Single-Dose Anti-PD-L1/IL-15 Fusion Protein KD033 Generates Synergistic Antitumor Immunity with Robust Tumor-Immune Gene Signatures and Memory Responses.

Immunocytokines hold great potential as anticancer agents, as they use a specific antitumor antibody to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME). We have developed a novel immunocytokine (KD033) composed of a fully human, high-affinity antiprogrammed death-ligand 1 (PD-L1) linked to the sushi-domain of the human IL-15/IL-15 receptor alpha (IL-15/IL-15Rα) complex. A murine PD-L1 cross-reactive KD033 surrogate (srKD033) and a nontargeting antibody (ntKD033) were also developed to investigate mechanism of action in murine tumor models. Efficacy analyses showed a robust antitumor effect of single-dose srKD033 in several diverse syngeneic murine tumor models. In a CT26 murine colon tumor model, single-dose srKD033 produced durable antitumor immunity as evidenced by resistance to subsequent tumor rechallenges. Mice responding to srKD033 treatment showed increased retention of PD-L1/IL-15 in the TME which likely facilitated prolonged IL-15-induced expansion of cytotoxic cells. Importantly, target-based PD-L1/IL-15 delivery via srKD033 was well-tolerated and induced significant antitumor activity in murine carcinoma models that are non- or minimally responsive to IL-15 or anti-PD-L1/PD-1 monotherapy.

Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones.

Cloning by nuclear transfer remains inefficient but is more efficient when nuclei from embryonic cells or embryonic stem cells (ECNT) are employed as compared with somatic cells (SCNT). The factors determining efficiency have not been elucidated. We find that somatic and embryonic nuclei differ in their ability to organize meiotic and mitotic spindles of normal molecular composition. Calmodulin, a component of meiotic and mitotic spindle chromosome complexes (SCCs), displays sharply reduced association with the SCC forming after SCNT but not ECNT. This defect persists in mitotic spindles at least through the second mitosis, despite abundant calmodulin expression in the cell, and correlates with slow chromosome congression. We propose that somatic cell nuclei lack factors needed to direct normal SCC formation in oocytes and early embryos. These results reveal a striking control of SCC formation by the transplanted nucleus and provide the first identified molecular correlate of donor stage-dependent restriction in nuclear potency.

Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer.

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

Multiparameter analysis of human oocytes at metaphase II stage after IVF failure in non-male infertility.

BACKGROUND: Using fluorescence imaging, an a posteriori multiparametric analysis was performed of human oocytes which failed to give pronucleated zygotes after IVF in cases of very low rates of fertilization or complete fertilization failure. METHODS: The analysis included: (i) the state of the maternal and paternal chromatin; (ii) quality of the metaphase II oocytes; and (iii) cortical granule (CG) distribution. RESULTS: Most oocytes were arrested in metaphase II, but they were abnormal in 50% of cases. The incidence of spindle and chromosome aberrations was strongly influenced by maternal age (69% for 40- to 45-year-old women versus 35% for 26- to 33-year-olds), and sperm chromatin was always condensed in immature oocytes, and fully decondensed only in normal metaphase II. The migration of CGs appeared to be associated with achievement of nuclear maturation at the time of puncture. CONCLUSIONS: These factors, when analysed on a complete set of oocytes from the same patient, provided information about potential causes of IVF failure, and also represented part of an 'oocyte quality evaluation' to select the assisted fertilization technique most suitable for each patient. For example, when the majority of oocytes were judged non-fertilizable at a first attempt, no pregnancy was registered at any subsequent attempt.

Chromatin configuration and transcriptional control in human and mouse oocytes.

In vitro maturation of human oocytes at the germinal vesicle (GV) stage could offer an alternative in several cases of female infertility. It however rests on a better knowledge of the quality of human oocyte. Using fluorescence imaging of DNA and of the transcription sites, combined with electron microscopy, we show that human oocytes follow size-dependent changes in chromatin configuration, transcription sites distribution and nuclear ultrastructure that follow those observed in mouse GV oocytes. We thus analyzed in mouse GV oocytes the phosphorylation dependence of the transcriptional activity. We show by Western blot that, while active GV oocytes have approximately the same proportion of hypo- and hyperphosphorylated forms of the RNA polymerase II (RNAP II), the hyperphosphorylated form is almost absent from inactive oocytes. We also show that (1) RNAP II-dependent transcription is much less sensitive to various kinase inhibitors in mouse oocytes than in somatic cells or mouse one-cell embryos, although the phosphorylation equilibrium of RNAP II was largely shifted towards the hypo-phosphorylated form upon treatment with these inhibitors (2) RNAP I is completely insensitive to kinase inhibitors in GV oocytes.