Strategies to reduce glucose exposure in peritoneal dialysis patients.

Glucose has been used successfully for more than two decades in peritoneal dialysis, and in this regard, must be considered a safe and effective osmotic agent. Recently, however, insight has been growing about the potential for metabolic and peritoneal effects arising from long-term exposure to high glucose concentrations--for example, hyperlipidemia and loss of peritoneal ultrafiltration. Clinical concerns over exposure to excessive glucose and glucose degradation products (GDPs) during peritoneal dialysis can be significantly ameliorated by the use of non-glucose-based peritoneal dialysis (PD) solutions, in combination with more biocompatible glucose-based formulations. Peritoneal exposure to GDPs can be reduced by using low-GDP-containing glucose formulations and non glucose solutions such as amino acids and icodextrin. Peritoneal glucose exposure, hyperosmolar stress, and carbohydrate absorption can be reduced by using a combination of icodextrin and amino acids.

The potential of gene therapy in the peritoneal cavity.

Gene therapy is a promising new treatment modality based on molecular genetic modification to achieve a therapeutic benefit. We believe that gene therapy in the peritoneal cavity holds considerable promise, and we describe strategies by which genetic modification can be used to treat a variety of disease states or conditions. First, we can envision a strategy, based on genetic modification of the peritoneal membrane, to improve the practice of peritoneal dialysis through the production of proteins that would be of therapeutic value in preventing membrane damage and in preserving or enhancing its function as a dialyzing membrane. Second, the membrane could be genetically modified for either local or systemic delivery of therapeutic proteins. This approach could be applied to a variety of pathologies or conditions that require either sustained or transient delivery of therapeutic proteins, such as enzymes or growth factors. Third, gene transfer has already been incorporated into several strategies for the treatment of intra-abdominal carcinomas, and it has been effective in animal models of ovarian and bladder cancer and of peritoneal mesothelioma. Finally, gene transfer can be a valuable tool in increasing our understanding of the biology of the peritoneal membrane. By being able to manipulate the expression of specific genes through gene transfer, their role in various (patho)physiological processes can be identified. In summary, gene therapy in the peritoneal cavity has significant potential to address a variety of diseases or pathophysiological conditions, and to further our knowledge of peritoneal cavity biology.

New solutions for peritoneal dialysis in adult and pediatric patients.

The standard PD solutions used today contain physiological electrolyte profiles similar to that of interstitial fluids and are supplemented with glucose as the osmotic agent. Improvements in solution composition during the last 20 years have been largely restricted to minor changes in buffer and electrolyte levels. Newer PD solutions, on the other hand, are designed to manage comorbidities associated with patients on maintenance dialysis, to tailor the ultrafiltration profile based upon dwell time, and to better preserve peritoneal membrane function and host defenses. The evidence to date indicates that, in malnourished PD patients (children and adults), IP amino acids improve protein nutritional status, particularly if low protein intakes are a cause of the malnutrition. The availability of glucose polymers allows the clinician to complement standard glucose-based formulations with one that can provide improved ultrafiltration in both CAPD and APD patients for long dwells, and in patients experiencing ultrafiltration loss owing to a large effective peritoneal surface area. Owing to the reduced calorie and carbohydrate load, glucose polymers may also offer long-term metabolic advantages. Although the control of acid-base balance can be well managed in the vast majority of patients with a 35-40 mmol/L lactate solution, the development and clinical evaluation of bicarbonate-based solutions is underway as a result of concern over the potentially bioincompatible nature of acidic lactate formulations. To date, in vitro, ex vivo, and limited clinical studies show that such formulations, and in particular bicarbonate/lactate combinations are efficacious and well tolerated, and show improved peritoneal cell function versus conventional solutions. In conclusion, ongoing research and development has produced a new generation of PD solutions that, to various degrees, meet different criteria established for an ideal PD solution for chronic adult and pediatric patients on PD. These criteria include good clearance and ultrafiltration, supply of nutrition, iso-osmolality, physiologic pH, bicarbonate buffer, and minimal absorption of the osmotic agent. Several of the new solutions have already demonstrated clinical utility in controlled clinical trials and are commercially available in Europe. Wider clinical use will further add to our understanding of the impact of these formulations on patient outcomes.

