A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies

Summary The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes ¹²⁵I and ¹³¹I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with ¹³¹I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with ¹²⁵I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.     

Accessible hyaluronan receptors identical to ICAM-1 in mouse mast-cell tumours

Immunohistochemical studies of the hyaluronan (HA)-receptor (R), originally found on liver endothelial cells (LEC) and related to the intercellular adhesion molecule 1 (ICAM-1), showed that polyclonal antibodies against HARLEC (HA receptor on LEC) also stain structures in mouse mastocytomas, mainly vessels. To test if intravenously administered HA might target the tumour receptorsin vivo, mice carrying an inoculated mastocytoma in one hind leg muscle were injected in the tail vein with¹²⁵I-tyrosine (T)-labelled HA and killed 75 min after injection when organs and tissues were checked for radioactivity. When doses exceeding the binding capacity of the liver were injected, a significant increase in radioactivity (up to five-fold) within the tumour tissue was found. The weight adjusted difference between control and tumour tissue was greater for smaller tumours, probably due to necrosis in the larger. HA-staining of tumours from animals receiving¹²⁵I-T-HA, showed HA in areas that also stained weakly for ICAM-1 using monoclonal antibodies. ICAM-1 staining was dramatically increased after hyaluronidase treatment of the sections, indicating that the HA is bound to these receptors and thereby blocks antibody recognition.Abbreviations ICAM-1 intercellular adhesion molecule 1 - HA hyaluronan - HARLEC hyaluronan receptor on liver endothelial cells - MW molecular weight     

Pharmacokinetics of a radioiodinated monoclonal antibody F(ab′)2 fragment in a xenograft model with circulating antigen

The pharmacokinetics of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment were investigated in athymic mice bearing OVCAR-3 ovarian carcinoma xenografts, a model in which the CA 125 antigen is present in serum. Nine antibody doses between 0.1 and 650 μg were studied. Optimal tumor to normal tissue ratios were obtained at 100–200 μg of F(ab′)₂. At most antibody doses, the pre-injection level of circulating CA 125 appeared to influence the localization of ¹³¹I activity in tumor, liver and spleen.     

Influence of circulating antigen on blood pool activity of a radioiodinated monoclonal antibody

Athymic mice with and without circulating CA 125 antigen were injected with 0.1–100μg of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment. Both the blood clearance of ¹³¹I activity and the change in serum CA 125 were monitored over 24 h. Influence of CA 125 on blood pool activity could be avoided only at the 100 βg dose. In patient studies, circulating CA 125 levels decreased for the first 2 h after injection of OC 125 F(ab′)₂ but generally returned to preinjection levels shortly thereafter. In vitro binding studies using the sera from patients injected with ¹³¹I-labeled OC 125 F(ab′)₂ suggest that circulating CA 125 could interfere with the tumor uptake of the labeled antibody.     

Disposition of125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [¹²⁵I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. Afteri.v. administration, [¹²⁵I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [¹²⁵I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [¹²⁵I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [¹²⁵I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [¹²⁵I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4⁺ and CD8⁺ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4⁺ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8⁺ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4⁺ and CD8⁺ T cells were also observed in Methl-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4⁺ T cells and the maintenance of the suppressive activity of CD8⁺ T cells.     

Quantitative autoradiographic evaluation of the influence of protein dose on monoclonal antibody distribution in human ovarian adenocarcinoma xenografts

Summary We studied the effect of monoclonal antibody protein dose on the uniformity of radioiodinated antibody distribution within tumor masses using quantitative autoradiography. Groups (n = 11–13/group) of athymic nude mice with subcutaneous HTB77 human ovarian carcinoma xenografts were injected intraperitoneally with an¹²⁵I-labeled anticarcinoma-associated antigen murine monoclonal antibody, 5G6.4, using a high or a low protein dose (500 µg or 5 µg). At 6 days post-injection the macroscopic and microscopic intratumoral biodistribution of radiolabeled antibody was determined. The degree of heterogeneity of the labeled antibody distribution within each tumor was quantified and expressed as thecoefficient of variation (CV) of the activity levels in serial histological sections. Tumors from mice given the 500-µg protein doses had substantially lower CV values, 0.327±0.027, than did tumors from animals given 5-µg protein doses, 0.458±0.041, (P = 0.0078), indicating that the higher protein dose resulted in more homogeneous distribution of radioactivity in tumors than did the lower dose. While the percentage of the injected dose reaching the tumor was comparable between groups, injecting the higher dose of protein resulted in significantly lower tumor to non-tumor uptake ratios than those obtained for the lower protein dose. These data indicate, in this system, that to achieve more uniform intratumoral antibody (and radiation for radioimmunotherapy) delivery, a relatively high protein dose must be administered. However, to obtain this increased uniformity, a substantial drop in tumor/background uptake ratios was seen. Quantitative autoradiographic evaluation of human tumor xenografts is a useful method to assess the intratumoral distribution of antibodies.     

