Antitumor activity of CTLA-4 antibody blockade is thought to be mediated by interfering with the negative regulation of T-effector cell (Teff) function resulting from CTLA-4 engagement by B7-ligands. In addition, a role for CTLA-4 on regulatory T cells (Treg), wherein CTLA-4 loss or inhibition results in reduced Treg function, may also contribute to antitumor responses by anti-CTLA-4 treatment. We have examined the role of the immunoglobulin constant region on the antitumor activity of anti-CTLA-4 to analyze in greater detail the mechanism of action of anti-CTLA-4 antibodies. Anti-CTLA-4 antibody containing the murine immunoglobulin G (IgG)2a constant region exhibits enhanced antitumor activity in subcutaneous established MC38 and CT26 colon adenocarcinoma tumor models compared with anti-CTLA-4 containing the IgG2b constant region. Interestingly, anti-CTLA-4 antibodies containing mouse IgG1 or a mutated mouse IgG1-D265A, which eliminates binding to all Fcg receptors (FcgR), do not show antitumor activity in these models. Assessment of Teff and Treg populations at the tumor and in the periphery showed that anti-CTLA-4-IgG2a mediated a rapid and dramatic reduction of Tregs at the tumor site, whereas treatment with each of the isotypes expanded Tregs in the periphery. Expansion of CD8þ Teffs is observed with both the IgG2a and IgG2b anti-CTLA-4 isotypes, resulting in a superiorTeff to Treg ratio for the IgG2a isotype. These data suggest that anti-CTLA-4 promotes antitumor activity by a selective reduction of intratumoral Tregs along with concomitant activation of Teffs.

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Induction of apoptosis by death receptors such as Fas or tumour necrosis factor (TNF) R1 leads to distinct changes in cell morphology, activation of the caspase protease cascade, and the degradation of nuclear chromatin by activated nucleases. Here, we describe the purification and cDNA cloning of a novel 40 kDa endonuclease from Jurkat cells that is activated by caspases. This protein, designated caspase-activated nuclease (CPAN), is sufficient to degrade naked DNA and to induce apoptotic morphology and DNA fragmentation in naive nuclei. CPAN is highly homologous to a recently described mouse nuclease, CAD [1], and may represent the human homologue. Our data on the human cDNA as well as additional data on the mouse homologue suggest that a 30 amino-acid portion of the recently published mouse sequence [1] is incorrect. We show that the activity of human CPAN is regulated by DFF45 [2], an inhibitor necessary for CPAN expression and stabilization in an inactive state in living cells. Proteolytic cleavage of DFF45 by caspases in vitro leads to dissociation of DFF45 fragments from CPAN and activation of CPAN as an endonuclease. CPAN is a tightly regulated endonuclease with unique characteristics that might represent a distinctive family of endonucleases.

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating factor (GM-CSF)^secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1blockade, which has been shown to augment cellular immune responses. Experimental Design: Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model. GM-CSF^secret-ing tumor cell immunotherapy was administered s.c. and the anti^PD-1 antibody was administered i.p. Results:The studies reported here show that combining PD-1blockade with GM-CSF^secret-ing tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with strong antigen-specific T-cell responses detected by tetramer staining and an in vivo CTL assay, higher secretion levels of proinflammatory cytokines by splenocytes, and the persistence of functional CD8 + T cells in the tumor microenvironment. Furthermore, in the biweekly multiple treatment setting, repeated antigen-specificT-cell expansion was only observed following administration of the cellular immunotherapy with the PD-1 blockade and not when the cellular immunotherapy or PD-1 blockade was used as monotherapy. Conclusion:The combination of PD-1 blockade with GM-CSF^secreting tumor cell immunotherapy leads to significantly improved antitumor responses by augmenting the tumor-reactive T-cell responses induced by the cellular immunotherapy. Readministration of the cellular immunotherapy with the anti^PD-1antibody in subsequent immunotherapy cycles was required to reactivate theseT-cell responses.