Quantitation of dextran 70 in peritoneal dialysate from patients administered 7.5% polyglucose.

A method using gel permeation chromatography was evaluated for the quantitation of dextran 70 in dialysate samples containing polyglucose. Dialysate samples containing dextran 70 and polyglucose were pretreated using the enzyme alpha-amylase to selectively hydrolyze the alpha(1-4)-linked polyglucose, while leaving the alpha(1-6)-linked dextran 70 intact. Following sample deproteinization with trichloroacetic acid, dextran 70 was quantitated using gel permeation chromatography with refractive index detection. This method was evaluated for accuracy, precision, specificity, linearity, range, and analyte stability. Adequate method linearity with a correlation of >0.999 was established over the range of dextran 70 concentration from 1 to 0.025 mg/ml. Method precision was approximately 2% R.S.D. and accuracy (% recovery) was approximately 98-100% in the typical sample concentration range (1-0.5 mg/ml). This method was applied to the determination of intraperitoneal fluid kinetics in continuous ambulatory peritoneal dialysis (CAPD) patients administered daily night-time intraperitoneal exchanges with either 7.5% polyglucose or 4.25% dextrose. Dextran 70 was added to the dialysis solutions to yield an initial concentration of 1 mg/ml. Dialysate samples were collected at various times over a 10-h dwell-time and assayed for dextran 70. Intraperitoneal volume profiles based on dextran 70 concentrations and drain volumes were then calculated for each dialysis solution.

Enhancement of the functional repertoire of the rat parietal peritoneal mesothelium in vivo: directed expression of the anticoagulant and antiinflammatory molecule thrombomodulin.

We have used our previously described ex vivo mesothelial cell (MC)-mediated gene therapy strategy (Gene Ther. 2:393-401, 1995) to modify the functional properties of the rat parietal peritoneal mesothelium in vivo by expression of a membrane-bound recombinant protein on the MC surface. Rat primary MCs were stably transfected (using strontium phosphate DNA coprecipitation) with a plasmid containing the gene for rat thrombomodulin (TM), a transmembrane glycoprotein that functions as an essential cofactor for the physiological activation of the anticoagulant protein C by the enzyme thrombin. As demonstrated by immunohistochemistry and by direct equilibrium binding with radiolabeled thrombin, genetically modified MCs expressed high levels of TM antigen on their surface in vitro. As judged by a thrombin-dependent protein C activation assay, such MC membrane-bound TM was biologically active. Once reseeded on the denuded parietal peritoneal surface of syngeneic recipients, these TM-transfected MCs continued to express TM antigen in vivo for at least 90 days. Moreover, the recombinant TM expressed on the reconstituted parietal mesothelium retained its ability to activate protein C in a thrombin-dependent manner. Our data indicate that MC-mediated expression of TM can be used to augment the anticoagulant properties of the parietal peritoneal surface. In general, our results suggest that ex vivo MC-mediated gene therapy can be used to deliver other therapeutic transmembrane proteins to the MC surface to enhance the functional repertoire of the parietal mesothelium in vivo.

Modulation of transgene expression in mesothelial cells by activation of an inducible promoter.

