Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1997-09-29
2001-09-25
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388200, C530S403000
Reexamination Certificate
active
06294654
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the activation of T-cells, and particularly for stimulating the body's cell-mediated immune system to deal with infection or cancerous mutations in cells.
BACKGROUND
One of the mechanisms by which the body's immune system operates involves the engulfed of foreign protein by antigen presenting cells (APCs), where the proteins are broken down by proteases into peptide fragments, associated with the cell's MHC II protein in a peptide loading compartment, and then transported to the surface of the APC, where the peptide is presented in association with the MHC II to the T-cell receptors (TCRs) of T-helper cells (CD4
+
T-cells). The TCRs of CD4 T-cells only recognise the antigenic peptides in association with MHC II, and the TCRs have a repertoire of recognition sites, so that only those helper cells with the appropriate TCRs will recognise a given antigenic peptide. This activates the helper cell to stimulate cytotoxic T-cells (CD8
+
or Tc cells) and B-cells with the corresponding antigen specificity, which then mount an attack on the original source of foreign protein, either directly by CD4
+
T-cells themselves, or by the production of antibody (B-cells) or by the Tc-cells. In the case of the latter, receptors on the Tc-cells recognise the original antigen presented in association with MHC I on the surface of cells in which they are endogenously produced, for example as a result of infection or through the generation of a mutated protein within the cell.
Vaccination has been of great importance in the protection against infectious diseases. Even so there is still a need to develop more safe and more effective vaccines. It is also necessary to develop vaccines for diseases for which there until now have been no such preventive measures. For effective vaccination, one needs to activate T-cells of the CD4 type because CD4
+
T-cells direct activation of cytotoxic T-cells and B-cells.
Many cancer cells are the result of mutation, for example mutation in the p21 ras gene, and in consequence, an antigenic peptide fragment bearing the mutation is presented at the surface of the cell. The body's immune system may normally deal with such potential cancer cells by receptors on CD4
+
T-cells recognising the apparently “foreign” antigen on MHC II and subsequently providing “signal 2” for specific cytotoxic T-cells which may kill the tumour cells. In this way, we probably fight off potential cancers by treating the cancer cells as though they had been infected with a foreign organism. However, in some cases tumours are established, and then it is beneficial to alter the balance between the T-cells and the tumour cells in favour of the T-cells by inducing or increasing specific T-cell activity.
The antigen peptide which locates in the MHC II molecule is typically about 11-20 amino acids in length, and one way of trying to efficiently vaccinate against infectious agents or to activate T-cells specific for tumour specific antigens, is to provide the peptide artificially. However, it has not proved very successful, probably because the peptide is easily degraded.
One group of workers (Zaghouani et al, Science (1993), 259:224-227, and Brumeanu et al, J Exp Med (1993), 178:1795-1799) disclosed the substitution of DNA encoding a viral epitope peptide into the DNA encoding the CDR3 loop of the heavy chain of an immunoglobulin molecule. This gene was co-expressed with a light chain to produce a complete Ig type of molecule, which was taken up by the Fc receptor (FcR) of an APC, and the viral epitope was presented with MHC II on the surface of the APC in vitro. The authors suggested that antigenized self Ig molecules could represent an effective carrier for delivery of peptides to MHC II molecules, as in vaccination or (tolerization) protocols, and a carrier that has a potential to be long-lived and devoid of side-effects.
We have studied antigen presentation of the 91-101 amino acid fragment from the &lgr;2 light chain of M315 antibody (Bogen et al, Eur J Immunol (1986), 16:1373). We have further used in vitro mutagenesis to move the epitope to loops in the human IgG3 heavy chain. Three different mutants were made, each with one of the loops of the CH1 domain replaced with the 91-101 peptide. The mutant genes were transfected into a fibroblast cell line which had previously been transfected with genes encoding the E
&agr;
k
E
&bgr;
d
MHC II. The resulting clones were assayed for the ability to stimulate &lgr;2
315
specific T-cell clones. It appears that the mutated heavy chains are retained intracellularly in the transfected fibroblasts, but nevertheless the peptide is processed and presented on MHC II.
SUMMARY OF THE INVENTION
We have found that altered antibodies can be made in which a peptide antigen can be incorporated into a non-CDR loop of an antibody (Ab), and the resulting Ab can be taken up in an APC so that the peptide antigen is presented on the surface of the APC in the context of MHC II, and thereby produce an immune response.
The invention may be used in vaccination against infectious diseases, in which case the peptide antigen, inserted into Ig molecules, should be derived from proteins of the infectious agent. For vaccination purposes, the modified Ig will be targeted to dendritic cells which are especially potent at stimulating naive T-cells.
The invention can also be used in connection with certain types of therapy, the object being to stimulate the immune system which has apparently become tolerant of a particular antigen. For example, many cancers derived from mutations in normal cell proteins may arise because the body's immune system is no longer activated by the mutant protein. Furthermore, the cancer cells are often poor APCs because of a lack of co-stimulatory molecules. A strategy to deal with that, therefore, is to stimulate the body's immune system by presenting it with the mutant protein. Preferably the antigen should be presented on dendritic cells, which are especially potent at stimulating T-cells.
Another aspect of the approach is that peptide antigens presented with MHC II on the surface of APCs stimulate either or both of two types of T-helper cells, Th1 and Th2 respectively. Th1 stimulation results in the production of the cytokines IFN-gamma and IL-2 and the stimulation of cytotoxic T-cells (Tc). Th2 stimulation, on the other hand, results in the production of the cytokines IL-4, IL-5 and IL-10, leading to activation of B-lymphocytes and the production of antibodies to the antigen. Of the two, the Th1 route is preferred for the present approach, since Tc cells are strong antiviral agents. Also, Tc cells (CD8
+
T-cells) with specificity for mutant ras peptide have been detected in cancer patients, and such cancer cells should be susceptible to an enhanced Tc immune response.
The preference for the Th1 or Th2 route depends on the dosage and nature of the antigen administered. A stable antigen will have a longer half-life. As Ig molecules seem to be stable inside an APC, its peptides will be produced at a slow rate at the surface of the APC, giving a low steady state concentration of epitope at the surface, but the antigen will be expected to persist in the APC for a long time. Moreover, an Ig molecule is stable in the organism itself, and therefore the APC may be exposed to the antibody for a long time. Thus, the overall effect of our approach should be a long-lasting low level exposure of the immune system to the antigen carried by the Ig, which may be important for eliciting potent T-cell responses.
We have found that immunoglobulin (Ig) molecules, when engulfed by dendritic cells, stimulate specific naive T-cells to IL-2 production. Moreover, we have found that a modified Ig incorporating an antigen peptide can do likewise.
We have demonstrated that a peptide from a CDR loop of an Ig light chain can be transferred to a loop on the heavy chain constant region, and the Ig will still be expressed.
It is crucial that the main outline of the constant do
Bogen Bjarne
Fossum Sigbjørn
Lunde Elin
Mjaaland Siri
Rasmussen Ingunn B.
Akin Gump Strauss Hauer & Feld L.L.P.
Sandlie Inger
Saunders David
Tung Mary Beth
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