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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Immunomedics, Inc., Morris Plains, New Jersey and 2 Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey
Requests for reprints: Puja Sapra, Immunomedics, Inc., 300 American Road, Morris Plains, NJ 07950. Phone: 973-605-8200, ext. 240; Fax: 973-605-1340; E-mail: psapra{at}immunomedics.com.
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Abstract |
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8:1 (mol/mol). CD74 is a rapidly internalizing molecule associated with HLA-DR, which has high expression by several tumor types. Here, we describe safety evaluations of IMMU-110 in mice and monkeys as well as efficacy studies in a xenograft model of the human multiple myeloma cell line, MC/CAR. Experimental Design: In vitro binding of IMMU-110 was determined by a cell-based ELISA and cytotoxicity of IMMU-110 assayed with a tetrazolium assay. Pharmacokinetics and biodistribution of radiolabeled IMMU-110 were examined in tumor-free BALB/c mice, and the therapeutic effectiveness was evaluated in severe combined immunodeficient mice bearing MC/CAR cells. Acute toxicity of IMMU-110 was studied in CD74-positive cynomolgus monkeys (Macaca fascicularis).
Results: In vitro, IMMU-110 specifically binds to CD74 and is cytotoxic against MC/CAR cells. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile identical to that of unconjugated hLL1 monoclonal antibody, except for higher kidney uptake. Treatment with a single dose of IMMU-110 as low as 50 µg antibody/mouse (or 1.4 µg doxorubicin/mouse), 5 days postinjection of the multiple myeloma cells, resulted in cure of most mice. In mice, no host toxicity of IMMU-110 was observed at the highest protein dose tested (125 mg/kg). In cynomolgus monkeys, bone marrow toxicity was observed at 30 and 90 mg/kg doses.
Conclusions: The excellent safety and efficacy profile of IMMU-110 supports clinical testing of this immunoconjugate in the treatment of CD74-positive B-cell malignancies.
and ß chains and directs the transport of class II molecules to lysosomal and endosomal compartments, where the invariant chain is degraded (1). The invariant chain-free MHC class II chains subsequently can bind antigenic peptides and appear on the cell surface for presentation to CD4-positive T lymphocytes. Besides having a role in the trafficking of MHC class II, CD74 is involved in B-cell maturation through activation of transcription mediated by the nuclear factor-
B p65/RelA homodimer and the coactivator TAFII105 (2). CD74 was shown recently to bind to a cytokine, macrophage migration inhibitory factor, and to play a role in macrophage migration inhibitory factor–induced activation of signaling events, such as phosphorylation of extracellular signal-regulated kinase-1/2 and prostaglandin E2 production (3).
CD74 is a particularly appealing target for the development of isotope, drug, and toxin immunoconjugate therapy, because it is internalized extremely rapidly into the target cells following antibody binding; CD74-positive cells internalize and catabolize 8 x 106 molecules of anti-CD74 monoclonal antibody (mAb) per cell per day (4). Indeed, in prior studies, we have shown improved therapeutic effects in lymphoma models expressing CD74 and given anti-CD74 mAb, LL1, conjugated to diverse radionuclides, including low-energy emitters (5, 6). Prior studies with a conjugate of LL1 with doxorubicin also showed potent activity in a lymphoma model (7). In these initial studies, we showed excellent therapeutic efficacy of IMMU-110 in a xenograft model of human B-cell lymphoma, effecting cures of severe combined immunodeficient (SCID) mice given previously a lethal i.v. injection of Raji lymphoma cells, with a single 350 or 117 µg protein dose of the conjugate (7). To further develop this drug immunoconjugate into a candidate clinical product, we have generated a humanized form of anti-CD74 mAb, hLL1, by complementarity-determining region grafting, which exhibits comparable antigen binding and internalization properties to the parental murine antibody (8, 9).