BACKGROUND: The efficacy of peritoneal dialysis and its success as a long-term treatment depends on the preservation of the integrity of the peritoneal membrane. With increasing time on dialysis, the membrane may become compromised resulting in decreased dialysing capacity. We have pursued an innovative strategy, i.e. genetic modification of the mesothelial cell to change the properties of the membrane to potentially improve its dialysing capacity and longevity, and have demonstrated the feasibility of this approach in a rat model of ex vivo gene transfer. The potential to regulate transgene expression in this model is examined here. METHODS: Rat peritoneal mesothelial cells (MCs) were stably modified to express human growth hormone (hGH) under control of the heavy metal ion and glucocorticoid-regulatable murine metallothionein-1 promoter. The effect of zinc and the synthetic glucocorticoid dexamethasone on hGH expression was analysed in MC clones maintained in continuous passage or stationary phase, and in our rat model of ex vivo gene transfer. RESULTS: Exposure of these clones to zinc and dexamethasone, either singly or in combination, resulted in significant (i.e. 2-200-fold) increases in hGH production. Zinc-induced modulation of hGH production was demonstrated in cells in continuous passage and stationary culture. Regulation was also demonstrated after ex vivo gene transfer by both the intraperitoneal administration of zinc ions or the systemic administration of dexamethasone. CONCLUSIONS: Our results demonstrate the modulation of transgene expression in MCs in vitro and in vivo, and suggest the potential for the regulation of gene expression in a genetically modified mesothelium that may ultimately be used for the delivery of therapeutic proteins to maintain peritoneal membrane viability in the peritoneal dialysis patient.

Maltose and isomaltose in uremic plasma following icodextrin administration.

The presence of mixed disaccharides (maltose and isomaltose) in plasma from uremic patients has been previously investigated using gel-permeation chromatography. However, this method is unable to separate maltose (linked alpha-1-4) from isomaltose (linked alpha-1-6). We describe an alternative method using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) for the direct determination of maltose and isomaltose in uremic plasma. We measured maltose and isomaltose using HPAE-PAD in 6 normal subjects and in 15 uremic patients before and after once-daily icodextrin administration for at least 4 weeks. Both maltose and isomaltose were below limits of detection (< 1.0 mg/L) in plasma from normal controls. Patients with end-stage renal disease treated by continuous ambulatory peritoneal dialysis had elevated levels of isomaltose (23.6 +/- 8.3 mg/L) but low levels of maltose (< 3.0 mg/L). Treatment with icodextrin resulted in elevated plasma levels of maltose (range: 500-1600 mg/L), while levels of isomaltose declined to 9.8 +/- 5.2 mg/L (P < 0.0001 vs. baseline levels). We conclude that isomaltose (not maltose) is the primary disaccharide isomer that is elevated in the plasma of uremic patients, whereas maltose is the primary disaccharide isomer that is elevated following icodextrin administration. Furthermore, icodextrin administration results in an apparent reduction of isomaltose. Additional investigation will be required to address the mechanism for the reduction of isomaltose in patients treated by icodextrin.

Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) was evaluated for the quantitation of polyglucose metabolites (DP2-DP7) in human plasma. The method was investigated for accuracy, precision, specificity, linearity, range and analyte stability. Samples were prepared by dilution into the standard range (0.1-10 microg/ml) followed by deproteinization using a 30,000 molecular mass cut-off filtration device. The limit of detection was 0.05 microg/ml for all metabolites. Method precision for DP2-DP7 varied from approximately 2% R.S.D. in the upper range to approximately 15% R.S.D. at the limit of quantitation. Samples were stable following one or two freeze-thaw cycles and, after preparation, they could be refrigerated for up to 72 h. Application of this method to clinical plasma samples from continuous ambulatory peritoneal dialysis (CAPD) patients administered one daily night-time intraperitoneal exchange of 2 l of 7.5% polyglucose solution for four weeks indicated that plasma levels of DP2, DP3 and DP4 increased from baseline levels of <0.01 g/l to steady-state levels of 1.2+/-0.3, 1.2+/-0.3 and 0.4+/-0.1 g/l (mean+/-S.D.), respectively. These steady state plasma levels for DP2 and DP3 are comparable to previously reported levels in patients administered daily overnight 7.5% polyglucose dialysis solution.

Future clinical research with icodextrin-containing solutions.

