease related antigen, several conditions have to be met. Firstly, it is necessary to identify molecules whose expression is tumour or disease specific (or at least selective), and which can therefore serve as the target for an immune response. This task has proved very difficult for the majority of common tumours, but is solved in for example the case of cervical cancer by the presence, in most cases, of the viral oncogenes E6 and E7, and for other tumours, good candidate antigens are beginning to be identified. For example the MUC-1 gene product is over, expressed in a number of tumours, including 90% of ovarian cancers. Various other tumour associated antigens have also been identified, any of which might be used in an immunotherapy treatment of cancer. Secondly, following the identification of the antigen/antigens, it is necessary to deliver the antigens in an immunogenic form to the immune system. To generate the cellular immune response critical for tumour rejection, this means the proteins must either be delivered inside the cytoplasm of a host cell (a difficult task for high molecular weight protein antigens) or synthesized by the host cells themselves after gene delivery or DNA immunisation. Viral vectors which have been considered for this purpose include vaccinia, adenoviruses, or retroviruses.
The cell-type which is now widely recognised as providing the optimal immune stimulus is the lymphoid dendritic cell (DC; see for example Girolomoni and Ricciardi-Castagnoli, 1997). Indeed the DC appears to be the only cell-type capable of stimulating a primary immune response in vivo, and moreover has even been shown to be capable of breaking established tolerance in certain circumstances. A number of groups are exploring the use of DCs in autologous adoptive immunotherapy protocols to stimulate immune responses against tumours in the hope that they may show a therapeutic effect. Such protocols involve culture and/or enrichment of DCs from peripheral blood, in vitro loading of DCs with antigen, followed by reintroduction of the DCs to the patient However this approach has been hampered by the absence of efficient means by which to load these cells with antigens. Recent work has however shown that presentation of antigens by peptide pulsed DCs has produced anti-tumour responses in vivo (Celluzzi et al., 1996; Zitvogel et al., 1996). As regard to viral vectors, retroviruses do not give high efficiency gene delivery to dendritic cells (Reeves et al., 1996; Aicher et al., 1997), and in our hands, unlike work reported by others (Arthur et al., 1997), adenoviruses only give low efficiency gene delivery.
We have previously tested and reported that herpes simplex viruses (HSV) can efficiently infect and deliver genes to dendritic cells (Coffin et al., 1998), although associated toxicity was not quantified. HSV has a number of advantages over other vector systems for this purpose, in that it can efficiently infect a wide variety of cell-types (including some very hard to infect with other vector systems e.g. Dilloo et al., 1997; Coffin et al., 1998), is easy to manipulate, and can accept large DNA insertions allowing the expression of multiple genes (reviewed by Coffin and Latchman 1996). Delivery of multiple antigens to dendritic cells followed by re-introduction into the body may be a particularly promising approach to the treatment of some cancers and infectious diseases.
SUMMARY OF THE INVENTION
We have previously found that, unlike other vectors in our hands, HSV can efficiently transduce dendritic cells (Coffin et al., 1998), but any toxic effects and the functional capabilities of the transduced dendritic cells were not tested. We have now tested a variety of HSV mutants, from essentially wild type viruses to viruses with mutations either preventing replication in some cell types (mutations in non-essential genes) or all cell types (mutations in essential genes), and some mutants with combinations of deletions in both essential and non-essential genes. We have found considerable differences in both the gene delivery efficiencies of these various mutants and the level of toxicity exhibited (as estimated by dendritic cell death).
Surprisingly we have found that: (i) a virus with inactivating mutations in ICP34.5, VMW65, vhs, and UL43 provides a virus with lower toxicity and higher gene delivery efficiency for dendritic cells than other replication competent viruses tested, including the virus reported previously (Coffin et al., 1998) and also a number of the replication incompetent viruses tested. This is particularly surprising as ICP34.5, VMW65, vhs, and UL43 are all usually regarded as non-essential genes. For example a similar virus including the deletion of one essential gene (mutations in ICP34.5, VMW65, vhs, and ICP27) showed a much lower gene delivery efficiency than this virus. Moreover, unlike the other virus mutants tested, results indicating that efficient antigen processing was occurring could be demonstrated within the target cells. This suggested that transduced dendritic cells retain a functional capability which would allow the stimulation of an effective immune response in vivo. We have also found that (ii) a virus disabled such that only very minimal levels of immediate early genes are expressed in target cells (and thus from which only very low level HSV gene expression generally would be expected) give similar results, unlike other mutants with only a single immediate early gene removed.
These results show that while efficient gene delivery to dendritic cells can be achieved relatively easily using HSV vectors, for the cells to retain their antigen processing capabilities the particular combination of mutations in the virus must be carefully chosen. The invention thus for the first time provides viral vectors for dendritic cells which provide highly efficient gene delivery, without adversely affecting the antigen processing capabilities of the infected dendritic cells.
Thus the present invention provides an attenuated herpes virus capable of efficiently infecting dendritic cells without preventing efficient antigen processing within the infected cell. Preferably said herpes virus is a human herpes simplex virus. More preferably said virus is a herpes simplex virus (HSV), for example HSV1 or HSV2 or a homologous viral strain.
In one embodiment (embodiment (i) above), the herpes virus of the invention typically lacks a functional UL43 gene and a functional vhs gene, if an HSV strain, or their functional equivalents in other viral species. Preferably the virus of the invention also lacks a functional HSV ICP34.5 gene, or its functional equivalent in other viral species. The virus of the invention may also lack a functional VMW65 gene or its functional equivalent in another viral species, especially due to a mutation in said gene which abolishes its transcriptional-activation activity. In a second embodiment (embodiment (ii) above), the herpes virus of the invention contains mutations minimising immediate early gene expression in cells not contemplating the
Chain Benjamin
Coffin Robert Stuart
Biovex Limited
Mosher Mary E.
Nixon & Vanderhye P.C.
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