Recently, we showed using immunohistochemical, flow cytometry, and PCR analyses that CD74 is expressed in a majority (19 of 22) of multiple myeloma clinical specimens and cell lines (10). The high prevalence of CD74 antigen expression in multiple myeloma clinical specimens, together with frequently developed resistance to anthracyclines, prompted us to perform a comprehensive preclinical evaluation of IMMU-110 conjugate in a new multiple myeloma xenograft model. Extensive safety and efficacy preclinical testing were conducted, which included in vitro binding, cytotoxicity, in vivo pharmacokinetics, tissue biodistribution, in vivo therapy in a SCID mouse model of a human multiple myeloma cell line, MC/CAR, and acute toxicity evaluation of IMMU-110 in nonhuman primates (cynomolgus monkeys) that express hLL1-reactive CD74. Because, in contrast to lymphomas, there is a paucity of antibody targets in multiple myeloma and yet no drug immunoconjugate has been reported to be active in this tumor type, we reasoned that this would be of particular clinical interest.
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Materials and Methods |
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Animals, cell line, and antibodies. Seven- to 8-week-old female BALB/c homozygous or SCID (C.B-17) mice were purchased from Taconic Farms, Inc. (Germantown, NY). All animal studies were approved by the Center for Molecular Medicine and Immunology's Institutional Animal Care and Use Committee and conducted in compliance with Association of Assessment and Accreditation of Laboratory Animal Care, U.S. Department of Agriculture, and Department of Health and Human Services regulations. The tolerability study in cynomolgus monkeys (Macaca fascicularis; 2-5 kg) was done at the Charles River Laboratories, Inc., Sierra Division (Sparks, NV). Treatment of the animals was in accordance with regulations outlined in the U.S. Department of Agriculture Animal Welfare Act (9 CFR, Parts 1-3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research publication, National Academy Press, 1996).
A CD38-positive human multiple myeloma cell line, MC/CAR (ATCC CRL 8083), was purchased from American Type Culture Collection (Manassas, VA) and cultured in suspension in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 20% (v/v) fetal bovine serum, penicillin G (100 units/mL), and streptomycin sulfate (50 µg/mL). Flow cytometry analysis showed that the cells were positive for CD138 (syndecan-1), a prognostic marker for multiple myeloma (data not shown), and 92% of the cells were positive for hLL1 staining. MC/CAR cells produce, but do not secrete, immunoglobulin (IgG1). For experimentation, only cells in the exponential phase of cell growth were used. Daudi and Raji Burkitt's B lymphoma cells were also obtained from American Type Culture Collection and grown as suspension cultures in RPMI 1640 with 10% (v/v) fetal bovine serum, penicillin G (100 units/mL), and streptomycin sulfate (50 µg/mL). hLL1 and a negative isotype-matched control, humanized IgG1 antibody, hRS7 (mAb against epithelial glycoprotein-1), were manufactured at Immunomedics as described previously (9, 12).
Preparation of doxorubicin immunoconjugates. Doxorubicin conjugates of mAbs (hLL1 or hRS7) were prepared in a similar manner as published already (7). Briefly, doxorubicin was reacted with an equimolar amount of the commercially available cross-linker SMCC-hydrazide (4-[N-maleimidomethyl] cyclohexane-1 carboxylhydrazide). The doxorubicin-SMCC intermediate was isolated by repeated extraction of starting materials from the reaction mixture with acetonitrile followed by diethyl ether washing and drying to obtain a red powder. The mAb was reacted with a 40-fold molar excess of DTT to reduce disulfide bonds of the IgG, predominately the interchain disulfide bonds. The reduced antibody was separated from low molecular weight materials by diafiltration, and the number of available free thiol groups on the antibody was determined by the Ellman reaction (13), resulting in an average of eight free thiol groups per molecule of IgG. The doxorubicin-SMCC was dissolved in DMSO and reacted with mAb at 4-fold molar excess to the number of available thiol groups. Residual doxorubicin derivatives bound by hydrophobic bonding with protein hydrophobic groups were removed by passage through a column of BioBeads (Bio-Rad, Hercules, CA). The efficacy of purification was shown by purifying hLL1 that was reacted with doxorubicin in the absence of the SMCC linker. The antibody eluted from the column with no detectable absorbance at 496 nm, indicating that BioBeads are effective at removing noncovalently bound doxorubicin.
