Preclinical evaluation of light-activatable,bispecific anti-human CD3 antibody conjugates as anti-ovarian cancer therapeutics

The administration of anti-CD3 antibodies, either unmodified or in bispecific formats, has been shown to kill tumors. However, their activity needs to be carefully controlled. We have approached this problem by inhibiting their anti-CD3 activity until it is required. Folated anti-human CD3 antibody bispecific conjugates were therefore synthesised in which the folate portion of the conjugates remained free to bind to folate receptor (FR) expressing cancer cells, whilst their anti-CD3 activity was reversibly inhibited. On irradiation with UV-A light, the T-cell binding activity of the anti-CD3 antibody can be restored only when and where it is required, i.e., adjacent to a tumor. Conjugate bound to FR expressed on normal tissues in other parts of the body remains inactive. This report describes the preclinical in vivo testing of these conjugates in transgenic mice whose T-cells express human CD3 molecules. When the ‘cloaked’ conjugates were reactivated in the region of the primary tumor, both primary tumor growth and liver metastasis were markedly reduced. That the deliberate targeting of T-cell activity locally to the primary tumor also resulted in reduced distant metastatic growth was a key finding. Light-activatable bispecific antibody conjugates similar to those described here offer a means to control T-cell targeting with a much higher degree of specificity to tumors because they minimize potentially dangerous and unwanted side effects in non-illuminated areas. The addition of light-specific targeting to the inherent tumor specific targeting of therapeutic antibody conjugates could result in the development of safer treatments for patients.     

Preclinical evaluation of light-activatable,bispecific anti-human CD3 antibody conjugates as anti-ovarian cancer therapeutics

The administration of anti-CD3 antibodies, either unmodified or in bispecific formats, has been shown to kill tumors. However, their activity needs to be carefully controlled. We have approached this problem by inhibiting their anti-CD3 activity until it is required. Folated anti-human CD3 antibody bispecific conjugates were therefore synthesised in which the folate portion of the conjugates remained free to bind to folate receptor (FR) expressing cancer cells, whilst their anti-CD3 activity was reversibly inhibited. On irradiation with UV-A light, the T-cell binding activity of the anti-CD3 antibody can be restored only when and where it is required, i.e., adjacent to a tumor. Conjugate bound to FR expressed on normal tissues in other parts of the body remains inactive. This report describes the preclinical in vivo testing of these conjugates in transgenic mice whose T-cells express human CD3 molecules. When the ‘cloaked’ conjugates were reactivated in the region of the primary tumor, both primary tumor growth and liver metastasis were markedly reduced. That the deliberate targeting of T-cell activity locally to the primary tumor also resulted in reduced distant metastatic growth was a key finding. Light-activatable bispecific antibody conjugates similar to those described here offer a means to control T-cell targeting with a much higher degree of specificity to tumors because they minimize potentially dangerous and unwanted side effects in non-illuminated areas. The addition of light-specific targeting to the inherent tumor specific targeting of therapeutic antibody conjugates could result in the development of safer treatments for patients.Key words: T-cells, bispecific antibody, caging, photo-activation, UV-light, folate receptor, tumor targeting     

Selective localization of radioiodinated alkylphosphocholine derivatives in tumors

We have designed and synthesized two radioiodinated analogs of hexadecylphosphocholine in order to evaluate their tumor imaging potential. 12-(m[¹²⁵I]iodophenyl)dodecyl phosphocholine (NM-324) and hexadecyl-2-[N,N-dimethyl-N-(m[¹²⁵I]iodobenzyl-ammonium]ethyl phosphate (NM-326) demonstrated the ability of such compounds to localize in and thereby visualize the Walker 256 tumor in rats. However, the tumor avidity of NM-324 was far superior to NM-326. In addition, NM-324 showed excellent tumor localization in athymic mice bearing subcutaneous human tumors.     

In vivo studies of unlabeled and radioiodinated rhodamine-123

Radioiodinated rhodamine-123 (Rh123), potential tumor imaging agent, was injected in mice bearing experimentally-induced tumors to investigate its tissue distribution. Some accumulation of radioactivity was found in tumors; most of it cleared rapidly from the blood after injection. Also, the radioiodinated Rh123 had metabolized to water-soluble species which was excreted in urine and feces.Unlabeled Rh123, on the other hand, accumulated only marginally in the tumors. However, it was found to accumulate significantly in the heart; as much as seventy times the level in blood at 4 h post-injection. Accumulation of unlabeled Rh123 increased steadily even at 24 h post-injection; whereas, it cleared rapidly from the blood via the kidney.This finding of selective accumulation of Rh123 in heart could be exploited in synthesizing ¹¹C- and ¹⁸F-labeled Rh123 for use in PET studies of the myocardium.     

