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Keywords:

  • extracorporeal;
  • rat;
  • colon carcinoma;
  • toxicity;
  • auristatin;
  • immunoconjugate

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

BACKGROUND:

Antibody-drug conjugates, comprising monoclonal antibodies (MoAbs) that bind to tumor-associated antigens, display different toxicity profiles compared with radiolabeled MoAbs. Dose-limiting toxicities may include damage to the liver and myelotoxicity. The drug component is the antimitotic agent auristatin, which is 100-1000 times more potent than doxorubicin. Consequently, auristatin antibody-drug conjugates require a high selectivity in tumor targeting to display pronounced activity at well-tolerated doses. We have evaluated the possibility of increasing the therapeutic index of BR96-auristatin by combining the administration of conjugates with subsequent extracorporeal affinity adsorption treatment.

METHODS:

Rats were injected with biotinylated, monomethyl auristatin F (MMAF)-conjugated monoclonal antibody BR96. The conjugate was then removed from the circulation by extracorporeal affinity adsorption treatment, 24 hours postinjection using an avidin affinity column. By analyzing blood parameters for 100 days, myelotoxicity, hepatotoxicity, and nephrotoxicity were assessed. Body weight, general status, and tumor size were also recorded. The toxicity-reducing effect of extracorporeal affinity adsorption treatment was evaluated.

RESULTS:

Extracorporeal affinity adsorption treatment removed 85%-90% of BR96-MMAF from the circulation. Early toxicity-related death was seen in nontumor-bearing animals that were given MMAF-conjugated BR96, in contrast to animals that were given a higher amount of BR96-MMAF with subsequent extracorporeal affinity adsorption treatment, in which all survived 100 days postinjection. Extracorporeal affinity adsorption treatment reduced the loss of body weight, myelotoxicity, and hepatotoxicity.

CONCLUSIONS:

Extracorporeal affinity adsorption treatment can be used to reduce the toxicity associated with administration of BR96-MMAF conjugates, making it possible to increase the amount of conjugates administered. The combined treatment will be further optimized in future studies. Cancer 2010;116(4 suppl):1033–42. © 2010 American Cancer Society.

One of many approaches being evaluated in experimental models and in the clinic for the treatment of cancer is the use of antibody-drug conjugates. The strategy is to target a tumor-associated marker with a highly specific antibody that can deliver a toxic payload to the cancer cell. The specific targeting of cancer cells means that the toxic payload has much greater potency than conventional chemotherapeutics, while reducing the risk of toxic side effects.

The auristatins are synthetic antimitotic agents that destabilize microtubules and are structurally related to the potent marine cyclic pentapeptide dolastatin-101. These drugs are much more potent than conventional chemotherapeutic drugs. A dipeptide (valine-citrulline) linker, cleavable by lysosomal proteases (eg, cathepsin B), has recently been used to link auristatins to different monoclonal antibodies.2 Monomethylauristatin F (MMAF; Fig. 1) is an auristatin that possesses a negatively charged C-terminal phenylalanine residue that limits cell permeability, in contrast to monomethylauristatin E (Fig. 1), which is uncharged and freely cell permeable. Intracellular linker proteolysis of monomethylauristatin E conjugates may, therefore, expose surrounding normal tissues to free drug that may induce toxicity. The risk of toxicity is reduced when MMAF is used. However, the “bystander effect” of monomethylauristatin E, ie, the exposure of nonconjugate-binding tumor cells to extracellular free drug, will also be lost. MMAF is of great interest for targeted delivery because the free drug has higher cell cytotoxicity, lower toxicity, and much higher aqueous solubility than monomethylauristatin E.2

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Figure 1. Chemical structure of monomethyl auristatin F and monomethyl auristatin E.

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Conjugated antibodies can remain in the circulation for several days after administration, resulting in a low target-to-normal tissue ratio. As a consequence, dose-limiting toxicity often prevents administration of the doses needed to eradicate solid tumors. Removal of the antibody conjugates from the circulation has been applied for radioimmunoconjugates as a strategy to overcome the problem of insufficient targeting and to reduce the toxicity to normal organs. One technique developed for the removal of circulating conjugates is extracorporeal affinity adsorption treatment, which removes the conjugate from the blood in a very selective way.3-5 In the case of drug-conjugated antibodies, it may be of advantage to reduce the amount of conjugates in the body to reduce the catabolic breakdown, as metabolites often result in toxic effects. Other means of clearance, such as the use of anti-IgG or anti-idiotypes, result in complexes that are trapped in the liver, which may result in toxicity to this organ.