The amount of doxorubicin linked to the mAb was determined by UV-visible absorbance of the conjugate at 280 and 496 nm, indicating the concentration of the antibody and doxorubicin, respectively. The doxorubicin conjugates of mAbs were shown to be homogeneous by size-exclusion high-performance liquid chromatography when compared with unsubstituted mAb. The amount of free doxorubicin (doxorubicin not covalently bound to antibody) present in the final formulation was determined to be <7% by solid-phase extraction followed by high-performance liquid chromatography (14).
In vitro cell binding. Cell binding of IMMU-110 to MC/CAR cells was determined by an indirect cell surface binding ELISA. Briefly, cells were placed in 96-well plates (2 x 105 per well) and subsequently incubated with serial dilutions of IMMU-110, naked hLL1, nonspecific negative control mAb-drug conjugate (hRS7-doxorubicin), or nonspecific mAb (hRS7) on ice for 1.5 hours. After incubation, the plates were washed thrice with PBS and further incubated with a secondary antibody, horseradish peroxidase goat anti-human IgG Fc specific (The Jackson Laboratory, West Grove, PA), for an additional 1 hour at 4°C. At the end of the incubation, plates were washed with PBS as before, and the bound antibody was detected with a luminescent horseradish peroxidase substrate (LumiGLO). The plates were read in an Envision plate reader (Perkin-Elmer Life Sciences), using a luminescence protocol.
In vitro cytotoxicity. The in vitro cytotoxicity of IMMU-110 was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt dye reduction assay. Briefly, MC/CAR, Daudi, or Raji cells were placed in 96-well plates (2 x 105 per well) and treated with IMMU-110, hRS7-doxorubicin, free doxorubicin, or naked hLL1 for 4 hours at 37°C. Cells were then washed twice with PBS before replacing with fresh medium and incubated for an additional 44 hours at 37°C. At the end of the incubation, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt dye was added, and following an additional 2 to 3 hours at 37°C, the plates were read in an Envision plate reader at 490 nm.
Pharmacokinetics and biodistribution. The pharmacokinetics and biodistribution of radiolabeled hLL1 or IMMU-110 were evaluated in naive (tumor-free) BALB/c mice. The benzyl-DTPA conjugate of hLL1 was prepared using 2-(4-isothiocyanatobenzyl)DTPA as described previously for similar humanized antibodies (15). Matrix-assisted laser desorption/ionization time-of-flight analysis of the benzyl-DTPA-hLL1 conjugate indicated an average of 0.98 benzyl-DTPA per unit of hLL1. The doxorubicin conjugate of benzyl-DTPA-hLL1 was made in an identical manner to the doxorubicin conjugate of hLL1 and was analyzed to contain 8 molecules of doxorubicin per unit of benzyl-DTPA-hLL1 by UV-visible absorption dual wavelength analyses at 280 and 496 nm. Benzyl-DTPA-hLL1-doxorubicin was radiolabeled with buffered 111In (2.5 mCi/mg) for 30 minutes at room temperature, analyzed by instant TLC on silica gel impregnated glass fiber sheets (Gelman Sciences, Ann Arbor, MI) developed in 10 mmol/L EDTA, and showed an incorporation of 95.9% 111In into the conjugate. It was purified on two successive G-50 spin columns to yield an injectable conjugate that was found to be 99.9% and 99.5% pure by instant TLC and size-exclusion high-performance liquid chromatography analyses, respectively. Benzyl-DTPA-hLL1 was radiolabeled with 88Y at 0.1 mCi/mg for 30 minutes at room temperature. After two successive spin column purifications, instant TLC and size-exclusion high-performance liquid chromatography indicated 99.0% and 98.9% level purity of 88Y-benzyl-DTPA-hLL1, respectively. Naive (tumor-free) BALB/c mice were injected i.v. with a mixture of 0.001 mCi 88Y-DTPA-hLL1 and 0.02 mCi 111In-DTPA-IMMU-110 supplemented with unlabeled DTPA conjugates of hLL1 or IMMU-110, so that each animal received a total dose of 10 µg each of hLL1 and IMMU-110. At selected times after dosing (1, 2, 4, 16, 48, 72, and 168 hours), groups of five mice were anesthetized and a blood sample was withdrawn by cardiac puncture. Major tissues were removed, weighed, and placed in containers. Blood samples and tissues were counted in a calibrated gamma counter, Minaxi 
Auto-Gamma 5000 series gamma counter (Packard Instrument Co., Downers Grove, IL) for 111In (channels 120-480) and 88Y (channels 600-2,000). A crossover curve was generated to correct for the back scatter of 88Y energy into the 111In counting window.
In vivo survival experiments. SCID female mice weighing 18 to 22 g (8-10 mice per group) were pretreated with a chemotherapeutic cocktail of fludarabine phosphate (0.4 mg/mouse) and cyclophosphamide (2 mg/mouse) to abrogate any innate immunity in the mice 3 days before an i.v. injection of 1 x 107 MC/CAR cells. IMMU-110 and other therapeutics were injected i.v. 5 days after tumor cell inoculation. Mice were monitored daily and euthanized when they developed hind leg paralysis or lost 20% of their pretreatment weight.
Tolerability of IMMU-110 in severe combined immunodeficient mice. Groups of SCID mice were dosed with IMMU-110 ranging from a protein dose of 350 µg/mouse (17.5 mg/kg) to 2.5 mg/mouse (125 mg/kg). This translates to a doxorubicin equivalent dose ranging from 10 to 70 µg/mouse. Mice were monitored for visible signs of toxicity and body weight loss. The maximum tolerated dose (MTD) was defined as that at which no deaths occurred, and body weight loss was 
20% of pretreatment animal weight. On termination of the study (60 days), animals were sacrificed and tissues from selected mice were harvested and subjected to histopathologic analysis.
Tolerability of IMMU-110 in cynomolgus monkeys. A single-dose pilot tolerability study was done to characterize the acute toxicity of IMMU-110 following i.v. infusion to cynomolgus monkeys. A total of 10 monkeys were assigned to five treatment groups (vehicle and IMMU-110 at doses of 3, 10, 30, and 90 mg/kg; n = 2 per group). This translates to a doxorubicin equivalent dose ranging from 0.09 to 2.6 mg/kg. All animals were dosed once as a 1-hour i.v. infusion. The animals were evaluated for changes in clinical observations, body weight, ophthalmic and electrocardiographic variables, and clinical pathology indices [i.e., serum chemistry, hematology, coagulation, and cardiovascular variables (troponins T and I)]. All animals were euthanized following 14 days of observations. At termination, a full necropsy was conducted on all animals, and tissues were examined microscopically.
Data analysis. In vitro binding data were analyzed by nonlinear regression to determine the equilibrium dissociation constant (Kd). For in vitro cytotoxicity studies, dose-response curves were generated from the mean of triplicate determinations, and IC50 values were obtained using the GraphPad Prism software (Advanced Graphics Software, Encinitas, CA). Pharmacokinetic data were analyzed using the standard algorithms of noncompartmental analysis program WinNonlin version 4.1 (Pharsight, Mountain View, CA). In the biodistribution study, comparisons were made between the tissues of mice injected with 88Y-DTPA-hLL1 and 111In-DTPA-IMMU-110 using Student's t test after determination of equality of variance using the f test and a Grubbs' Critical Z test for any outliers. Survival studies were analyzed using Kaplan-Meier plots (log-rank analysis) with GraphPad Prism software. Differences were considered significant at P < 0.05.
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Results |
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8 molecules of doxorubicin per molecule of antibody did not interfere with the specificity and avidity of hLL1 to CD74-positive MC/CAR cells.
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) lasting 5 hours and a later slower clearance (ß) phase. Data points beyond 5 hours were used to compute the terminal half-life (t1/2 ß) of hLL1 (136 hours) and IMMU-110 (121 hours). IMMU-110 had a mean residence time similar to that of hLL1 (222 hours for IMMU-110 versus 210 hours for hLL1). The rate of clearance of both IMMU-110 and hLL1 was 0.015 mL/h. Thus, the coupling of doxorubicin to hLL1 did not significantly alter the pharmacokinetic profile of hLL1 mAb. At 1-hour postinjection, there was significantly higher IMMU-110 versus hLL1 in the blood (P < 0.05; Table 2); however, at all the remaining time points, there were no significant differences. In biodistribution studies, at early time points, there were some instances of significant differences between the two reagents in a few of the tissues. However, there were no trends, except for the kidneys, which were consistently higher for IMMU-110 uptake (P < 0.05).