90Y Labeling of monoclonal antibody MOv18 and preclinical validation for radioimmunotherapy of human ovarian carcinomas

The monoclonal antibody (mAb) MOv18 binds the membrane alpha isoform of the folate receptor (FR) which is overexpressed in human ovarian carcinoma cells. Exploiting the targeting capacity of this mAb, we developed and preclinically validated a protocol for the stable labeling of the mAb with ⁹⁰Y, an isotope which has shown promise in cancer radioimmunotherapy. MOv18 was derivatized with the stable macrocyclic ligand p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (Bz-DOTA). MOv18-Bz-DOTA conjugates were labeled with ⁹⁰Y or ¹¹¹In under metal-free and good laboratory practice conditions. At the optimal Bz-DOTA/mAb derivatization ratio of 4–5, conjugates maintained binding activity up to 6 months, were efficiently labeled with ⁹⁰Y or ¹¹¹In (mean labeling yield 85 and 64%, associated to a final mean specific activity of 74 and 37 MBq/mg) and displayed a mean immunoreactivity of 60 and 58%, respectively. The radiolabeled preparations were stable in human serum, with >97% radioactivity associated to mAb at 48 h after labeling. The ability of ⁹⁰Y- and ¹¹¹In-MOv18 to localize FR on tumors in vivo was analyzed in nude mice bearing tumors induced by isogenic cell lines differing only in the presence or absence of the relevant antigen [A431FR (FR-positive) and A431tMock (FR-negative)]. In vivo biodistribution in organs other than tumor was comparable in non-tumor-, A431tMock- and A431FR-bearing mice, whereas the median tumor uptake of the radiolabeled reagents, expressed as area under the curve (AUC) and maximum uptake (U_max), was significantly higher (sixfold to sevenfold) in A431FR than in A431tMock tumors (P=0.0465 and P=0.0332, respectively). Mean maximum uptake (% ID/g) for ⁹⁰Y-MOv18 was 53.7 and 7.4 in A431FR and A431tMock respectively; corresponding values for ¹¹¹In-Mov18 were 45.0 and 11.3. These data demonstrate the feasibility of ⁹⁰Y-labeling of MOv18 without compromising antibody binding ability and the immunoreagent-specific localization in vivo on FR-expressing tumors, suggesting the suitability of ⁹⁰Y-MOv18 for clinical studies.Angela Coliva and Alberto Zacchetti contributed equally to this work.     

Preclinical and clinical studies on cancer-associated cachexia

Background

Cancer cachexia is the wasting condition that is often seen in advanced stage cancer patients. This wasting is largely attributable to a systemic and progressive loss of skeletal muscle mass that greatly hinders performance of normal daily activities, resulting in reduced quality of life. Moreover, it negatively influences the prognosis of cancer patients. A general consensus in the field is that the loss of muscle mass is due both to an increase in protein degradation and a decrease in protein synthesis. Recent studies using preclinical models for studying cachexia have been useful in identifying the contribution of inflammatory cytokines (e.g. tumor necrosis factor-α and Interleukin-6), and myostatin receptors (e.g. the type IIB activin receptor) to cachexia development, and have led to several clinical trials. However, many questions remain about the molecular mechanisms thought to play a role in the development of cachexia.

Methods

We conducted a literature search using search engines, such as PubMed and Google Scholar to identify publications within the cancer cachexia field.

Results

We summarized our current knowledge of: 1) the driving mechanisms of cancer cachexia, 2) the preclinical models available for studying the condition, and 3) the findings of recent clinical trials.

Conclusion

Cancer cachexia is a complex and variable condition that currently has no standard effective therapeutic treatment. Further studies are desperately needed to better understand this condition and develop effective combination treatments for patients.