Antibody-directed enzyme prodrug therapy (ADEPT)

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with¹²⁵I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab′)₂ fragment showed localization in these xenografts. The AB57-F(ab′)₂ fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with¹²⁵iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon). A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab′)₂-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress.     

Regional and systemic distribution of anti-tumor x anti-CD3 heteroaggregate antibodies and cultured human peripheral blood lymphocytes in a human colon cancer xenograft.

Anti-tumor antibody (317G5) covalently coupled to an anti-CD3 antibody (OKT3) produces a heteroaggregate (HA) antibody that can target PBL to lyse tumor cells expressing the appropriate tumor Ag. The i.v. and i.p. distribution of radiolabeled HA antibody 317G5 x OKT3 and of radiolabeled cultured human PBL were studied in athymic nude mice bearing solid intraperitoneal tumor established from the human colon tumor line, LS174T. Mice were injected with 125I-labeled HA antibody, 125I-labeled anti-tumor mAb, or 111In-labeled PBL, and at designated timepoints tissues were harvested and measured for radioactivity. 125I-317G5 x OKT3 localized specifically to tumor sites. Tumor radioactivity levels (percent injected dose/gram) were lower with 125I-317G5 x OKT3 HA antibody than with 125I-317G5 anti-tumor mAb, but were similar to levels reported for other anti-tumor mAb. The major difference in radioactivity levels observed between i.v. and i.p. administration of 125I-317G5 x OKT3 was an increase in hepatic radioactivity after i.v. HA antibody administration. HA antibodies produced from F(ab')2 fragments, which exhibit decreased m. w. and decreased Fc receptor-mediated binding, demonstrated improved tumor:tissue ratios as compared to intact antibody HA. 125I-317G5 F(ab')2 x OKT3 F(ab')2 antibody levels were equivalent to intact HA antibody levels in tumor, but were lower than intact HA antibody levels in the blood, bowel, and liver. Tumor:bowel ratios (20:1 at 48 h) were highest when 317G5 F(ab')2 x OKT3 F(ab')2 was injected i.p. Autoradiography confirmed that anti-tumor x anti-CD3 HA antibodies localized specifically to intraperitoneal tumor; that i.p. administered HA antibodies penetrated tumor directly; and that i.v. administered HA antibodies distributed along tumor vasculature. Cultured human PBL distributed in moderate concentrations to intraperitoneal tumor when administered i.p., but not when administered i.v. The poor localization of i.v. injected PBL to tumor may reflect species disparity in homing receptors and/or endothelial ligands, a problem which may be overcome with a syngeneic model. These results suggest that regional therapy with HA antibodies and PBL may offer advantages over systemic therapy for initial clinical trials.     

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies

The HER2 protooncogene encodes a receptor tyrosine kinase, p185^HER2. The overexpression of p185^HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185^HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185^HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185^HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185^HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185^HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 antibody), was also tested in soft-agar growth assays for activity against p185^HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185^HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a humanized 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185^HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185^HER2.     

Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/- mice

A neutralizing antibody to the receptor for the advanced glycation end products (anti-RAGE Ab) was developed as a potential treatment of acute and chronic inflammatory conditions. Previous pharmacology studies demonstrated efficacy of the anti-RAGE antibody in a mouse model of sepsis. We examined pharmacokinetics and lung distribution of [¹²⁵I]anti-RAGE Ab in RAGE^-/- and wild-type (129S5) mice following single IV administration. Serum pharmacokinetics of [¹²⁵I]anti-RAGE Ab was similar in RAGE^-/- and 129S5 mice, with the total body clearance of 0.3 mL/hr/kg and the elimination half-life of 11–12 days, suggesting the target expression had limited impact on overall elimination of [¹²⁵I]anti-RAGE Ab from mice. [¹²⁵I]Anti-RAGE Ab accumulated in the lung of 129S5 mice, with ∼4% of total dose retained in the lung at days 6–27 and the lung AUC_0-∞ of ∼300% of that in serum. The SDS-PAGE analysis suggested that most of retained lung radioactivity was attributed to intact antibody. No accumulation of radioactivity was observed in the lung of RAGE^-/- mice, indicating that lung uptake of [¹²⁵I]anti-RAGE Ab was target-dependent in wild-type mice. These data suggest that the anti-RAGE Ab was able to localize to the site of RAGE expression, the lung, and support the findings in the previous pharmacology studies.Key words: monoclonal antibody, ADME, tissue distribution, sepsis, pharmacokinetics, advanced glycation end products, RAGE, autoimmunity     

Improved monoclonal antibody tumor/background ratios with exchange transfusions

Blood exchange transfusions were performed in nude rats with subcutaneous HTB77 human ovarian carcinoma xenografts in an attempt to improve specific monoclonal antibody (MoAb) tumor/non-tumor uptake ratios. Animals were injected intravenously with both ¹³¹I-5G6.4 specific and ¹²⁵I-UPC-10 non-specific MoAb. Twenty-four hours later 65–80% of the original blood was exchanged with normal heparinized rat blood and then these rodents were sacrificed. Exchange transfusion significantly (P < 0.05) decreased normal tissue activities of ¹³¹I (except for muscle) by 63–85%. while tumor activity decreased only 5%. Tumor to background ratios increased from 0.1–0.8 to 2.3–6.3. Exchange transfusions substantially enhance tumor/normal tissue antibody uptake ratios and, along with plasmapheresis, may be useful in enhancing antibody localization in vivo, particularly for therapy.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.     

ImmunoPET and biodistribution with human epidermal growth factor receptor 3 targeting antibody 89Zr-RG7116

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 (⁸⁹Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of ⁸⁹Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of ⁸⁹Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg ⁸⁹Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent ⁸⁹Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of ⁸⁹Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of ⁸⁹Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, ⁸⁹Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.     

A new method for faster blood clearance in radioimmuno-guided surgery

A major inconvenience of intraoperative tumor detection with radiolabeled monoclonal antibody (MoAb) by means of a probe is the long interval needed between the injection of MoAb and surgery (at least 20 days). We describe a new method which entails injection of 125I-labeled biotinylated monoclonal antibodies and cold avidin in order to obtain a rapid blood clearance of the radiotracer. The first clinical application of this protocol has been carried out in a colon cancer patient operated on six days after the administration of 125I-labeled biotinylated MoAb and cold avidin, with an optimal tumor-to-normal tissue ratio and with 90% of the circulating radioactivity cleared in two days.     

A13C NMR study of the hinge region of a mouse monoclonal antibody

Summary A¹³C NMR study is reported of the hinge region of an intact mouse monoclonal antibody with a molecular weight of 150 K. Cys, Ile, and Pro analogs of the antibody labeled with¹³C at the carbonyl carbon were prepared by growing hybridoma cells in the serum-free media. Resonance assignments have been performed as described previously [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I. and Arata, Y. (1991)Biochemistry,30, 270–278]. The spectral data obtained show that¹³C NMR can give detailed information about the structure of the hinge region of the intact antibody molecule. Prospects for the future role of¹³C NMR in the structural analyses of larger proteins are briefly discussed.Dedicated to the memory of Professor V.F. Bystrov     

Scintigraphic detection of xenografted tumors producing human basic fibroblast growth factor

A murine monoclonal antibody 3H3 recognizes the basic fibroblast growth factor (FGF) and inhibits the growth of human glioblastoma cells both in vitro and in vivo. We studied the potential of a scintigraphic technique using the 3H3 antibody to detect tumors that produce basic FGF.¹²⁵I- and¹¹¹In-labeled 3H3 bound to U87MG human glioblastoma cells in vitro. U87MG cells were inoculated subcutaneously into nude mice. After development of the tumor, radiolabeled 3H3 was injected into the subcutaneous space surrounding the tumor. A high level of radioactivity from 3H3 was retained at the tumor, whereas an irrelevant antibody cleared rapidly from the injected site. Radiolabeled 3H3 was not retained in tumors that did not produce basic FGF. Scintigraphic detection of tumors expressing basic FGF would be valuable for the therapeutic application of the antibody.     

Dose dependent pharmacokinetics,tissue distribution,and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of ¹²⁵Iodide and ¹¹¹Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.     

Dose dependent pharmacokinetics,tissue distribution,and anti-tumor efficacy of a humanized monoclonal antibody against DLL4 in mice

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of ¹²⁵Iodide and ¹¹¹Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.