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To determine if cynomolgus monkeys were a relevant model for preclinical toxicology studies, cross-reactivity studies of hLL1 with tissues from healthy human, cynomolgus, and rhesus monkey tissues were done. The staining pattern of hLL1 for rhesus and cynomolgus monkey tissues was observed to be similar with that for human tissues (data not shown). hLL1 specifically stained CD74-positive cells in lymph nodes but did not cross-react with fibroblasts. hLL1 reacted with potentially CD74-expressing cells (lymphocytes and dendritic cells) in all tissues examined and hematopoietic precursors in bone marrow and mononuclear cells in the spleen. Some endothelial cell staining also was observed in the heart, liver, kidney, lymph node, and spleen. Acute safety studies in CD74-positive cynomolgus monkeys revealed no IMMU-110-related morbidity or mortality at any dose. The clinical observations, physical examinations, body weight, ophthalmic examination, and electrocardiograms were normal. Clinical pathology indices, including serum chemistry, hematology, coagulation variables, and urinalysis, also were normal, except as noted below. In addition, there were no IMMU-110-related effects on the specific markers of cardiac injury, troponin T or troponin I. However, at the dose of 30 mg/kg, bone marrow hypocellularity was observed histologically, but without depletion of circulating blood cells, whereas both bone marrow and peripheral blood cell reductions were observed for those animals given the 90 mg/kg dose. Both animals treated at this dose exhibited decreased indicators of circulating RBC mass (i.e., RBC and hemoglobin concentration). By day 14, RBC and hemoglobin concentration were 25% lower than day 1 (predose). WBCs diminished by 25% of their predose value, including a significant drop in neutrophils. Platelet counts dropped for both animals, although the degree was highly variable. The bone marrow toxicity was considered most likely due to the cytotoxic effects of the doxorubicin component of IMMU-110. Another histopathologic finding potentially related to IMMU-110 administration was the presence of pigment in lymph nodes, particularly in animals dosed at 10, 30, and 90 mg/kg. However, because there was no evidence of tissue damage, the presence of this pigmentation was not considered to be adverse. Due to the small number of monkeys used, no statistical analysis could be done.
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Discussion |
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8 drug molecules per molecule of antibody did not significantly alter the pharmacokinetic profile of the antibody component in the conjugate. However, the experiments reported here only represent the pharmacokinetics and biodistribution profile of the antibody component of IMMU-110, because the radioisotope is exclusively attached to the DTPA chelate that is coupled to the hLL1 mAb. Although there were some instances of transient difference in normal tissue uptake at the earliest time points (1-4 hour postinjection), there were no consistent trends, with the exception of kidneys. It is not clear at this time why we observed higher renal uptake of IMMU-110 versus naked hLL1 in these studies. It should be noted that IMMU-110 has a isoelectric point of 0.06 pH units higher than naked hLL1, indicating that it is a molecule with greater positive charge (data not shown). In addition to differences in charge, there are also differences in the overall hydrophobicity of the protein on addition of 8 doxorubicin groups on to the molecule. These changes in charge, structure, and hydrophobicity could contribute to altered renal clearance. Because hLL1 does not cross-react with murine CD74, it is likely that the differences observed in renal uptake of hLL1 versus IMMU-110 will not affect the toxicity profile of the immunoconjugate. Further, in both SCID mice and monkeys, no acute IMMU-110-related toxicity was observed in the kidneys on histopathologic examination, and no abnormal urinalysis variables were observed.
Excellent therapeutic results with IMMU-110 were obtained in a mouse xenograft model of multiple myeloma, which is likely due to the specific binding and extremely rapid rate of internalization of IMMU-110. The internalization rate of hLL1 is 100 times faster than that observed with other mAbs that are generally considered to be rapidly internalized, such as mAbs to CD19, CD22, and the transferrin receptor (4). The literature suggests that internalization is a requirement for improved therapeutic efficacy of other mAb-based therapeutics, such as immunoliposomal doxorubicin (16) or similar doxorubicin-mAb conjugates (17). We hypothesize that following internalization into the low pH compartments of the cell (lysosomes and endosomes) the hydrazone linkage between hLL1 and doxorubicin is hydrolyzed to release free doxorubicin. Free doxorubicin can survive the acid-labile environment and passively diffuse to the nucleus and other organelles of the cell to elicit antitumor activity. The CD74 antigen is most likely degraded in lysosomes. A nonspecific drug conjugate (hRS7-doxorubicin) dosed at up to 2 mg protein/mouse failed to show any therapeutic effect, thus confirming the specific targeting of IMMU-110.