The primary aim of the present study was to evaluate the possibility of reducing the toxicity of an auristatin-conjugated monoclonal antibody, BR96, when used in combination with subsequent extracorporeal affinity adsorption treatment in a syngeneic immunocompetent rat tumor model. This syngeneic tumor model in immunocompetent rats is relevant to the clinical situation in humans because the Lewis Y antigen recognized by BR96 is also expressed in sensitive normal tissues, such as the gastrointestinal epithelium. In addition, Lewis Y-positive tumors in rats develop distant metastases, as is the case in humans. By using a subperitoneal tumor model, the tumors mimic the clinical situation by growing invasively in the surrounding tissue. Furthermore, the tumor size can be easily assessed by palpation. Radiolabeled MoAbs (eg, 90Y- and 177Lu-conjugated BR96) have previously been evaluated with subsequent extracorporeal affinity adsorption treatment in humans6, 7 and in animal models by us and others.8, 9 To the best of the our knowledge, this is the first study to evaluate the feasibility of combining an antibody-drug conjugate with subsequent extracorporeal affinity adsorption treatment.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Monoclonal Antibody and Drug-Immunoconjugate

The chimeric (mouse/human) monoclonal IgG1 antibody BR96 (Seattle Genetics Inc., Seattle, Wash), binding the tumor-associated Lewis Y glycoprotein, was used. Lewis Y is expressed on the majority of human epithelial tumors, including breast, gastrointestinal tract, nonsmall-cell lung, cervical, and ovarian, cancers, as well as some melanomas. As with the majority of tumor-associated MoAbs, BR96 also reacts with some normal tissues, primarily the epithelial cells of the GI tract.

To 500 mg BR96 at a concentration of 10 mg/mL in phosphate buffered saline, was added 50 mL of a solution of 500 mM sodium borate/500 mM NaCl, pH 8.0, and 110 μL 0.5 M diethylenetriamine pentaacetic acid (Aldrich, Steinheim, Germany). The mixture was heated to 37°C, and 85.1 μL 100 mM tris-carboxyethyl phosphine (Aldrich, Steinheim, Germany), 2.5 equivalents) was added. The mixture was maintained at 37°C for 2 hours. To the reduced antibody, 5.3 mL dimethylsulfoxide and 897 μL of a dimethylsulfoxide solution of maleimidocaproyl-valine-citrulline-p-aminobenzyl carbamoyl-monomethyl auristatin F (20.5 mM) were added. When a test using Ellman's reagent (5,5′-dithio-bis(2 nitrobenzoic acid) showed that antibody thiols had been consumed, the maleimido reagent was quenched by the addition of 817 μL of a neutralized aqueous solution of N-acetylcysteine. The conjugate was diluted 1:10 using 25 mM NaOAc buffer, pH 5.0 (loading buffer), and then it was applied to a sulfopropyl cation-exchange column, washed with loading buffer, and eluted with 3 times standard concentration of phosphate buffered saline. Late eluting fractions were analyzed separately and then pooled. The collected product was diluted 3 times with water and concentrated. A total of 385 mg of antibody-drug conjugate was obtained in a volume of 37 mL.

To the isolated conjugate, 4 mL of 500 mM sodium borate/500 mM NaCl solution, pH 8.0, was added, followed by 100 μL of a 100 mM dimethylsulfoxide solution of NHS-LC-biotin (Pierce; 3.8 equivalents). The reaction mixture was left at room temperature for 3 hours and then purified using cation-exchange chromatography, in the same manner as the original conjugate. The eluate collected was diluted 3 times in water, concentrated, and filtered through a 0.2 μm membrane, yielding 21.5 mL of biotinylated conjugate at a concentration of 14.9 mg/mL in phosphate buffered saline. Analysis indicated that the conjugate, designated b-BR96-MMAF (biotin-BR96-monomethyl auristatin F), contained 4.4 MMAF/MoAb and 3.2 biotin/MoAb.

