Anti-PD-1 monoclonal antibody MEDI0680 in a phase I study of patients with advanced solid malignancies.

BACKGROUND: The safety, efficacy, pharmacokinetics, and pharmacodynamics of the anti-programmed cell death-1 antibody MEDI0680 were evaluated in a phase I, multicenter, dose-escalation study in advanced solid malignancies. METHODS: MEDI0680 was administered intravenously once every 2 weeks (Q2W) or once every 3 weeks at 0.1, 0.5, 2.5, 10 or 20 mg/kg. Two cohorts received 20 mg/kg once a week for 2 or 4 weeks, then 20 mg/kg Q2W. All were treated for 12 months or until progression. The primary endpoint was safety. Secondary endpoints were efficacy and pharmacokinetics. Exploratory endpoints included pharmacodynamics. RESULTS: Fifty-eight patients were treated. Median age was 62.5 years and 81% were male. Most had kidney cancer (n = 36) or melanoma (n = 9). There were no dose-limiting toxicities. Treatment-related adverse events occurred in 83% and were grade ≥ 3 in 21%. Objective clinical responses occurred in 8/58 patients (14%): 5 with kidney cancer, including 1 with a complete response, and 3 with melanoma. The relationship between dose and serum levels was predictable and linear, with apparent receptor saturation at 10 mg/kg Q2W and all 20 mg/kg cohorts. CONCLUSIONS: MEDI0680 induced peripheral T-cell proliferation and increased plasma IFNγ and associated chemokines regardless of clinical response. CD8+ T-cell tumor infiltration and tumoral gene expression of IFNG, CD8A, CXCL9, and granzyme K (GZMK) were also increased following MEDI0680 administration. TRIAL REGISTRATION: NCT02013804 ; date of registration December 12, 2013.

Human recombinant Fab fragments with sub-nanomolar affinities for acetylated histones.

Acetylation of lysines at different sites in the N-terminus of core histones is a common mode of chromatin modification; different combinations of such modifications are associated with distinct patterns of gene expression, replication and repair. Antibodies are usually used to identify and localize particular histone modifications and to correlate their presence with transcription or other cellular processes. This requires antibodies of sufficient specificity and affinity for each of the many modifications that have now been observed. In most instances, polyclonal antibodies have been used but monoclonal antibodies can also be effective. Here we report that a phage-displayed repertoire of rearranged antibody genes from splenic B cells from a patient with systemic lupus contain Fab fragments that can bind native acetylated lysine 8 histone H4. This finding represents the first selection of human antibodies specific for acetylated histone and suggests that lupus antibodies may contribute to dissection of the histone code.

Peptide epitopes from the Wilms' tumor 1 oncoprotein stimulate CD4+ and CD8+ T cells that recognize and kill human malignant mesothelioma tumor cells.

PURPOSE: Wilms' tumor 1 protein (WT1), a transcription factor overexpressed in malignant mesothelioma, leukemias, and other solid tumors, is an ideal target for immunotherapy. WT1 class I peptide epitopes that were identified and shown to stimulate CD8(+) T cells are being tested as vaccine candidates in several clinical trials. The induction and maintenance of a robust memory CD8(+) cytotoxic T-cell response requires CD4(+) T-cell help. EXPERIMENTAL DESIGN: Three HLA class II peptide epitopes of WT1 with high predictive affinities to multiple HLA-DRB1 molecules were identified using the SYFPEITHI algorithm. Due to the highly polymorphic nature of the HLA class II alleles, such reactivity is critical in the development of a broadly useful therapeutic. One of the WT1 CD4(+) peptide epitopes, 122-140, comprises a previously identified CD8(+) peptide epitope (126-134). By mutating residue 126 from an arginine to a tyrosine, we embedded a synthetic immunogenic analogue CD8(+) epitope (126-134) inside the longer peptide (122-140). This analogue was previously designed to improve immunogenicity and induce a potent CD8(+) response. RESULTS: WT1 peptides 328-349 and 423-441 are able to stimulate a peptide-specific CD4(+) response that can recognize WT1(+) tumor cells in multiple HLA-DRB1 settings as determined by IFN-gamma enzyme-linked immunospot assays. The mutated WT1 peptide epitope 122-140 is able to induce CD4(+) and cytotoxic CD8(+) WT1-specific T-cell responses that can recognize the native WT1 epitopes on the surface of human WT1(+) cancer cells. Cross-priming experiments showed that antigen-presenting cells pulsed with either mesothelioma or leukemia tumor lysates can process and present each of the CD4(+) peptides identified. CONCLUSIONS: These studies provide the rationale for using the WT1 CD4(+) peptides in conjunction with CD8(+) peptide epitopes to vaccinate patients with WT1-expressing cancers.

Peptide binding motif predictive algorithms correspond with experimental binding of leukemia vaccine candidate peptides to HLA-A*0201 molecules.

The ability to reliably identify the peptides that can bind to MHC molecules is of practical importance for rapid vaccine development. Several computer-based prediction methods have been applied to study the interaction of MHC class I/peptide binding. Here we have compared the binding of peptides predicted by three algorithms (BIMAS, SYFPEITHI and Rankpep) to the binding of the peptides to HLA-A*0201 molecules in vitro, assessed using a MHC stabilization assay on live T2 cells. Fifty HLA-A*0201 peptides were selected from several target oncoproteins: Wilms' tumor protein (WT1), native and imatinib-mutated bcr-abl p210, JAK2 protein and Ewing's sarcoma fusion protein type 1. The sensitivity and specificity of BIMAS, SYFPEITHI and Rankpep respectively, were: 86%, and 82%; 75% and 73%; 64% and 82%. Combining two or more computer methods did not appear to significantly improve the predictive value.

Variable-region-identical antibodies differing in isotype demonstrate differences in fine specificity and idiotype.

A central tenet of the current understanding of the relationship between Ab structure and function is that the variable region domain is solely responsible for Ag specificity. However, this view was recently challenged by the observation that families of mouse-human chimeric Abs with identical V regions demonstrate differences in fine specificity and by reports of changes in Ab Id structure with isotype switching. Here we revisited this question by evaluating the reactivity of two families of murine IgG switch variants that differed in V region usage for Cryptococcus neoformans glucuronoxylomannan, glucuronoxylomannan peptide mimetics, and anti-Id mAbs. The results reveal isotype-related differences in fine specificities and Id for two mAb isotype switched families, thus establishing the validity of this observation with sets of homologous Abs. The results suggest that the C region affects V region protein conformation, leading to differences in fine specificity and Id. The finding that isotype can affect fine specificity has major implications for current concepts of the generation of secondary responses, idiotypic network regulation, and isotype function. Given that isotype class switching and Ig gene somatic hypermutation share molecular mechanisms, these observations unify these processes in the sense that both can alter specificity and affinity.

Antibodies to keyhole limpet hemocyanin cross-react with an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development.

Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags.

High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library.

Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT.