We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-γ in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.

ABSTRACT

We evaluated recurrent NXS2 neuroblastoma tumors that developed following NK- or T-cell-mediated immunotherapy in tumor-bearing mice. Recurrent tumors developed following an NK-dependent antitumor response using a suboptimal dose of hu14.18-IL2, a humanized IL-2 immunocytokine targeted to the GD(2)-ganglioside. This treatment initially induced complete resolution of measurable tumor in the majority of mice, followed, however, by delayed tumor recurrence in some mice. These recurrent NXS2 tumors revealed markedly enhanced (> fivefold) MHC class I antigen expression when compared with NXS2 tumors growing in PBS-treated control mice. A similar level of enhanced MHC class I antigen-expression could be induced on NXS2 cells in vitro by culturing with interferon gamma, and was associated with reduced susceptibility to both NK-cell-mediated tumor cell lysis and antibody-dependent cellular cytotoxicity in vitro. In contrast, Flt3-ligand treatment of NXS2-bearing mice induced a protective T-cell-dependent antitumor memory response. Recurrent NXS2 tumors that developed following Flt3-L therapy revealed a decreased expression of MHC class I antigens. While NXS2 tumors are susceptible to in vivo destruction following either hu14.18-IL2 or Flt3-ligand immunotherapies, these results suggest that some tumor cells may be selected to survive and progress by expressing either higher or lower levels of MHC class I antigen in order to resist either NK- or T-cell-mediated antitumor responses, respectively.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

The hu14.18-IL2 (EMD 273063) IC, consisting of a GD(2)-specific mAb genetically engineered to two molecules of IL-2, is in clinical trials for treatment of GD(2)-expressing tumors. Anti-tumor activity of IC in vivo and in vitro involves NK cells. We studied the kinetics of retention of IC on the surface of human CD25(+)CD16(-) NK cell lines (NKL and RL12) and GD(2)(+) M21 melanoma after IC binding to the cells via IL-2R and GD(2), respectively. For NK cells, ∼ 50% of IC was internalized by 3 h and ∼ 90% by 24 h of cell culture. The decrease of surface IC levels on NK cells correlated with the loss of their ability to bind to tumor cells and mediate antibody-dependent cellular cytotoxicity in vitro. Unlike NK cells, M21 cells retained ∼ 70% of IC on the surface following 24 h of culture and maintained the ability to become conjugated and lysed by NK cells. When NKL cells were injected into M21-bearing SCID mice, IT delivery of IC augmented NK cell migration into the tumor. These studies demonstrate that once IC binds to the tumor, it is present on the tumor surface for a prolonged time, inducing the recruitment of NK cells to the tumor site, followed by tumor cell killing.

