Augmentation of antitumor activity of an antibody-interleukin 2 immunocytokine with chemotherapeutic agents.

PURPOSE: Immune-based therapies, such as the immunocytokine huKS-IL2, exert potent antitumor responses in some animal models by targeting cytokine activity to the tumor microenvironment. We found that certain chemotherapy agents in the appropriate dose and schedule can augment the antitumor activity of huKS-IL2. EXPERIMENTAL DESIGN: Chemotherapy agents were given in a single dose followed 1 day (paclitaxel) or 3 days (cyclophosphamide) later with five daily doses of huKS-IL2 in mice bearing established s.c. tumors, liver metastases, or lung metastases. Tumor models used were CT26/KSA colon, 4T1/KSA mammary, or LLCKSA Lewis lung carcinomas. To measure huKS-IL2 distribution, radiolabeled protein was given to CT26/KSA tumor-bearing mice 1 or 24 h after paclitaxel. huKS-IL2 levels in the tumors were evaluated. RESULTS: Both paclitaxel and cyclophosphamide followed by huKS-IL2 resulted in enhanced antitumor responses compared with either of the treatments alone in the three different tumor models. Results from studies to determine whether the role of the cytotoxic agents in antitumor activity enhancement was related to tumor uptake indicated that a larger fraction of the radiolabeled huKS-IL2 penetrated the tumors when it was administered 24 h after cytotoxic drug "sensitization." CONCLUSION: These data support the idea that prior drug therapy serves to decompress the tumor and lower the diffusion barrier for macromolecules, thus allowing for increased uptake of the huKS-IL2 immunocytokine into the tumor microenvironment. Because the toxicity of the immunocytokine is relatively low at optimal doses, the therapeutic index would likely be greater with the combination treatments.

Antibody-IL-12 fusion proteins are effective in SCID mouse models of prostate and colon carcinoma metastases.

IL-12 is a complex cytokine in both its structure and its range of biologic activities. Fusions of this heterodimeric molecule with an intact antitumor Ab were made to test the feasibility and efficacy of targeting IL-12 to tumors to elicit a local immune response. Fusion proteins composed of the human p35 and p40 subunits had IL-12 bioactivities that were nearly as potent on human immune cells as the rIL-12 standard, but were inactive on mouse cells. Hybrid IL-12 fusion proteins composed of mouse p35 and human p40, fused to Ab, were capable of inducing IFN-gamma, but were much less active on mouse spleen cells than a mouse IL-12 standard. Despite this relatively low activity, the hybrid fusion protein was as effective in a SCID mouse model as a fully active Ab-IL-2 fusion protein in eliminating established pulmonary metastases of CT26 colon carcinoma. Specific targeting of a human IL-12 fusion protein to metastatic prostate carcinoma xenografts was also shown to be effective in SCID mice transplanted with human lymphocyte-activated killer cells. These results demonstrate the importance of directing this potent cytokine to the tumor microenvironment and suggest an important alternative to systemic IL-12 administration or gene therapy for increasing its therapeutic index.

Role of inter-heavy and light chain disulfide bonds in the effector functions of human immunoglobulin IgG1.

The role of inter-heavy and light chain disulfide bonds in the effector functions of human IgG1 was investigated. This was accomplished by mutating appropriate sites in IgG1 such that the disulfide bond pattern now resembled that of IgG4. The effector functions of the mutant antibody were then compared to native IgG1 and IgG4. The antibody-dependent cell cytotoxicity activity was completely abolished in the mutant and the complement-dependent cytotoxicity assay was reduced fifteen-fold. The results suggest that the inter-heavy and light chain disulfide bond pattern of an antibody molecule play a role in its effector functions.

Targeting human cytotoxic T lymphocytes to kill heterologous epidermal growth factor receptor-bearing tumor cells. Tumor-infiltrating lymphocyte/hormone receptor/recombinant antibody.

A genetically engineered conjugate between an anti-CD3 antibody and epidermal growth factor (EGF) was tested for its ability to mediate the lysis of receptor-bearing cells by human CTL. This construct was made by fusing an EGF coding sequence to the 3' end of the human gamma-1 H chain gene sequence and expressing the modified gene in transfected cells together with the V regions of a mouse antibody specific for the human T cell marker, CD3. The resulting conjugate was able to compete with EGF for its receptor and, at extremely low concentrations, was able to mediate the lysis of receptor-bearing tumor targets by a tumor-infiltrating lymphocytes line or by a CTL line established from peripheral blood. The construction of such conjugates by genetic engineering represents a general approach to the direct expression of highly specific hetero-bifunctional reagents without the necessity of further in vitro manipulations.

The use of microcapsules for high density growth of human tumor infiltrating lymphocytes and other immune reactive T cells.