Icodextrin-based solutions have been investigated for over ten years and are commercially available in some countries in Europe. Although many studies have been described using icodextrin, we believe that there are many interesting areas remaining for clinical research with icodextrin-based PD solutions. Included in these are the careful examination of the kinetics of icodextrin during the 14-16 hour daytime exchange in CCPD, new studies investigating fluid absorption during icodextrin exchanges, and finally, the potential use of icodextrin in an early start dialysis regime. We look forward to seeing the results from these very interesting studies.

Ultrafiltration and solute kinetics using low sodium peritoneal dialysate.

Low sodium peritoneal dialysate has been reported to enhance sodium loss and alleviate signs of fluid overload in continuous ambulatory peritoneal dialysis patients. To elucidate the mechanisms involved, we compared ultrafiltration and solute kinetics using low sodium dialysate (LNaD; 105 mEq/liter sodium, 2.5% glucose, 348 mOsm/liter), conventional dialysate with equal osmolality (CD1.5; 132 mEq/liter sodium, 1.5% glucose, 348 mOsm/liter) and conventional dialysate with equal glucose concentration (CD2.5; 132 mEq/liter sodium, 2.5% glucose, 403 mOsm/liter). A 2 liter, six hour exchange of each dialysate was performed on separate days in 10 chronic peritoneal dialysis patients. Transperitoneal solute diffusion was assessed by calculating the permeability-area product (PA) of the peritoneal membrane from the dependence of plasma and dialysate solute concentrations on tie. Net fluid removed using LNaD of 190 +/- 90 (SEM) ml was similar to that using CD2.5 (250 +/- 90 ml) but higher (P < 0.01) than that using CD1.5 (-200 +/- 60 ml). Sodium loss was higher using LNaD (72 +/- 11 mEq, P < 0.01) and CD2.5 (41 +/- 12 mEq, P < 0.05) than using CD1.5 (-18 +/- 8 mEq). Changes in plasma sodium concentration were small during each dwell and were not different among the study dialysates. PA values for urea (23.4 +/- 1.6 ml/min), creatinine (10.0 +/- 1.0 ml/min), and glucose (10.3 +/- 1.3 ml/min) were similar when determined in each dialysate. The PA value for sodium (7.6 +/- 1.5 ml/min) could only be accurately determined in LNaD. We conclude that: (1) net fluid removed is greater using LNaD than CD1.5 despite similar osmolalities because LNaD has a higher glucose concentration and glucose is a more effective osmotic solute than sodium; (2) sodium loss when using LNaD is enhanced by both diffusion and convection; and (3) sodium diffuses across the peritoneum slower than urea, creatinine and glucose. These data suggest that LNaD alleviates signs of fluid overload by increasing net fluid removal and enhancing sodium loss.

Mesothelial cell-mediated gene therapy: feasibility of an ex vivo strategy.

We have developed a model system in the rat to test the feasibility of recombinant protein expression by genetically modified peritoneal mesothelial cells following autologous peritoneal implantation. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transduced (using a Moloney murine leukemia virus (MoMLV)-derived retroviral vector, BAG, expressing the Escherichia coli lacZ gene) to mark the cells with a reporter protein (beta-galactosidase, beta-gal). Such transduced mesothelial cells, tagged with DiO, a fluorescent lipophilic dye used for long-term tracing of transplanted cells, were then reseeded on the denuded peritoneal surface of syngeneic recipients. DiO-labeled, BAG-transduced mesothelial cells were observed to repopulate the denuded areas and remain attached there for > 90 days. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo (ie beta-gal activity was present for at least 1 month). Our results demonstrate the feasibility of ex vivo gene therapy using peritoneal mesothelial cells.

Systemic delivery of a recombinant protein by genetically modified mesothelial cells reseeded on the parietal peritoneal surface.

