Abstract. Integrin Otv~3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, ave3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca++-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the Otv-integrin subunit and could be significantly inhibited by an antibody to the av~33 heterodimer. M21 cells also displayed some av133-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and e~vl33 was also observed to promote significant haptotactic cell migration.To map the site of ~v~33 ligation we used recombinant L1 fragments comprising the entire extraceUular domain of human L1. Significant av133-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Iglike domain of L1 abrogated M21 cell adhesion. We conclude that av133-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, av133 may recognize L1 in a cellcell or cell-substrate interaction.

SummaryInduction of a T-cell mediated antitumor response is the ultimate goal for tumor immunotherapy. We demonstrate here that antibody-targeted IL2 therapy is effective against established pulmonary and hepatic melanoma metastases in a syngeneic murine tumor model. The effector mechanisms involved in this tumor eradication are not dependent on NK cells, since the therapeutic effect of antibody-IL2 fusion protein was not altered in NK cell-deficient mice. In contrast, T cells are essential for the observed antitumor effect, since therapy with antibody-IL2 fusion proteins is unable to induce tumor eradication in T cell-deficient SCID mice. In vivo depletion studies characterized the essential effector cell population further as CD8+ T cells. Such CD8+ T cells, isolated from tumor bearing mice after antibody-directed IL2 therapy, exerted a MHC class I-restricted cytotoxicity against the same tumor in vitro. These data demonstrate the ability of antibody-targeted IL2 delivery to induce a T cell--dependent host immune response that is capable of eradicating established melanoma metastases in clinically relevant organs. Most progressively growing neoplasms, e.g., melanoma, do not provoke antitumor immune responses that are capable of controlling the growth of malignant cells, in spite of the fact that these cells express tumor-associated, T cell epitopes. Ahnost two decades ago, Talmage et al. (l) first described a tumor cell line that failed to induce an allogeneic T cell response despite its apparently normal expression of MHC molecules. Furthermore, they demonstrated that this defective T cell activation could be restored by cytokines (1); thereby providing the rationale for cytokine based forms of immnnotherapy.A therapeutic approach currently receiving much attention is the ex vivo genetic modification of tumor cells to express various cytokines (2, 3). When produced by tumors, many of these cytokines induce a local inflammatory response which results in elimination of the injected tumor cells. In some cases, systemic immune responses are generated against challenge with the wild-type parental tumor. Since most cytokines are paracrine factors working physiologically at high concentrations within a few cell diameters from their cell of origin, this gene transfer approach, in general can not be replaced by systemic cytokine administration unless the cytokine is directed preferentially to the tumor site (4).We recently demonstrated the feasibility of an alternative therapeutic approach for cancer that combines high local concentrations of cytokines in the tumor microenvironment, low systemic toxicities, and a technically simple modus operandi (5, 6). This goal was achieved by the construction of fusion proteins consisting of tumor-specific monoclonal antibodies and cytokines; thereby, employing the unique targeting ability of antibodies to direct cytokines to the tumor site. Here, we demonstrate the effectiveness of antibody-targeted IL2 therapy for established pulmonary and hepatic melanoma metastases in a syngen...

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (chl4.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The chl4.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The chl4.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment ofGD2-expressing human tumors in an adjuvant setting.

(8-10).An alternative means of cytokine delivery is to target them to tumor sites with mAbs directed against tumor associated antigens (11, 12). Thereby, effective local cytokine concentrations can be achieved at the tumor site without resorting to patient-specific therapy. A number of groups have reported the construction of such fusion proteins combining the antigen-binding capacity of antibodies or their fragments with the cytokine activity of IL-2 using different strategies for plasmid construction and protein expression (11)(12)(13) Mouse-human chimeric antibodies directed against the EGF receptor (ch225) or GD2 (chl4.18) were constructed by joining the cDNA for the variable region of the murine antibodies with the constant regions of the yl heavy chain and the K light chain as described (19). The antibody-IL-2 fusion

We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 ϫ 10 9 plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t 1/2 of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME 359 ), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME 359 expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.

A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the C-gamma1 gene of chl4.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumour load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases.

No abstract

Human lymphocytes secreting tumor cell-specific IgM antibodies were enriched in vitro following the stimulation of allogeneic human splenocytes from nontumor-bearing donors with cytostatic tumor cells or tumor cell plasma membrane fractions. The antibodies were generally of the IgM class and displayed low intrinsic affinity (K(d) > 100 nM). Nonetheless, the avidity arising from multivalent binding sites permitted the identification of multiple monoclonal antibodies (MAbs) displaying specificity for cultured tumor cells. Five antibodies were cloned from the B cells and two of these were expressed as human Fabs with IgG(1) constant regions. Although the avidity of the human IgM antibodies was sufficient to permit detection in the original screening, the monovalent Fabs displayed low binding activities, consistent with their low intrinsic affinity. Thus, in vitro affinity maturation was used to rapidly generate multiple variants of both antibodies displaying greater than 100-fold higher affinity. Two of the antibodies were characterized further and shown to have distinct specificities. One of the targets, LH11238, is associated both with the plasma membrane and with lysosomes and is rapidly internalized following incubation of the antibody with intact live cell monolayers. The second antigen, designated LH13, is a secreted antigen that has been enriched 200-fold from conditioned media and consists of two reactive bands at 42 and 45 kDa on denaturing Western blots. The stimulation and enrichment of human lymphocytes in culture coupled with rapid in vitro affinity maturation of low affinity antibodies potentially enables the discovery of human antibodies to a broader range of epitopes, including those that might be of greater therapeutic relevance.