Development of herpes simplex virus-1 amplicon-based immunotherapy for chronic lymphocytic leukemia.

Herpes simplex virus (HSV)-based vectors have favorable biologic features for gene therapy of leukemia and lymphoma. These include high transduction efficiency, ability to infect postmitotic cells, and large packaging capacity. The usefulness of HSV amplicon vectors for the transduction of primary human B-cell chronic lymphocytic leukemia (CLL) was explored. Vectors were constructed encoding beta-galactosidase (LacZ), CD80 (B7.1), or CD154 (CD40L) and were packaged using either a standard helper virus (HSVlac, HSVB7.1, and HSVCD40L) or a helper virus-free method (hf-HSVlac, hf-HSVB7.1, and hf-HSVCD40L). Both helper-containing and helper-free vector stocks were studied for their ability to transduce CLL cells, up-regulate costimulatory molecules, stimulate allogeneic T-cell proliferation in a mixed lymphocyte tumor reaction, and generate autologous cytotoxic T lymphocytes (CTLs). Although helper-containing and helper-free amplicon stocks were equivalent in their ability to transduce CLL cells, a vigorous T-cell proliferative response was obtained using cells transduced with hf-HSVB7.1 but not with HSVB7.1. CLL cells transduced with either HSVCD40L or hf-HSVCD40L were compared for their ability to up-regulate resident B7.1 and to function as T-cell stimulators. Significantly enhanced B7.1 expression in response to CD40L was observed using hf-HSVCD40L but not with HSVCD40L. CLL cells transduced with hf-HSVCD40L were also more effective at stimulating T-cell proliferation than those transduced with HSVCD40L stocks and were successful in stimulating autologous CTL activity. It is concluded that HSV amplicons are efficient vectors for gene therapy of hematologic malignancies and that helper virus-free HSV amplicon preparations are better suited for immunotherapy.

A RANTES-antibody fusion protein retains antigen specificity and chemokine function.

The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.

Recognition of nonconserved bases in the P22 operator by P22 repressor requires specific interactions between repressor and conserved bases.

The ability of P22 repressor protein to distinguish between the six naturally occurring operator binding sites is critically important in determining whether the bacteriophage chooses to grow lytically or lysogenically. We have shown that changes in the highly conserved bases at P22 operator positions 3, 5, 6, and 7 prevent specific binding of P22 repressor. Moreover, studies of mutant proteins identified the three repressor amino acids that directly contact these conserved bases. The pattern of operator sequence conservation permits these direct amino acid-base pair interactions to occur in all except one of the 12 operator half-sites in the phage chromosome. Therefore, repressor differential affinity for these sites cannot be due to these highly conserved base pair-amino acid interactions. Our binding studies show that the nonconserved bases at positions 2 and 4 also play an important role in determining the relative affinity of the naturally occurring P22 operators for P22 repressor. Our data indicate that the direct contacts between the three solvent-exposed amino acids and the conserved bases in the binding site lock these amino acids in place, forming a scaffold allowing the rest of the amino acids side chains to form weaker interactions with the nonconserved bases in the binding site.

DNA-based loss of specificity mutations. Effects of DNA sequence on the contacted and non-contacted base preferences of bacteriophage P22 repressor.

Although the two central bases of the P22 operator are not contacted by the P22 repressor, changes in these bases alter the affinity of operator for repressor. Previous studies (Wu, L., and Koudelka, G. B. (1993) J. Biol. Chem. 268, 18975-18981) show that the structure of the P22 repressor-operator complex varies with central base sequence. Here we show that central base sequence composition affects the strength of two, and likely all, specific amino acid-base pair contacts between synthetic P22 operators and P22 repressor. However, altering a specific protein-DNA contact via a loss-of-contact mutation in repressor results in a loss of specificity at only one contacted position. Thus, only changing the sequence of non-contacted bases affects repressor's global base specificity. The observed effects of ionic concentration on the affinities of various operators for repressor and the DNase I patterns of protein complexes with these binding sites indicate certain central base sequences facilitate optimal juxtaposition of repressor with its contacted bases, while others prevent it. The existence of different structural forms of the repressor-operator complexes explains how the relative energetic importance of specific amino acid-base pair edge contacts is modulated.