To evaluate the ability of genetically modified peritoneal mesothelial cells to deliver recombinant proteins to the systemic circulation, we used our previously described mesothelial cell-based ex vivo gene therapy strategy. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transfected (using strontium phosphate DNA co-precipitation) with the plasmid pSVTKgh to express a secreted reporter gene product, human growth hormone (hgh). Such hgh-secreting mesothelial cells were reseeded on the denuded peritoneal surface of syngeneic recipients and delivery of the reporter gene product to the systemic circulation was monitored by analysis of serum samples for the presence of hgh at various times after mesothelial cell implantation. Polymerase chain reaction (PCR) analysis demonstrated that the hgh-transfected mesothelial cells repopulated the denuded areas and remained attached there for at least 12 weeks. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo and secreted hgh in sufficient quantity to be detected in the systemic circulation (ie statistically significant amounts of hgh could be measured in the serum of cyclosporine A-treated rats for at least 2 months; Mann-Whitney test, P < 0.05). Our results demonstrate the successful, sustained, systemic delivery of a recombinant protein by genetically modified peritoneal mesothelial cells following their reattachment to the peritoneal surface, and suggest the potential of ex vivo mesothelial cell-mediated gene therapy for the treatment of inherited or acquired disorders requiring delivery of therapeutic proteins to the circulation.

Compartmental distribution of tumor-specific monoclonal antibodies in human melanoma xenografts.

Monoclonal antibodies (MAb) are attractive for tumor therapy because of their exquisite specificity. Although a majority of tumor cells in small (< or = 20 mg) solid tumors can be labeled following systemic administration of antitumor cell MAbs, little quantitative information is available as to the distribution of these MAbs within the several compartments that comprise solid tumors. Our goal was to provide such data in a well-characterized melanoma xenograft system. In accord with earlier work, i.v.-injected, melanoma-specific MAbs 436 and IND1, directed, respectively, against the 125 kD and HMW-melanoma-associated antigens, accumulated in M21 and SK-MEL-2 tumor xenografts in amounts of approximately 20% of injected dose/g. However, only 20-24% of the MAbs present in tumor xenografts was bound to tumor cells; the great majority (76-80%) was in the tumor extracellular fluid (ECF) and collagenous residue fractions. These results could not be accounted for by MAb degradation or release of MAbs from tumor cells during xenograft dissociation. Rather, they reflected in large part interactions of MAbs with antigens which tumors had shed into the ECF. Thus, 48 h after i.v. injection of 20 micrograms of melanoma-specific, biotin-tagged MAb, 46-66% of that present in the tumor ECF was complexed with melanoma-associated antigens. Overall, 61-73% of the MAbs recovered from tumor xenografts were bound to tumor antigens (either to tumor cells themselves or to tumor-shed antigens). In contrast, only approximately 4% of a melanoma-nonspecific MAb (B72.3) accumulated per g tumor after i.v. injection and nearly all of this was free in the ECF. Consistent with these data, fluorescence microscopy revealed that i.v.-injected, fluorescein-tagged MAbs achieved highest concentrations in tumor stroma, particularly at the tumor-host interface. Flow cytometry of dissociated solid tumors revealed that both the fraction of MAb-labeled tumor cells and the amount of MAb/tumor cell could be increased by increasing the administered i.v. dose of melanoma-specific MAb. Nonetheless, even at the highest i.v. injected dose (300 micrograms), 15-37% of tumor cells lacked detectable MAb labeling. Taken together, the data indicate that delivery of tumor cell-specific MAbs to solid tumors cannot be equated with their delivery to tumor cells. This distinction is important for immunotherapeutic approaches that require MAb contact with tumor cells.

Experimental peritoneal dialysis solutions.

A survey of the literature suggests continued interest in modifying the composition of peritoneal dialysis solutions. Osmotic agents such as glucose polymers and short-chain polypeptides may find a role in peritoneal dialysis fluids as partial substitutes for dextrose. New solutions containing amino acids should serve as more than sinks for uremic toxins by providing nutritional support and by ameliorating uremic lipid abnormalities. Increasing emphasis is being paid to biocompatibility during the development of new peritoneal dialysis fluids.

Pathways for fluid loss from the peritoneal cavity.