Syngeneic Rat Tumor Model

Immunocompetent rats of the Brown Norway strain (Harlan, Horst, the Netherlands) were used. As demonstrated by immunohistochemistry, Brown Norway rats express the BR96-binding epitope in sensitive normal tissues, such as the gastrointestinal tract (esophagus, stomach, intestines, pancreas), hence mimicking the human situation.10 BN7005-H1D2 is a single-cell clone of a rat colon carcinoma originally induced by 1,2-dimethyl-hydrazine in a Brown Norway rat. BN7005-H1D2 cells were cultured in RPMI 1640 medium (Euroclone, Devon, UK) supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 10 mM HEPES buffer solution, and 29.3 μM gentamicin at 37°C in a humidified atmosphere containing 5% CO2. Cells were washed in saline, trypsinized and washed in medium plus 10% fetal calf serum. Animals were inoculated subperitoneally with 3 × 105 cells (in 50 μL medium). Experiments were initiated 12-14 days after inoculation (tumor size 10 × 15 mm). The animals were kept under standard conditions and fed with standard pellets and fresh water. Studies were conducted in compliance with Swedish legislation on animal rights and protection and were approved by the Animal Ethics Committee of Lund University.

Extracorporeal Affinity Adsorption Treatment

The extracorporeal system included a 403U/C12 pump (W-Marlow Alitea AB, Stockholm, Sweden) with a 15 cm silicone tube (1.6 of 6.35 mm inner/outer diameter). The column housing comprised a modified 2 mL polypropylene syringe (9 × 30 mm) with a 72 μm filter at the bottom. The syringe was packed with 1.5 mL avidin-agarose5 with about 0.5 mL NaCl above it as an extra air trap. PVC tubing (1 mm inner diameter) was used as medical lines. An air trap that comprised PVC tubing (9.5 mm inner diameter) was connected to trap any air bubbles before the blood was returned to the animals. The extracorporeal circuit had a volume of about 3.5 mL (corresponding to approximately 20% of the animal's blood volume). Before starting extracorporeal affinity adsorption treatment, the system was flushed with a heparin solution (20 IU/mL heparin in 9 mg/mL NaCl) as an anticoagulant. The system is illustrated in Figure 2.

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Figure 2. Illustration of the extracorporeal affinity adsorption treatment. A cannula is inserted into one of the lateral tail veins (1) for the return of blood, and this is connected to the extracorporeal system (regulated by the pump [2]). Another cannula is inserted into the ventral tail artery for blood access (3). When the cannulas have been correctly inserted, the extracorporeal circulation is started in bypass mode (4) without the column connected. Once the circuit is filled with blood and any air bubbles in the circuit have been collected in the air trap (5), the column (6) is connected to the circuit and affinity adsorption is started.

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Thirty minutes before insertion of the cannulas (Neoflon 0.7 × 19 mm, Becton Dickinson, Helsingborg, Sweden) a 2% glyceryl nitrate salve (The National Cooperation of Swedish Pharmacies) was applied to the entire tail of each rat to dilate the blood vessels. The animals were anesthetized with Isofluran using a U-400 anesthesia unit (AgnTho's, Stockholm, Sweden). The rats were first anesthetized in a 1.4 l induction chamber (3.3% Isofluran, 575 mL/min air flow) and then placed on a heating pad (30°C). Anesthesia was sustained through anesthesia masks connected to the same anesthesia unit as the induction chamber. A cannula was carefully inserted into 1 of the lateral tail veins (1-2 cm from the tip of the tail) for the return of blood. The cannula was secured to the tail with adhesive tape and connected to the extracorporeal system. To prevent coagulation and to confirm that the cannula was correctly inserted, heparin solution from the extracorporeal circuit was infused for a few seconds and then stopped (regulated by the pump). Another cannula was inserted into the ventral tail artery, approximately 5 cm from the tip of the tail, for blood access. This cannula is accurately inserted when there is a continuous flow of blood through the cannula. Before connecting the cannula to the extracorporeal circuit, a blood sample was collected. As soon as the artery cannula was connected to the system, extracorporeal circulation was started in bypass mode (column not connected). The heparin solution present in the system was infused to prevent clotting, and the whole circuit was filled with blood, bypassing the column. When the circuit was filled with blood and any air bubbles in the circuit had been collected in the air trap, the adsorption column was connected to the circuit and affinity adsorption started. Blood was pumped through the column at a rate of 0.4 mL/min. During extracorporeal affinity adsorption treatment, the rats were anesthetized at a lower level of anesthesia (2.0% Isofluran, 575 mL/min air flow) and kept on electric heating pads (30°C) to keep them warm. After about 2 hours of affinity adsorption (approximately 3 blood volumes; blood volume estimated to be 65 mL/kg body weight), the procedure was stopped, the tubing connected to the arterial cannula was placed in a bottle containing saline solution, and the blood in the circuit was returned to the rat. A blood sample was collected from the arterial cannula before withdrawal of the cannula. The tail was compressed to stop bleeding.

