Methods and compositions to induce antitumor response

Details Methods and compositions to induce antitumor response Methods and compositions to induce antitumor response

1999-10-13

2003-11-18

Crouch, Deborah (Department: 1632)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

C435S069100, C435S083000, C435S320100, C435S325000

Reexamination Certificate

active

06649158

ABSTRACT:

BACKGROUND OF THE INVENTION
The immune system has evolved to process and recognize intracellular antigens via a class I MHC mediated antigen presentation pathway. Class I MHC restricted antigens are targeted by CD8 positive T cells largely consisting of cytotoxic T cells (CTL). An essential property of tumor antigen recognition by CD8 positive CTL cells is the requirement of the TCR to engage class I MHC/peptide complexes. Class I MHC restricted peptides are customarily derived from intracellular proteins. Thus, stimulation of immunity following in vivo administration of recombinant proteins preferentially stimulates antibody responses and only weak CTL responses.
Based on these fundamentals of CTL recognition, several methods have traditionally been utilized to stimulate CTL responses. Peptides with conanical sequences optimized for MHC class I binding can displace non-covalently bound peptide on cell surface MHC class I in a concentration dependent manner. Peptides have successfully been utilized to stimulate CTL responses in vivo but generally require the use of adjuvants such as IFA. Alternatively, genetic approaches such as viral vectors or naked DNA have been utilized to introduce gene sequences directly into cells to expression intracellularly to deliver antigen directly into the endogenous class MHC antigen processing machinery. However, a limitation to targeting specific antigens limited to a relatively small subset of tumors is that mutations or overexpression of specific tumor associated antigens must be determined and applied on an individual basis.
An optimal immunotherapeutic strategy would allow treatment of a broad spectrum of human malignancies with a common pharmaceutical product. Stimulating immune responses to tumors with p53 mutations may enable treatment of a broad spectrum of tumors as approximately 50% of tumors have mutations in the p53 tumor suppresser gene. The p53 tumor associated antigen is characterized as a mutant TAA. Initially, strategies were designed to elicit CTL responses to “unique peptide antigens” generated by the p53 mutant sequences. This strategy was based on the premise that tumor specific CTL recognize peptides, derived from endogenously synthesized cellular proteins, presented by class I major histocompatibility complex (MHC) molecules. However, targeting such tissue-specific antigens may restrict immunotherapies to a very limited set of tumors as the mutations occur in many different loci within the p53 gene.
A distinguishing characteristic of p53 tumor associated antigens is that mutations within the p53 tumor associated antigen occur in approximately 50% of human malignancies. Moreover, most of the mutations result in an increase in the half-life of the p53 proteins resulting in a marked overexpression in tumor cells. The extended expression of p53 in tumor cells may modify processing and presentation of wt p53-derived peptides by MHC class I molecules. Since most MHC class I receptors are occupied by endogenously derived cellular proteins, a shift in p53 expression may result in a disproportionate number of MHC molecules presenting p53 derived peptides. This may permit development of T cell responses to p53 by T cells expressing T cell receptors (TCR) with low to moderate affinity. Indeed, several lines of evidence support the premise that tolerance to p53 can be overcome resulting in an immune response to tumor cells overexpressing p53. Anti-p53 antibodies have been found in sera of patients with several types of cancers and T cell lymphoproliferative responses to p53 have been detected in breast cell cancer patients. Moreover, subcutaneous immunization with canarypox viral vectors expressing p53 protected mice from challenge with tumors overexpressing p53.
Another distinguishing feature of utilizing p53 transgenes to stimulate tumor immunity is that transduction of tumors cells bearing p53 mutations with wt p53 transgenes generally induces apoptosis. Recent reports indicate that phagocytosis of apoptotic cells by dendritic cells may be an important mechanism for antigen transfer to dendritic cells and subsequent stimulation of specific T cell immunity to antigens expressed by the apoptotic cell. Thus, the induction of apoptosis may promote antigen loading of unknown TAA following phagocytosis by dendritic cells resulting in a more broad spectrum of tumor associated antigenic stimuli as described in the methods of use section below.
An important issue for strategies to induce immunity to self antigens that are differentially expressed in tumor cells is to assess the potential for autoimmune responses to normal cells. Several preclinical studies in mice induced to respond to p53 using peptide antigens and viral vectors have been reported in which specific cytotoxic T cell responses to tumor cells overexpressing p53 were observed while cells expressing normal levels of p53 were not killed. This has been attributed to the induction of T cell responses by CTL cells bearing T cell receptors (TCR) with low to moderate affinity for self MHC/p53 peptide complexes; T cells bearing TCR with high affinity are presumed to be eliminated during thymic negative selection. Moreover, in recent clinical trials, autoimmune responses were assessed in patients undergoing ex vivo dendritic cell immunotherapy to induce immune responses to melanoma antigens. Specific responses to melanoma antigens were successfully induced in the patients, yet no clinically overt sign of autoimmunity were observed.
Genetic immunization has proven to be an effective means to stimulate CTL mediated immunity to viruses and tumors. Expression of transgenes or minigenes (encoding antigenic peptides) intracellularly results in delivery of the immunogen directly into the MHC class I peptide processing and antigen loading system. Moreover, the entire antigenic protein or multiple proteins can be expressed allowing natural processing and loading of the antigenic peptides onto numerous allelic MHC class I and class II molecules permitting a more broad immune response. DNA vaccines and viral vectors show significant promise as effective vehicles for genetic immunization.
In vivo CTL activation may be mediated by dendritic cells. Dendritic cells are professional antigen presenting cells. Dendritic cells exist in distinct functional states. Immature dendritic cells corresponding to those found in peripheral tissues, exhibit a phenotype in which most class II molecules are intracellular and localized to lysosomes. In this phase, they are capable of uptake of antigen. These immature dendritic cells “patrol” the peripheral tissues in search of foreign antigens. Culturing dendritic cell precursors in vitro with GM-CSF and IL-4 induces differentiation into immature dendritic cells capable of highly efficient antigen uptake. Further differentiation into mature DC with highly developed antigen presentation functions can be induced by CD40 ligation, TNF&agr; or LPS. Moreover, antigen pulsed dendritic cells under these culture conditions are extremely efficient at stimulating lymphoproliferative and CTL responses both in vitro and in vivo (Current estimates indicate that dendritic cells may be 10-100 times more potent as antigen presenting cells than other APCs such as macrophage and B cells). These immature dendritic cells mature into an intermediate phenotype in which intracellular class II molecules are found in peripheral non-lysosomal vesicles. The intermediate cells then differentiate into late dendritic cells which express almost all of their class II molecules on the plasma membrane. The maturing dendritic cells migrate to the lymph nodes where they present the processed peptides to T-cells.
The ability of dendritic cells to present tumor antigens to the immune system to recruit an immune response to tumor cells has been suggested as an anticancer therapy. The majority of dendritic cell therapeutic studies to date have utilized ex vivo strategies to load dendritic cells with antigen and to activate them to stimulate T cell immunity following reinfusion. The antigen pulsed dendrit

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