     

Improvement of pharmacokinetics of radioiodinated Tyr(3)-octreotide by conjugation with carbohydrates

Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high tumor/background ratios and thus signal intensity in diagnostic imaging by single photon emission tomography (SPECT) or positron emission tomography (PET). To overcome the unfavorable pharmacokinetics of radiohalogenated octreotide analogues, we evaluated three carbohydrated conjugates of Tyr(3)-octreotide (TOC). Glucose ([(125)I]Gluc-TOC), maltose ([(125)I]Malt-TOC), and maltotriose ([(125)I]Mtr-TOC) derivatives of [(125)I]TOC were synthesized via Maillard reaction and subsequent radioiodination. In cells transfected with sst1-sst5, I-Malt-TOC, and I-Mtr-TOC show sst-subtype binding profiles similar to I-TOC with high affinity for sst2. Comparative biodistribution studies 10, 30, and 60 min pi in nude mice bearing rat pancreatic tumor xenografts showed fast blood clearance for all glycosylated derivatives. Due to their markedly increased hydrophilicity, [(125)I]Gluc-TOC and [(125)I]Malt-TOC were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine (5.3 and 1.4 versus 10.6%ID/g for [(125)I]TOC in the liver, 1.7 and 1.0 versus 3.8%ID/g for [(125)I]TOC in the intestine 60 min pi). For all compounds, hydrophilicity and uptake in liver and intestines correlate. Uptake of the carbohydrate conjugates in the kidney was comparable. Compared to the parent compound, the accumulation of the carbohydrated compounds in sst-rich tissues (pancreas, adrenals) was increased by a factor of 1.5-3.5. While tumor uptake of [(125)I]TOC (6.7 +/- 2.6%ID/g), [(125)I]Malt-TOC (5.3 +/- 1.9%ID/g), and [(125)I]Mtr-TOC (4.9 +/- 2.2%ID/g) at 30 min postinjection was comparable, accumulation of [(125)I]Gluc-TOC was significantly increased (10.1 +/- 2.8%ID/g at 30 min pi). Somatostatin receptor specificity of tumor uptake was confirmed by pretreatment, competition, and displacement experiments in vivo using 0.8 mg TOC/kg and gamma-camera imaging. Glycosylation proved to be a powerful tool for the development of high affinity sst ligands with excellent excretion profiles and improved tumor accumulation.     

Modulation of pharmacokinetics of radioiodinated sugar-conjugated somatostatin analogues by variation of peptide net charge and carbohydration chemistry

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr(3)-octreotate (TOCA) with z = 0 were evaluated in a comparative study using [(125)I]Mtr-TOCA (z = +1), the maltotriose-Amadori analogue of [(125)I]TOCA, as a reference, [(125)I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [(125)I]Gluc-S- and [(125)I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst(1)-sst(5), all three new analogues show sst-subtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst(2). In contrast, internalization into sst(2)-expressing cells (in % of [(125)I]Tyr(3)-octreotide ([(125)I]TOC)) as well as the EC(50,R) of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [(123)I]Gal-S-TOCA and [(123)I]Gluc-S-TOCA (internalization, 190% +/- 12% and 265% +/- 20%, respectively, vs 168% +/- 6% of [(125)I]TOC for [(123)I]Mtr-TOCA; EC(50,R), 2.62 +/- 0.07 and 2.96 +/- 0.14, respectively, vs 1.81 +/- 0.07 for [(123)I]Mtr-TOCA). The tumor accumulation of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 +/- 2.2 and 26.2 +/- 5.6%ID/g) and comparable to that of [(125)I]Mtr-TOCA (25.1 +/- 4.4%ID/g). [(125)I]Glucuron-TOCA showed lower uptake in sst-expressing tissues than did [(125)I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [(125)I]Gal-S-TOCA and [(125)I]Gluc-S-TOCA was also slightly higher than that of [(125)I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 +/- 0.3 and 2.2 +/- 0.4, respectively, vs 4.0 +/- 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([(125)I]Glucuron-TOCA vs [(125)I]Gal-S- and [(125)I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [(123)I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.     

Effect of 3-methyl-branching on the myocardial retention of radioiodinated 19-iodo-18-nonadecenoic acid analogues

The effect of methyl-branching at the 3(β-)-position on myocardial uptake and retention of fatty acids where radioiodide has been stabilized as a terminal trans-(E)-vinyl iodide has been evaluated in fasted rats. The syntheses of two new dimethyl-branched fatty acids, 17-iodo-3,3-dimethylheptadecanoic acid (14) and (E)-19-iodo-3,3-dimethyl-18-nonadecenoic acid (19), are described. Tissue distribution studies in fasted rats with [¹²⁵I]-19 showed significant heart uptake (2 min, 4.56% dose/g), and prolonged retention (60 min 4.10% dose-g). These results suggest that [¹²³I]-19 is a good candidate for further studies of regional myocardial fatty acid uptake patterns by SPECT.     