The use of microcapsules to achieve high density growth of tumor infiltrating lymphocytes (TIL) and other antigen-specific human T cells is described. Whereas human T cells in suspension cultures usually do not exceed 1-2 x 10(6) cells/ml, densities approaching that found in living tissues (greater than 10(8) cells/ml) have been observed for microcapsule cultures. TIL and human peripheral blood-derived T cells can be routinely recovered from microcapsules with viabilities greater than or equal to 90%. The recovered cells retain their antigen reactivities and bear cell surface phenotypes identical to their counterparts grown in suspension culture. These findings suggest that microcapsule technology could prove valuable in generating the vast numbers of cells required for TIL therapy and other forms of adoptive immunotherapy with T cells.

Antigen binding and biological activities of engineered mutant chimeric antibodies with human tumor specificities.

The constant region of the human gamma 1 chain was mutated either by deleting the second domain (CH2) or by mutating the two hinge region cysteine residues, normally involved in the inter-heavy chain disulfide bond formation, to serines. The effects of these mutations on chain assembly, antigen binding, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) were measured after expressing the human constant regions together with mouse variable regions encoding anti-tumor cell specificities. The CH2-deleted chimeric antibody was found to have increased antigen binding activity and little (ADCC) or no (CDC) biological activity. The cysteine to serine hinge region mutant antibody had normal or slightly reduced antigen binding activity, greatly reduced ADCC activity, and a reduced, but still significant, ability to mediate CDC. These results reflect the complexity of the interactions between the immunoglobulin domains and their role in balancing the antigen binding and effector functions of antibodies. They suggest further that such antibodies may be useful in applications, such as the in vivo imaging of tumors, where the loss of effector function (e.g., Fc receptor binding) is desired.

Development of a nonthrombogenic collagenous blood-prosthetic interface.

Investigations to develop an implantable assist pump for prolonged circulatory support have been impeded by accumulation of friable thrombus on the prosthetic interface, with subsequent embolization. To circumvent this problem, the textured, fibril surface of a polyurethane pump chamber (mat thickness 430 microns) was inoculated with cultured bovine fetal fibroblasts (labelled with thymidine-14C) prior to animal implantation. The pneumatically actuated device (stroke volume 75 ml), maintained a pulsatile blood flow throughout each study. In 20 calf experiments, extending up to 335 days, 30 X 10(6) fibroblasts (in 50 ml media) derived from a single Holstein fetus were distributed on the urethane surface (360 +/- 50 cells/mm2) by rotation of a sealed device for three hours (12 revolutions/hour). Following connection to the circulation, cell washout was minimal. Resultant biologic linings, examined after animal sacrifice, were densely adherent to the underlying polymer matrix, and varied in thickness from 250 micron-1.5 mm. Microscopically, fibroblasts were identified from the surface to base, accompanied by numerous collagen bundles and abundant ground substance. Amino acid analysis in 10/20 pumps implanted for 31--335 days, revealed 50 +/- 5 Hydroxyproline residues/1000 residues (50% collagen) and scant elastin. Donor fibroblasts were identified by radioautography and karyotyping. Lack of immunologic response in 12 Hereford pump recipients as confirmed by serial fibroblast cytotoxicity assays. In conclusion, an induced collagenous-blood interface permitted prolonged mechanical circulatory support in animals without thromboembolic complications.

An in vitro evaluation of a new plasticizer for polyvinylchloride medical devices.

The rate of Hatcol-200 (H-200) leaching from polyvinylchloride (PVC) by serum is one-hundredth that of di-2-ethylhexyl phthalate (DEHP) as determined by radioassay. There is a proportional decrease in leachability of DEHP but not of H-200 as the plasma proteins are diluted. Saline extracts of DEHP demonstrated a low but progressive rate of leaching with an accumulation of 11 microgram/ml after seven weeks whereas H-200 showed only a constant residue of 0.35 microgram/ml. Normal human serum incubated for three weeks at 37 C with PVC strips plasticized with DEHP produced significant growth-inhibition of human diploid fibroblasts. Human serum incubated in an identical manner with PVC plasticized with H-200 for up to four weeks demonstrated no effect on tissue culture. These studies indicate that H-200 deserves further evaluation as a replacement for DEHP in PVC biomedical devices.

Development of collagenous linings on impermeable prosthetic surfaces.

In an effort to accelerate development of a biologic lining on the fibrillar surface of a left ventricular assist pump, the blood-contacting interface was covered with bovine fetal fibroblasts immediately prior to implantation into the animal. Selection of these syngeneic cells was based on their demonstrated prolificacy and abundant collagen production. Comparative studies, carried out in 17 Holstein calves, indicated that an adherent, thin, collagenous lining developed on the fibroblast-seeded polyurethane pump chamber in nine animals. Similar implantations of eight non-cell-seeded (control) devices resulted in formation of a predominantly acellular, fibrinous membrane, varying in thickness from 1 to 8 mm. Pump chamber compliance was significantly reduced when the histologic surface exceeded 3 mm in thickness, resulting in impaired filling and an inadequate stroke volume. The use of 14C-thymidine-labeled fibroblasts permitted later identification of the donor cells in the collagenous linings by radioautography. Serial immunologic studies undertaken to detect evidence of rejection in recipient Holstein calves were negative.