No abstract

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. B-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of B-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. B-Lactamase was found to contain four CD4 + T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The B-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of B-lactamase variant with adjuvant. The B-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients. [Mol Cancer Ther 2005;4(11):1791-800]

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4+ T cell epitope peptide region in human IFN-β, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-β molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-β responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-β molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1–GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1–GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

Human CD4 þ T-cell epitopes were identified in interferon-beta (IFN-b)-1b. A prominent peptide epitope region was found that induced a proliferative response in 16% of all donors tested. Responses corresponded to the presence of the HLA-DR2 haplotype. Responsive donors expressing the HLA-DQ6 allele showed an increased level of proliferation to the epitope as compared to peptide-responsive HLA-DQ6 negative donors. A similar result was found for HLA-DR15-expressing donors. PBMC from donors expressing HLA-DR15 were more likely to proliferate in response to IFN-b in a whole-protein in vitro assay than donors who did not carry this haplotype. It is striking that the common DQ6 allele HLA-DQB1*0602 is found in linkage disequilibrium with HLA-DRB1*1501, and this combination defines the HLA genotype associated with the development of multiple sclerosis. The HLA association between a response to IFN-b and MS might explain the prevalence of neutralizing antibody development, and may underlie the etiology of the disease.

No abstract

The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ∼40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315−29). CD4+ T-cell proliferative responses to CH315−29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315−29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype.

A method to rank proteins based on their relative immunogenicity has been devised. A statistical analysis of peptide-specific responses in large human donor pools provides a structure index value metric that ranked four industrial enzymes in the order determined by both mouse and guinea pig exposure models. The ranking method also compared favorably with human sensitization rates measured in occupationally exposed workers. Structure index values for other proteins known to cause immune responses in humans were also determined and found to be higher than the value determined for human beta2-microglobulin. Using values from known immunogenic and putative nonimmunogenic proteins, a cut-off value was established. The structure index value calculation provides a comparative method to predict subsequent immunogenicity on a human population basis without the need to use animal models. Information provided by this assay can be used in the early development of protein therapies and other protein-based applications to select or create reduced immunogenicity variants.