Experimental Design

Immunocompetent rats, with and without subperitoneal tumors, were injected intravenously with 20 mg/kg (4.8 mg MMAF/m2) of b-BR96-MMAF (Groups 1 & 2) or 15 mg/kg body weight (3.6 mg MMAF/m2) b-BR96-MMAF (Groups 3 & 4), as specified in Table 1. The 8 rats injected with 20 mg/kg were subjected to extracorporeal affinity adsorption treatment 24 hours postinjection. Fifteen mg/kg of b-BR96-MMAF was chosen based on recommendations from the manufacturer of the auristatin-conjugate (Seattle Genetics Inc., Seattle, Wash). For rats treated with extracorporeal affinity adsorption treatment, we have chosen a higher dose with the intention of having the possibility of demonstrating a reduced toxicity but also an increased efficacy. Twenty mg/kg was chosen based on data from Junutula et al,14 which showed that a 50% increase of dose above maximal tolerable dose (MTD) resulted in severe toxicity with 50% survival at Day 12 postadministration. A control group with tumors (Group 5) was injected with 15 mg/kg unconjugated BR96.

Table 1. Experimental Design
GroupTumorDrug ConjugateExtracorporeal Affinity Adsorption Treatment
  • a

    Corresponds to 4.8 mg MMAF/m2.

  • b

    Corresponds to 3.6 mg MMAF/m2.

1 (n=5)Subperitoneal20 mg/kga b-BR96-MMAF24 h postinjection
2 (n=3)None20 mg/kga b-BR96-MMAF24 h postinjection
3 (n=6)Subperitoneal15 mg/kgb b-BR96-MMAF
4 (n=3)None15 mg/kgb b-BR96-MMAF
5 (n=6)Subperitoneal15 mg/kg BR96

The body surface area of the rats was calculated as 9.1 × (body weight in grams)0.6611 to make it possible to compare our results with those of other studies, where the administered dose is given in terms of mg/m2 body surface.

Evaluation of Toxicity

To evaluate myelotoxicity, blood samples were collected from the tail artery twice a week during the first 28 days postinjection and then once weekly up to Day 100 postinjection. White blood cell counts (WBC), red blood cell counts (RBC), and platelet counts (PLT) were analyzed in a Medonic Cell Analyzer–Vet CA530 (Boule Medical, Stockholm, Sweden).

In addition, plasma was collected, and the levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, bilirubin, and creatinine were measured to determine liver and kidney toxicity.

The body weight, tumor size, and general status of the animals were recorded at the time of blood sampling.

The toxicity was graded according to the National Cancer Institute Common Terminology for Adverse Events (version 3.0) to compare the severity of the toxicity with the various regimens used (Table 2).

Table 2. Toxicitya
Adverse EventTreatmentNo. of Rats With
Grade 1 MildGrade 2 ModerateGrade 3 Severe
  • a

    Grading according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0.

WeightBR96000
15 mg/kg b-BR96-MMAF015
20 mg/kg b-BR96-MMAF + extracorporeal affinity adsorption treatment050
Alanine aminotransferaseBR96100
15 mg/kg b-BR96-MMAF051
20 mg/kg b-BR96-MMAF + extracorporeal affinity adsorption treatment230
Aspartate aminotransferaseBR96010
15 mg/kg b-BR96-MMAF006
20 mg/kg b-BR96-MMAF + extracorporeal affinity adsorption treatment005
BilirubinBR96000
15 mg/kg b-BR96-MMAF000
20 mg/kg b-BR96-MMAF + extracorporeal affinity adsorption treatment000
CreatinineBR96500
15 mg/kg b-BR96-MMAF600
20 mg/kg b-BR96-MMAF + extracorporeal affinity adsorption treatment600
Gamma-glutamyltransferaseBR96000
15 mg/kg b-BR96-MMAF024
20 mg/kg b-BR96-MMAF + extracorporeal affinity adsorption treatment131

Therapeutic Efficacy and Endpoints of the Study

The size of the primary tumor (ie, at the site of inoculation) was measured in 2 perpendicular directions using a caliper, twice a week up to Day 28 postinjection and then once weekly. The tumor volume was calculated using the formula: 0.4 × (L × W2), where L = the measurement of the longest axis and W = the measurement of the axis perpendicular to L. Complete remission is used to describe a tumor that regressed completely and was not palpable for at least a week.