Free doxorubicin given at equivalent doses as present in the immunoconjugate or given at the MTD were not effective in this model possibly due to its rapid redistribution leading to low plasma drug concentrations. Treatment with naked hLL1 mAb also resulted in significant improvements in the life span of mice implanted with MC/CAR cells. In vitro, we have shown previously that hLL1 inhibits cell proliferation and induces apoptosis in the presence of an appropriate cross-linking agent but does not show antibody-dependent, cell-mediated cytotoxicity or complement-mediated cytotoxicity (9). The hLL1 component of the conjugate may thus be contributing to the overall activity of IMMU-110 in these studies. It should be noted, however, that the doxorubicin conjugate is significantly more effective than the naked antibody alone or the mixture of naked hLL1 and free doxorubicin in equivalent concentrations as the drug conjugate, thus confirming the improved and specific efficacy of this targeted drug immunoconjugate.
The therapeutic index, or the ratio of toxic dose to the effective dose, of any therapeutic agent is an essential criterion in considering its clinical potential. In SCID mice, IMMU-110 failed to show any toxicity, including myelotoxicity and cardiotoxicity, up to the maximum single dose tested of 2.5 mg/mouse (protein dose = 125 mg/kg; doxorubicin equivalent dose = 3.6 mg/kg), although the MTD of free doxorubicin was reached at 3 mg/kg. Therefore, conjugation of doxorubicin to hLL1 mitigated the toxicity of free doxorubicin in SCID mice. Doses higher than 2.5 mg IMMU-110 could not be tested due to limitations in the volume of material that can be injected via the i.v. route. Further, in therapy experiments, the minimum dose tested of 50 µg/mouse (equivalent to 1.5 µg doxorubicin dose/mouse) cured 88% of the animals. These data indicate that the therapeutic index of IMMU-110 in the MC/CAR mouse xenograft model is >50-fold. Thus, the comprehensive dose escalation efficacy and toxicity evaluation studies described here confirm that, in addition to non-Hodgkin's lymphoma, IMMU-110 is well tolerated and is highly active in this CD74-positive multiple myeloma xenograft model. We appreciate that these experiments could underestimate the therapeutic index of IMMU-110 in SCID mice; therefore, future experiments will aim at determining the minimum effective and the MTDs.
Lack of cross-reactivity with the rodent CD74 makes safety studies in mice less relevant. Therefore, we did preliminary tolerability studies of IMMU-110 in nonhuman primates (cynomolgus monkeys), because hLL1 reacts with monkey CD74. Although these acute toxicity studies were done in a few animals, they suggest that IMMU-110 is well tolerated up to 30 mg/kg, at which level the first signs of bone marrow toxicity appeared. This translates to a human clinical infusional dose of 1110 mg/m2 mAb dose or a 32 mg/m2 doxorubicin dose. Although bone marrow toxicity was observed at 30 mg/kg, IMMU-110 had no acute cardiotoxicity and did not show any adverse effects to other major organs at doses up to 90 mg/kg. Future experiments will aim to determine the recovery rate of hematologic variables following IMMU-110 treatment, especially because significant bone marrow suppression was not observed at the doses tested.
The frequent and high-level expression of CD74 on multiple myeloma clinical specimens, together with very encouraging preclinical safety and efficacy data, suggest that this disease is an excellent target for therapy with this drug immunoconjugate. In recent clinical trials, substitution of Stealth liposomal doxorubicin for free doxorubicin in the vincristine-Adriamycin-dexamethasone regimen was shown to improve the safety profile and convenience of the induction regimen without compromising efficacy (18). In a similar fashion, IMMU-110 could replace free doxorubicin in the vincristine-Adriamycin-dexamethasone treatment schedule. In addition, LL1 has been conjugated to a liposome bearing an anticancer agent (dioleoylated derivative of the anticancer drug 3',5'-O-dioleoyl-FdUrd) and shown to have improved efficacy in vitro (19).