Preclinical studies of porfiromycin as an adjunct to radiotherapy

The bioreductive alkylating agent porfiromycin (POR) is more toxic to EMT6 cells that are hypoxic at the time of treatment than to aerobic cells. The toxicity of POR to hypoxic EMT6 cells in vitro was similar to that of mitomycin C (MC): the aerobic toxicity of POR was considerably less than that of MC. Treatment of cells in vitro with POR before and during irradiation did not sensitize either hypoxic or aerobic cells to X rays; instead, only additive cytotoxicity was produced. In contrast, treatment of solid EMT6 tumors in vivo with POR plus radiation produced supra-additive cytotoxicity, as assessed by analyses of the complete dose-response curves for the killing of tumor cells by radiation alone or by POR alone. The supra-additivity of the combination regimens appeared to reflect the preferential killing by each agent of those tumor cells which were in an environment conferring resistance to the other agent. In contrast, combinations of POR and X rays produced only additive cytotoxicities to marrow CFU-GM. Supra-additive antineoplastic effects were obtained at doses of POR which produced little hematologic or other host toxicity. The complementary cytotoxicities of radiation and POR to cells in different microenvironments in solid tumors and the absence of a similar effect in normal tissue make optimized regimens combining radiotherapy and POR unusually promising for the treatment of solid tumors.     

Preclinical pharmacokinetics and tissue distribution of a natural cardioprotective agent astragaloside IV in rats and dogs

Astragaloside IV, a natural product purified from the Chinese medical herb Astragalus membranaceus (Fisch) Bge, is now being developed as a cardioprotective agent for treating cardiovascular diseases. The purpose of the present study was to examine in vivo pharmacokinetics and tissue distribution in both rats and dogs by using an established high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry quantitative detection method. Astragaloside IV showed moderate to fast elimination; the elimination half-life of astragaloside IV was 98.1, 67.2 and 71.8 min in male rats, and 34.0, 66.9 and 131.6 min in female rats at doses of 0.75, 1.5 and 3.0 mg/kg, respectively. There was no significant difference in systemic clearance at three dose levels, suggesting that astragaloside IV may have linear pharmacokinetic characteristics in rats within the dose ranges tested. The highest concentration of astragaloside IV was detected in the lung and liver. However, limited distribution to the brain, indicates that astragaloside IV may have difficulty penetrating the blood brain barrier. In addition, only about 50% of the parent astragaloside IV was recovered in both urine and feces. These results indicate that there was about 83% astragaloside IV binding to plasma protein and that the binding to the plasma is linear at the concentration range of 250-1000 ng/ml. As in rats, astragaloside IV may have linear pharmacokinetic characteristics in dogs within the dose ranges tested. Astragaloside IV was slowly cleared via hepatic clearance with a systemic clearance (CL) of about 0.004 l/kg/min. Based on the favorable pharmacokinetic properties in both rats and dogs, astragaloside IV warrants further investigation for the prevention of cardiovascular diseases.     

Interleukin-18 (IL-18) mRNA expression and localization of IL-18 mRNA-expressing cells in the mouse uterus

Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and alpha subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17beta (E(2)) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E(2)-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.     

Tumor localization and radioimaging with mixtures of radioiodinated monoclonal antibodies directed to different colon cancer associated antigens

After demonstrating enhanced tumor cell binding with a mixture of monoclonal antibodies (MAbs) in vitro, biodistribution and immunoscintigraphy studies with 3 radioiodinated anti-colon cancer MAbs and a non-specific control MAb (MOPC) were conducted in a human colon cancer (GW-39)-hamster model system. Each of the specific MAbs, but not MOPC, demonstrated extensive tumor binding and in scintigrams affected visualization of all large tumors (>0.85 g) over background. Using single MAbs, few small tumors (0.19–0.50 g) were defined above background (0–29%). However, with combinations of these specific MAbs small tumors were more frequently defined in scintigrams (43–67%). Radioimages using higher doses of MAbs and small, younger tumors more clearly demonstrated the superiority of a MAb mixture. These results confirmed that combinations of MAbs to different antigens can detect smaller tumors with better tumor localization when compared to component MAbs used singly. This study supports the concept that tumor targeting and detection may be enhanced with appropriate mixtures of MAbs.     

Animal experimental studies on the pharmacokinetics of streptokinase]

By the use of native and radioactively labelled streptokinase its distribution and elimination were determined in rabbits and compared to the course of the fibrinolytic action. Particularities of the pharmacokinetics of the activator of fibrinolysis were studied and related to the therapeutic effect.     

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia

ABSTRACT

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.