Body weight, general status, toxicity based on plasma analyses, effects on the primary tumor, and outcome were monitored during a total of 100 days postinjection. The animals were sacrificed when the tumor burden became too large (>25 × 25 mm), a marked decrease in general status, or at the end of the study (100 days postinjection).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Preparation of Drug-Immunoconjugate

Antibody-drug conjugates were formed through controlled reduction of interchain disulfide bonds within the BR96 MoAb using tris-carboxyethyl phosphine as a reducing agent.12 The drug linker derivative was then added, and the antibody-drug conjugate was purified before biotinylation. The final antibody-drug conjugate had an average of 4.4 MMAF (21 μg MMAF/mg BR96) and 3.2 biotin molecules per MoAb. No aggregation or fragmentation was observed by size-exclusion high-performance liquid chromatography (HPLC), and no free drug was detected. The avidin-binding fraction of the drug-immunoconjugate exceeded 90% at the time of injection.

Extracorporeal Affinity Adsorption Treatment

The extracorporeal affinity adsorption procedure was conducted on 8 rats, 24 hours postinjection (Groups 1 and 2). Blood was pumped through the system at a rate of 0.4 mL/min for 2 hours (corresponding to 3 blood volumes). Blood samples taken just before and after the extracorporeal affinity adsorption treatment was analyzed to obtain levels of human IgG, using an enzyme-linked immunosorbent assay, to calculate the efficiency of removal. A mean of 87% (range, 85%-90%) of the drug-immunoconjugate in the blood was eliminated during extracorporeal affinity adsorption treatment.

Evaluation of Toxicity

Survival

All 3 rats without subperitoneal inoculated tumor cells injected with 20 mg/kg b-BR96-MMAF followed by extracorporeal affinity adsorption treatment at 24 hours postinjection (Group 2) survived for 100 days, whereas 2 of 3 rats without subperitoneal inoculated tumor cells injected with 15 mg/kg b-BR96-MMAF without subsequent extracorporeal affinity adsorption treatment (Group 4) died within 3 weeks (Fig. 3). No toxicity-related deaths were seen after Day 21. Thus, the abnormal results of plasma analyses seen before Day 21 were considered to be mainly attributable to toxic effects, and after Day 21, mainly because of disease progression. Tumor-bearing animals injected with 20 mg/kg b-BR96-MMAF and treated with extracorporeal affinity adsorption treatment 24 hours postinjection survived slightly longer than tumor-bearing animals injected with 15 mg/kg b-BR96-MMAF without extracorporeal affinity adsorption treatment (Groups 1 vs 3; mean 67 days compared with 62 days; not significant by Mantel-Haenszel test) (Fig. 3).

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Figure 3. Survival of animals (A) without tumor cell inoculations (Groups 2 and 4) and (B) with tumor cell inoculations (Groups 1, 3, and 5). Deaths before Day 21 were attributed to toxicity. Four of 6 animals treated with unconjugated BR96 (Group 5) died because of local tumor recurrence, and the other 2 because of distant metastases. All the late deaths (after Day 45) were attributed to metastatic disease.

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Body Weight Loss

All animals injected with b-BR96-MMAF (Groups 1-4) lost weight, reaching a nadir on Days 4-7. In rats injected with 15 mg/kg b-BR96-MMAF, not subjected to extracorporeal affinity adsorption treatment (Group 3), the mean peak weight loss was 22%. The rats injected with 20 mg/kg b-BR96-MMAF subjected to extracorporeal affinity adsorption treatment after 24 hours (Group 1) had a mean peak weight loss of 16%. This difference was statistically significant when analyzed with an unpaired t test (p < .05). After Day 7, the animals started to gain weight. The weight of tumor-bearing control animals injected with unconjugated BR96 (Group 5) was unaffected by the injections (Fig. 4). The body weight loss in rats not inoculated with tumor cells (Groups 2 and 4) was comparable to rats inoculated with tumor cells (data not shown).

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Figure 4. Body weights of animals with tumor cell inoculations (Groups 1, 3, and 5), normalized to their initial weight.