A major obstacle to the successful treatment of multiple myeloma is the emergence of drug resistance. Although 60% to 80% of patients treated with melphalan and prednisone or vincristine-Adriamycin-dexamethasone achieve an objective response to the induction therapy, almost all patients eventually relapse. The tumor cells in the majority of vincristine-Adriamycin-dexamethasone–refractory patients exhibit multidrug resistance due to the overexpression of P-glycoprotein (20–23). We hypothesize that P-glycoprotein-mediated efflux could be circumvented by delivering drugs (e.g., doxorubicin) via a rapidly internalizing antibody (hLL1) to CD74-positive tumor cells, thus bypassing the activity of multidrug resistance transporters. Indeed, a recent study reported that an immunoconjugate composed of doxorubicin conjugated to an antibody against insulin-like growth factor receptor allowed bypassing of the P-glycoprotein-mediated resistance ex vivo and in vivo (24). Hence, the role of IMMU-110 in reversing P-glycoprotein-mediated resistance will be the subject of future studies.
In conclusion, we have shown that IMMU-110 is an antibody-drug conjugate with a potent efficacy and excellent safety profile in an animal model of human multiple myeloma in addition to our previously shown efficacy in a non-Hodgkin's lymphoma model. IMMU-110 also showed an acceptable safety profile up to 30 mg/kg in pilot acute safety studies in cynomolgus monkeys. Therefore, this drug immunoconjugate represents a rational choice for clinical development as a potential new therapeutic for multiple myeloma, non-Hodgkin's lymphoma, and possibly other CD74-positive neoplasms.
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Acknowledgments |
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Footnotes |
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Received 1/31/05; revised 4/14/05; accepted 4/27/05.
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B-dependent transcription program. J Biol Chem 2001;276:27203–6.This article has been cited by other articles:
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  E. C. Attar, D. J. DeAngelo, J. G. Supko, F. D'Amato, D. Zahrieh, A. Sirulnik, M. Wadleigh, K. K. Ballen, S. McAfee, K. B. Miller, et al. Phase I and Pharmacokinetic Study of Bortezomib in Combination with Idarubicin and Cytarabine in Patients with Acute Myelogenous Leukemia Clin. Cancer Res., March 1, 2008; 14(5): 1446 - 1454. [Abstract] [Full Text] [PDF]
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R. Stein, M. J. Mattes, T. M. Cardillo, H. J. Hansen, C.-H. Chang, J. Burton, S. Govindan, and D. M. Goldenberg CD74: A New Candidate Target for the Immunotherapy of B-Cell Neoplasms Clin. Cancer Res., September 15, 2007; 13(18): 5556s - 5563s. [Abstract] [Full Text] [PDF]
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 R. M. Sharkey and D. M. Goldenberg Targeted Therapy of Cancer: New Prospects for Antibodies and Immunoconjugates CA Cancer J Clin, July 1, 2006; 56(4): 226 - 243. [Abstract] [Full Text] [PDF]
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 C.-H. Chang, P. Sapra, S. S. Vanama, H. J. Hansen, I. D. Horak, and D. M. Goldenberg Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin Blood, December 15, 2005; 106(13): 4308 - 4314. [Abstract] [Full Text] [PDF]
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), IMMU-110 (
), nonspecific control antibody hRS7 (
), or nonspecific control mAb-drug conjugate, hRS7-doxorubicin (
), for 1.5 hours on ice. After incubation, plates were washed thrice with PBS and further incubated with a secondary antibody, horseradish peroxidase goat anti-human IgG Fc specific, for an additional 1 hour at 4°C. Bound antibodies or drug conjugates were detected using a chemiluminescent horseradish peroxidase substrate. Points, average of three replicates from one representative experiment; bars, SD.
). Control animals were treated with saline (
), 350 µg naked hLL1 (x), or a mixture of 350 µg naked hLL1 and 10 µg free doxorubicin (
).