During peritoneal dialysis, fluid is transported out of the peritoneal cavity by lymphatic and nonlymphatic pathways, thereby decreasing net ultrafiltration by 40-50% and reducing small solute clearance by 15-20%. The direct lymphatic pathway consists of the diaphragmatic lymphatics, which directly connect the peritoneal cavity to the bloodstream. The interstitial lymphatic and direct blood entry pathways convey fluid that has been driven into the interstitial space of the tissue surrounding the peritoneal cavity by the increased intraperitoneal pressure, and return it to the bloodstream. Since flow through lymphatic pathways is only a portion of the flow through all pathways, total fluid loss is greater than lymph flow. The best technique for estimating lymph flow is direct measurement by cannulation of lymphatic vessels, a technique that is not clinically feasible. The tracer disappearance technique, which measures the rate at which macromolecules leave the peritoneal cavity, is an indirect measure of fluid loss. The tracer appearance technique, which measures the rate at which macromolecules reach the blood from the peritoneal cavity, slightly overestimates lymph flow because some tracer may enter the bloodstream directly from the tissues. Much of the previous controversy over the contribution of the lymphatic pathways to total fluid loss can be resolved by understanding the differences in what these techniques measure.

A quantitative analysis of tumor specific monoclonal antibody uptake by human melanoma xenografts: effects of antibody immunological properties and tumor antigen expression levels.

The time-dependent (5 min-72 h) localization of 3 radiolabeled anti-melanoma monoclonal antibodies (MAbs 436, IND1, and 9.2.27) was studied in paired label experiments in small (4-12 mg) s.c. human melanoma xenografts (SK-MEL-2 and M21) in athymic nude mice. MAb 436 recognizes a Mr 125,000 cell surface melanoma-associated glycoprotein antigen (125 kDa-MAA); MAbs IND1 and 9.2.27 recognize a high molecular weight melanoma-associated antigen, but with equilibrium association constants differing by 2 orders of magnitude (10(8)-10(10) M-1). The two tumors were found to differ in their antigen expression levels and in both interstitial and vascular volumes. Accumulation of MAbs in both tumors was determined primarily by antigen expression levels and also by physiological factors such as vascular permeability and vascular volume; at the dose administered (20 micrograms/mouse), differences in MAb affinity among specific MAbs had minimal effect on accumulation. Quantitative flow cytometry measurements showed that antigen expression in vivo differed from that of cultured tumor cells. In vivo, expression of the Mr 125,000 MAA decreased by a factor of about 2.5 in both tumors. In contrast, the in vivo expression of the high molecular weight MAA decreased in M21 tumors but increased by 2.0-3.5-fold in SK-MEL-2 tumors. Data were analyzed using a three-compartment pharmacokinetic model (C. Sung et al., Cancer Res., 52:377-384, 1992) to provide plasma-to-tissue transport constants (k), the interstitial fluid flow rate (L), and estimates of the in vivo interstitial MAb binding site concentration (B0). For all MAbs, the plasma-to-tissue transport constants were consistently greater for M21 tumors (0.44-0.85 microliter/min/g) than for SK-MEL-2 tumors (0.28-0.66 microliter/min/g), and values of k for both tumors were approximately 1 order of magnitude greater than those for skeletal muscle (0.06-0.08 microliter/min/g). The model-estimated binding site concentration of melanoma-specific antibodies was 15-70 times lower than that predicted by experimental measurements of tumor antigen concentrations. Factors that may contribute to this discrepancy include inaccessibility of tumor cell binding sites to MAb and MAb catabolism. In summary, these results indicate that, for the MAb dose used in this study, variables pertaining to the tumor target (i.e., antigen expression levels, vascular volume, and vascular permeability) are the most important for determining MAb accumulation in tumors.

Predicted and observed effects of antibody affinity and antigen density on monoclonal antibody uptake in solid tumors.