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Blood Parameters

Myelotoxicity was monitored by quantification of blood cell counts. Both tumor-bearing groups injected with b-BR96-MMAF (Groups 1 and 3) showed a decrease in platelets, reaching a nadir on Day 10, but the values had recovered in all animals 21 days postinjection (Fig. 5). The WBC increased temporarily after injection of b-BR96-MMAF. No major change was seen in RBC (data not shown). The control animals (Group 5) were sacrificed 42 days postinjection because of large tumor burden. Animals without inoculated tumor cells (Groups 2 and 4) showed comparable changes in blood cell counts (data not shown).

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Figure 5. White blood count (A) and platelet counts (B) in animals with tumor cell inoculations (Groups 1, 3, and 5).

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Hepatotoxicity

All rats injected with b-BR96-MMAF showed an increase in aspartate aminotransferase and alanine aminotransferase levels during the first 3 weeks postinjection (Fig. 6). The increase was similar to the group treated with 20 mg/kg b-BR96-MMAF and extracorporeal affinity adsorption treatment (Group 1) and the group treated with 15 mg/kg b-BR96-MMAF without extracorporeal affinity adsorption treatment (Group 3). The peak serum levels of gamma-glutamyl transferase and alkaline phosphatase in the group treated with 15 mg/kg b-BR96-MMAF without extracorporeal affinity adsorption treatment (Group 3) were almost double those in the group treated with 20 mg/kg b-BR96-MMAF and extracorporeal affinity adsorption treatment (Group 1). No bone metastases were detected at subsequent autopsies, indicating that alkaline phosphatase reflects liver toxicity. Serum bilirubin was not influenced by any of the treatments. Comparable increases were also observed during the first 3 weeks in rats not inoculated with tumor cells (Groups 2 and 4); after this period, the levels were normal for all animals in these 2 groups.

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Figure 6. Serum levels of aspartate aminotransferase and alanine aminotransferase in animals with tumor cell inoculations (Groups 1, 3, and 5).

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Nephrotoxicity

Any of the treatments did not influence the serum creatinine levels.

Therapeutic Effect

At the time of treatment, the size of the primary tumor was approximately 10 × 15 mm. All 11 animals injected with b-BR96-MMAF (Groups 1 - 4) showed persistent, local complete response within 10 days postinjection. Four of 6 rats injected with unconjugated BR96 (Group 5) showed a transient local complete response, but 3 were sacrificed because of local recurrence before Day 45 postinjection, and the fourth died because of distant metastases on Day 77 postinjection.

Two animals treated with b-BR96-MMAF (15 mg/kg) without subsequent extracorporeal affinity adsorption treatment (Group 3) died because of toxicity within 10 days postinjection (Fig. 3).

All late deaths (ie, after Day 50) were attributed to metastatic disease (Fig. 3). Five of 9 rats (4 in Group 1 and 1 in Group 3) treated with b-BR96-MMAF developed metastases. Metastases were often observed at multiple locations in the liver, kidneys, lungs, abdominal lymph nodes, and peritoneum. Only 2 of 6 rats in the control group (treated with unconjugated BR96) developed metastases, probably because of local tumor growth and the associated short survival time.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

The results presented indicate that the extracorporeal removal of b-BR96-MMAF conjugates from the circulation of rats given 20 mg/kg, reduced the body weight loss, and prolonged the survival of rats, compared with a group of rats given 15 mg/kg without subsequent extracorporeal affinity adsorption treatment. Although the administered dose was 5 mg/kg higher, the decrease in the number of platelets was lower in the group treated with 20 mg/kg in combination with extracorporeal affinity adsorption treatment, than in the group given 15 mg/kg without extracorporeal affinity adsorption treatment. The hepatotoxicity in terms of aspartate aminotransferase and alanine aminotransferase was similar.

Liver toxicity seems to be the dose-limiting toxicity of the b-BR96-MMAF conjugates. On the basis of experience gained during radioimmunotherapy studies with 90yttrium- and 177lutetium-labeled BR96 in the same syngeneic tumor model, the degree of myelotoxicity or loss of body weight in the present study should not be regarded as dose limiting or result in reduced survival.13 The catabolic breakdown of the auristatin conjugates in the liver resulted in severe hepatotoxicity of grade 3, according to the NCI toxicity criteria, in all rats treated with the conjugate. Bilirubin was not affected, but this toxicity is usually seen only when almost the whole liver parenchyma is involved.

A previous study of b-BR96-monomethylauristatin E (data not shown), in the same syngeneic rat tumor model, was terminated because of the finding by HPLC that a large proportion of the injected conjugate formed aggregates in the circulation, making the therapy less effective. We, therefore, changed to the newly developed MMAF drug, which is less hydrophobic and more selective in its action.