We have studied the clinical impact of elective brain irradiation (EBI) in patients with locally advanced, non-small cell lung cancer (LA-NSC). All patients received combination chemotherapy (cyclophosphamide + doxorubicin (Adriamycin) + cisplatin = CAP) or CAP plus radiotherapy as the initial treatment for their active tumor or as an adjuvant therapy. Of 97 evaluable patients, 46 were randomized to receive EBI (3 000 rad in 10 fractions given over two weeks). The characteristics of both groups were comparable by sex, age, performance status, pretherapy weight loss, histologic cell type, clinical staging, and type of prior therapy. EBI significantly decreased the incidence of central nervous system (CNS) metastasis in the treated group compared to the control group (4% vs 27%, p = .002). CNS involvement occurred in the treated group after failure at other sites whereas 12 of 14 control patients had CNS metastases as the first site of relapse. EBI decreased the incidence of CNS metastasis in all prognostic categories. Using multivariate analysis, the beneficial effect was shown to be significant in females, patients with good performance status, weight loss less than 6%, squamous cell histology, state III disease or no prior therapy. EBI significantly increased CNS metastasis-free interval with a beneficial effect that was significant in males, patients with weight loss less than 6%, squamous cell histology or responders. Although no survival benefit was observed for the treated group because of the adverse effect from other relapses, EBI will become more important as better treatment programs are developed.(ABSTRACT TRUNCATED AT 250 WORDS)

pp32 is a nuclear protein found highly expressed in normal tissues in those cells capable of self-renewal and in neoplastic cells. We report the cloning of cDNAs encoding human and murine pp32. The clones encode a 28.6-kDa protein; approximately two-thirds of the N-terminal predicts an amphipathic a helix containing two possible nuclear localization signals and a potential leucine zipper motif. The C-terminal third is exceptionally acidic, comprised of approximately 70% aspartic and glutamic acid residues; the predicted pl of human pp32 is 3.81. Human and murine pp32 cDNAs are 88% identical; the predicted proteins are 89% identical and 95% similar. Although the structure of pp32 is suggestive of a transcription factor, pp32 did not significantly modulate transcription of a reporter construct when fused to the Gal4 DNA-binding domain. In contrast, in cotransfection experiments, pp32 inhibited the ability of a broad assortment of oncogene pairs to transform rat embryo fibroblasts, including ras + myc, ras + jun, ras + Ela, ras + mutant p53, and E6 + E7. In related experiments, pp32 inhibited the ability of Rat la-myc cells to grow in soft agar, whereas it failed to affect ras-induced focus formation in NIH3T3 cells. These results suggest that pp32 may play a key role in self-renewing cell populations where it may act in the nucleus to limit their sensitivity to transformation.

Conditionally active COBRA™ (COnditional Bispecific Redirected Activation) T cell engagers are engineered to overcome the limitations of inherently active first-generation T cell engagers, which are unable to discern between tumor and healthy tissues. Designed to be administered as prodrugs, COBRAs target cell surface antigens upon administration, but engage T cells only after they are activated within the tumor microenvironment (TME). This allows COBRAs to be preferentially turned on in tumors while safely remaining inactive in healthy tissue. Here, we describe the development of the COBRA design and the characterization of these conditionally active T cell engagers. Upon administration COBRAs are engineered to bind to tumor-associated antigens (TAAs) and serum albumin (to extend their half-life in circulation), but are inhibited from interacting with the T cell receptor complex signaling molecule CD3. In the TME, a matrix metalloproteinase (MMP)-mediated linker cleavage event occurs within the COBRA construct, which rearranges the molecule, allowing it to co-engage TAAs and CD3, thereby activating T cells against the tumor. COBRAs are conditionally activated through cleavage with MMP9, and once active are highly potent, displaying sub-pM EC 50 s in T cell killing assays. Studies in tumor-bearing mice demonstrate COBRA administration completely regresses established solid tumor xenografts. These results strongly support the further characterization of the novel COBRA design in preclinical development studies.

UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in an integration independent manner. Efficient expression can be derived from a single copy of an integrated gene site resulting in a higher percentage of cells expressing the marker gene in the selected pool in comparison to standard non-UCOE containing vectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapid production of large quantities of protein in a short period of time. Utilizing this system more than 300 mg of a recombinant antibody has been produced in less than 1 month from transfection pools in shake flask. Selected subclones have been scaled into small bioreactors in less than 2 months, producing significant quantities of monoclonal antibody using a protocol generic for the parent cell line. The increased efficiency obtained with the UCOE vector reduces the number of transfectants which need to be screened in order to obtain high productivity subclones. Transfection of a standard host cell line, preadapted to grow in a largescale setting, allows for rapid cell line development decreasing the transition time from research into development and manufacturing. Alternatively, the traditional approach of using a parent cell line which requires serum-free and suspension adaptation after transfection further increases the need for screening a large number of subclones, because many of the subclones will not be able to grow under conditions that allow large-scale protein production. The use of a preadapted cell line can reduce the time required to develop a cell line from months to weeks.