Encephalomyocarditis virus (EMCV) and mengovirus are serotypically related cardioviruses of the picornavirus family. Among the unusual features of their positive-sense singlestranded RNA genomes are long 5Ј untranslated polypyrimidine tracts [poly(C)] with sequences consisting of C 115 UCUC 3 UC 10 and C 44 UC 10 for EMCV (strain R) and mengovirus (strain M), respectively (6, 13). These cardioviruses and their closely related cousins, the foot-and-mouth disease viruses of the Aphthovirus genus, are the only known eukaryotic or prokaryotic genomes to contain such poly(C) tracts, and the specific function of the homopolymer region remains a biological enigma.We have reported the construction of multiple cDNA-derived EMCV and mengovirus strains which differ from each other and from wild-type parental strains only in the lengths of their 5Ј poly(C) tracts (8, 9, 13). The mengovirus panel included nine viruses with poly(C) lengths that ranged from C 44 UC 10 (vMwt) down to a precise deletion of all the cytidine residues (vMC 0 ). The EMCV panel (vEC 20 , vEC 9 , and vEC 4 ) was less extensive, although it contained representative analogues for the best-characterized mengovirus strains. As a definitive phenotype, deletion of the mengovirus poly(C) tract clearly correlates with attenuation of virus virulence in animals. vMC 0 , for example, has a median 50% lethal dose (LD 50 ) of Ͼ2 ϫ 10 9 PFU after intracerebral inoculation of mice. In contrast, the LD 50 of vMwt by equivalent inoculation is only 10 PFU (7). All intermediate-tract mengoviruses show correspondingly diminished virulence. For viruses with poly(C) tracts between 25 and 35 bases, the LD 50 in mice increases about 1 log 10 PFU for every 3 C's that are removed from the tract (13,20). For EMCV strains, the correlation between tract length and murine virulence is weaker, however, and the poly(C) needs to be truncated substantially (i.e., ϽC 9 ) before the attenuation becomes measurable (e.g., the LD 50 of vEC 4 is 3 ϫ 10 3 PFU compared to 1 PFU for EMCV-R) (9). Our EMCV and mengovirus recombinant isolates have been extensively characterized for growth in HeLa cells, and all were found to plaque with equivalent plating efficiencies regardless of the poly(C) tract length. While some strains do exhibit subtle changes in plaque size that correlate with incremental tract deletion (9, 13), none of the isolates show tract-dependent variations in replication kinetics or end-point titers when measured directly in single-step growth experiments. The poly(C) tracts also have no apparent influence on genome translation, virion stability, or growth temperature sensitivity, and it is clear, at least for growth in HeLa cells, that the major vegetative life cycle requirements for EMCV and mengovirus are not strongly vested in this region of the viral RNA.However, in infected animals, especially those receiving mengoviruses, there must be some cellular or tissue determinant that rapidly detects subtle differences in poly(C) genotype and reacts in a manner that clearly means l...

Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used to induce antigen-specific antibodies in mice, rats, and rabbits. Both methods allowed the efficient generation of high-titer, antigen-specific antibodies with an overall success rate of Western detectable antibodies of 78% and 92%, respectively. High-titer antibodies were typically present after 3 hydrodynamic tail vein plasmid DNA deliveries, 5 weeks after the initial injection (i.e., prime). For hydrodynamic limb vein plasmid DNA delivery, two deliveries were sufficient to induce high-titer antibody levels. Tail vein delivery was less successful at generating antibodies directed against secreted proteins as compared with limb vein delivery. Material for screening was generated by,transfection of the immunization vector into mammalian cell lines. The cell line (COS-7) that produced the highest level of antigen expression performed best in Western blot analysis screens. In summary, intravenous delivery of antigen-expressing plasmid DNA vectors is an effective genetic immunization method for the induction of antigen-specific antibodies in small and large research animals.

Picornaviruses are a family of positive-strand RNA viruses that are responsible for a variety of devastating human and animal diseases. An attenuated strain of mengovirus (vMC 24 ) is serologically indistinguishable from the lethal murine wild-type mengovirus and encephalomyocarditis virus (EMCV). Immunogen-specific stimulation of vMC 24 -immune splenocytes in vitro demonstrates preferential activation of CD4 ؉ lymphocytes. vMC 24 -immune splenocytes adoptively transferred to naive recipients conferred protection against lethal EMCV challenge. Immune splenocytes, expanded in vitro, were >92% CD4 ؉ T lymphocytes. Interestingly, adoptive transfer of these expanded cells engendered protection against lethal challenge. In vivo depletion of CD4 ؉ T lymphocytes prior to lethal challenge abrogated survival of transfer recipients, confirming that CD4 ؉ T lymphocytes were essential for protection. Subsequent rechallenge of vMC 24 -immune splenocyte recipients with a greater EMCV dose elicited serum neutralizing antibody titers paralleling the high titers observed in vMC 24 -immunized mice. Unexpectedly, an augmented humoral response was absent in vMC 24 -specific CD4 ؉ T-cell recipients after the secondary challenge. Moreover, comparably low serum neutralizing antibody titers failed to protect passive transfer recipients when correspondingly challenged. vMC 24 -immune splenocytes expanded in vitro (>94% CD4 ؉ ) lysed vMC 24 -infected A20.J target cells. The ability to transfer protection with primed CD4 ؉ T cells, in the absence of primed B lymphocytes or immune sera, is novel for picornaviral infections. Consequently, mechanisms such as CD4 ؉ cytolytic T-lymphocyte activity are implicated in mediating protection.