The uptake and binding of monoclonal antibodies (MAbs) in solid tumors after a bolus i.v. injection are described using a compartmental pharmacokinetic model. The model assumes that MAb permeates into tumor unidirectionally from plasma across capillaries and clears from tumor by interstitial fluid flow and that interstitial antibody-antigen interactions are characterized by the Langmuir isotherm for reversible, saturable binding. Typical values for plasma clearance and tumor capillary permeability of a MAb and for interstitial fluid flow and interstitial volume fraction of a solid tumor were used to simulate the uptake of MAbs at various values of the binding affinity or antigen density for a range of MAb doses. The model indicates that at low doses, an increase in binding affinity may lead to an increase in MAb uptake. On the other hand, at doses approaching saturation of antigen or when uptake is permeation limited, an increase in the binding affinity from moderate to high affinity will have only a small effect on increasing MAb uptake. The model also predicts that an increase in antigen density will greatly increase MAb uptake when uptake is not permeation limited. Our experiments on MAb uptake in melanoma tumors in athymic mice after injection of 20 micrograms MAb (initial plasma concentration, about 120 nM) are consistent with these model-based conclusions. Two MAbs differing in affinity by more than 2 orders of magnitude (3.8 x 10(8) M-1 and 5 x 10(10) M-1) but with similar in vivo antigen densities in M21 melanoma attained similar concentrations in the tumor. Two MAbs of similar affinity but having a 3-fold difference in in vivo antigen density in SK-MEL-2 melanoma showed that the MAb targeted to the more highly expressed antigen attained a higher MAb concentration. We also discuss the model predictions in relation to other experiments reported in the literature. The theoretical and experimental findings suggest that, for high dose applications, efforts to increase MAb uptake in a tumor should emphasize the identification of an abundantly expressed antigen on tumor cells more than the selection of a very high affinity MAb.

Spatial distribution of tumor-specific monoclonal antibodies in human melanoma xenografts.

The time-dependent (1-72-h) spatial distribution of three biotinylated anti-melanoma monoclonal antibodies (MAbs), a control MAb, and several macromolecular tracers was studied in two small (4-12-mg), well-characterized human melanoma xenografts (SK-MEL-2, M21) growing in the s.c. space of athymic nude mice. The specific MAbs (436, IND1, and 9.2.27) recognize two different melanoma cell surface antigens (Mr 125,000 glycoprotein melanoma-associated antigen and high molecular weight melanoma-associated antigen) and have equilibrium association constants differing by two orders of magnitude (10(8)-10(10) M-1). SK-MEL-2 tumors were poorly vascularized and were composed of one or several collections of tumor cells with few intratumor blood vessels. In contrast, M21 tumors induced a strong angiogenic response and were organized into multiple small tumor cell nests separated from each other by fine blood vessels. Neither tumor developed extensive connective tissue stroma. In both tumors, hyperpermeable blood vessels were concentrated at the tumor-host interface but some intratumor vessels in M21 tumors were also leaky. Macromolecular tracers extravasated extensively from leaky vessels into tumor stroma but penetrated poorly into tumor parenchyma. All three tumor-specific MAbs stained tumor cell surfaces in a time-dependent fashion such that one-half or more of all tumor cells were stained by 24-48 h. Tumor cell staining was favored by increased density of tumor cell antigens but, at the doses studied, was little affected by differences in affinity among tumor-specific antibodies. The distribution of MAb staining was nonuniform in two respects: (a) peripherally situated tumor cells were more likely to be stained than centrally placed cells, and only in the smallest tumors did MAb reach centrally placed tumor cells; and (b) staining was nonuniform in different parts of the same tumor. The inhomogeneity of tumor cell staining by tumor-specific MAb was attributable to several factors, including: tumor blood vessel number, distribution, perfusion and permeability; distribution of tumor connective tissue stroma; small volume of the parenchymal interstitial space and relatively impaired diffusion of macromolecules in that space (low effective diffusivity of MAb); and interactions between specific MAbs and tumor cells. Of these factors, those associated with the parenchymal compartment apparently were rate limiting, and strategies that enhance parenchymal penetration are likely to improve solid tumor therapy with MAbs.