The toxicity of monomethylauristatin E conjugates in rats has also been reported by Junutula and coworkers.14 After a single dose corresponding to 1.5 mg/m2 monomethylauristatin E, these authors recorded a marked depletion of circulating neutrophils and other white blood cells on Day 5, followed by a compensatory rebound on Day 12. Also, a slight increase in serum aspartate aminotransferase (mean, 2.5 times) and a transient weight loss was observed. A 50% increase in dose (2.25 mg/m2) resulted in a more profound effect on aspartate aminotransferase levels, and only half of the rats survived until the end of the study (Day 12). They also stated that safety studies with a related cytotoxic compound, dolastatin-10, showed that mice, compared with rats, were relatively insensitive to this class of drugs. Therefore, rat models appear to be more suitable for safety assessments of drug conjugates based on this class of cytotoxic compounds. Our model is also relevant to the clinical situation, as the BR96-binding epitope is also expressed in sensitive normal tissues, such as the gastrointestinal epithelium, reflecting the toxicity seen in humans.

An initial transient increase in leukocyte counts in rats treated with MMAF-conjugates has also been demonstrated for conjugates with other monoclonal antibodies (Peter Senter, personal communication, 2006). The reason for this increase is not known, but it may be because of the redistribution of leukocytes from bone marrow because of exposure to a toxic antibody conjugate.

This is the first investigation of the therapeutic effects of BR96–auristatin conjugates in our immunocompetent syngeneic rat model. The primary aim of the study was to evaluate the toxicity-reducing effect of the extracorporeal affinity adsorption treatment procedure. To further assess the efficacy of the combined treatment, the model will be further optimized because all primary tumors showed complete regression after auristatin-BR96 at the MTD, and it is not possible to detect an improvement. Once extracorporeal affinity adsorption treatment has been used to remove the conjugate from the blood, no further uptake of the conjugate by the tumor can take place. The interval between injection and extracorporeal affinity adsorption treatment was based on previous investigations with various radiolabeled BR96 conjugates in the same animal model.8

Distant metastases are developed in this tumor model. Five of 9 evaluable rats administered b-BR96-MMAF developed metastases after complete remission of the transplanted tumor. The distant metastases occurred in the animals after Day 45 postinjection. If the distant metastases still express the Lewis Y epitope, repeated treatment after normalization of blood status (namely, approximately 3 weeks postinjection) with subsequent extracorporeal affinity adsorption treatment might increase the disease-free survival rate. Another therapeutic alternative would be a combined therapy including an agent with a different toxicity profile and/or target, such as radiolabeled MoAbs or tyrosine kinase inhibitors.

From the results presented in this study, it can be concluded that extracorporeal affinity adsorption treatment can be used to reduce side effects arising from the administration of monomethyl auristatin F-conjugated antibodies, making it possible to increase the amount of drug conjugates administered above the maximal tolerable dose.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

The articles in this supplement represent proceedings of the “12th Conference on Cancer Therapy with Antibodies and Immunoconjugates,” in Parsippany, New Jersey, October 16-18, 2008. Unrestricted grant support for the conference was provided by Actinium Pharmaceuticals, Inc., Bayer Schering Pharma, Center for Molecular Medicine and Immunology, ImClone Systems Corporation, MDS Nordion, National Cancer Institute, NIH, New Jersey Commission on Cancer Research, and PerkinElmer Life & Analytical Sciences. The supplement was supported by an unrestricted educational grant from ImClone Systems Corporation, a wholly-owned subsidiary of Eli Lilly and Company, and by page charges to the authors. Supported by grants from the Swedish Cancer Society; Mrs. Berta Kamprad's Foundation; Gunnar Nilsson's Foundation; Lund University Medical Faculty Foundation and The Lund University Hospital Funds.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES
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    Nilsson R, Lindgren L, Lilliehorn P. Extracorporeal immunoadsorption therapy on rats. In vivo depletion of specific antibodies. Clin Exp Immunol. 1990; 82: 440-444.
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    Kurkus J, Nilsson R, Linden O, Schonstrom N, Sandberg BE, Tennvall J. Biocompatibility of a novel avidin-agarose adsorbent for extracorporeal removal of redundant radiopharmaceutical from the blood. Artif Organs. 2007; 31: 208-214.
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