Antibody-drug conjugates - a perfect synergy.

INTRODUCTION: There is a great unmet need for effective new treatments in cancer, which continues to be a major cause of death. Antibody-drug conjugates (ADCs) are emerging, after a long gestation, as a class of biopharmaceuticals with the potential to address this need by directing highly potent cytotoxic drugs to their point of action. There is increasing interest in ADCs by major pharmaceutical companies and a growing pipeline of candidates for clinical use. This review summarises progress with development of this new class of drugs. AREAS COVERED: The authors describe separately the antibody and drug elements of ADCs and then examine the technology and consequences of linkage. The work is presented in the light of recent developments in the design, using clinical examples where possible. EXPERT OPINION: Since their emergence as independent drugs, antibodies and chemotherapy are being brought together in effective synergy. The conjunction is timely: many of the technical challenges in preparing antibodies have been addressed; potent new drugs are available and linker technology is advancing apace. ADCs however are not just a sum of their individual parts. The current challenge is in understanding the holistic nature of this exciting class of drugs that promise a new avenue for cancer treatment. Target selection, the interaction of ADC with tumour and off-tumour targets and the internalisation of ADCs, are critical to the effective maturation of ADC technology. Ongoing recent developments in attachment sites and linker chemistry can provide fine-tuning of drug loading, elements of ADC PK and off-target ADC toxicity.

VB4-845, a conjugated recombinant antibody and immunotoxin for head and neck cancer and bladder cancer.

Viventia Biotech Inc, under license from the University of Zurich, is developing VB4-845, comprising a Pseudomonas exotoxin fused to an anti-epithelial cell adhesion molecule single-chain antibody fragment, for the potential treatment of head and neck cancer (intratumoral) and bladder cancer (intravesical). VB4-845 is currently undergoing phase II and III clinical trials in patients with head and neck squamous cell carcinomas, as well as phase II clinical trials for the treatment of superficial transitional cell carcinoma of the bladder.

Preclinical studies in rats and squirrel monkeys for safety evaluation of the bivalent anti-human T cell immunotoxin, A-dmDT390-bisFv(UCHT1).

The bivalent anti-human T cell immunotoxin A-dmDT390-bisFv(UCHT1) for treatment of patients with T cell malignancies is a single chain fusion protein composed of the catalytic domain and translocation domains of diphtheria toxin fused to two tandem sFv molecules reactive with human CD3 epsilon. This immunotoxin selectively kills CD3 epsilon positive T cells. To determine the maximum tolerated dose (MTD), pharmacokinetics and immunogenicity of A-dmDT390-bisFv(UCHT1), rat and squirrel monkey studies were performed. In both animal studies, animals received either 0, 2.5 (low), 25 (medium), or 56.25 microg/kg (high) of A-dmDT390-bisFv(UCHT1) intravenously twice daily for four consecutive days. Although transient elevation of liver transaminases in the high groups was observed, the A-dmDT390-bisFv(UCHT1) administration did not affect liver function, renal function, the hemogram, or produce serious organ histopathology. Adverse events included transient lethargy, inappetence and weight loss in high groups. A-dmDT390-bisFv(UCHT1) plasma half life was 26.95 min in rats and 18.33 min in squirrel monkeys. Immune responses to A-dmDT390-bisFv(UCHT1) were minimal in squirrel monkeys and mild in rats. In vitro cytokine release, T cell activation and CD3 epsilon receptor occupancy assays using human PBMC were further performed since rat and squirrel monkey T cells do not react with A-dmDT390-bisFv(UCHT1). A-dmDT390-bisFv(UCHT1) did not induce cytokine release or T cell activation. The A-dmDT390-bisFv(UCHT1) concentration for 50% CD3 epsilon receptor occupancy was 7.4 nM. The MTD of 200 microg/kg total provides a dose level sufficient for anti-tumor activity in vitro and in a rodent model. Therefore, we propose that this agent is a promising drug for patients with surface CD3+ T cell malignancies.

Biodistribution and radioimmunotherapy of SCCHN in xenotransplantated SCID mice with a 131I-labelled anti-EpCAM monoclonal antibody.

BACKGROUND: The mortality from squamous cell carcinoma of the head and neck (SCCHN) remains high and almost unchanged throughout the last decades. Therefore, new therapeutic strategies are urgently needed. One promising approach is the application of radio-labeled antibodies directed against tumor-associated antigens. EpCAM is a transmembrane protein, which is overexpressed on almost all SCCHN, making it a suitable anchor molecule for targeted radioimmunotherapy (RIT). The aim of this study was to establish an animal model to investigate the biodistribution and the therapeutic effect of a radio-labeled EpCAM-specific monoclonal antibody (mAb). MATERIALS AND METHODS: The mAb C215 was labeled with 131I and tested for its antitumor effect against established SCCHN xenografts in SCID mice. Initially, the biodistribution of the mAb in the tumor and different organs was determined with a gamma counter and was calculated as % injected dose/gram tissue. For therapeutic approaches 5, 15 or 25 MBq 131I-labeled mAb was injected as a single bolus into tumor-bearing mice. Control animals received either sodium chloride or the unlabeled mAb. The tumor growth and body weight of the animals were measured at various times after administration of the antibody. RESULTS: Initially, high activity was seen in all organs after systemic administration of 13I-C215. Over time general activity decreased whereas an accumulation of activity was seen in the tumor. Tumor growth was delayed in the groups receiving either 15 MBq or 25 MBq 131I-C215 relative to control groups and the 5 MBq group. However, animals in the high-dose groups suffered from treatment-related toxicity, which led to body weight loss of more than 20%. CONCLUSION: Our data demonstrate that the EpCAM-specific radio-labeled mAb C215 is a promising tool to target SCCHN leading to significant tumor control. Further studies are necessary to increase efficacy and reduce toxicity of this new therapeutic approach.

Nigrina b: una proteína inactivadora de ribosomas no tóxica del saúco. Utilidad farmacéutica en la construcción de inmunotoxinas y conjugados para la terapia del cáncer* / Nigrin b: a ribosome-inactivating protein from elder. Pharmaceutical utility in the construction of inmmunotoxins and conjugates for cancer therapy

El saúco posee una colección de proteínas inactivadoras de ribosomas, en particular las nigrinas, que parecen ser responsables de su toxicidad. La nigrina b (corteza) posee isoformas en frutos (nigrina f) y hojas (nigrina l) que se encuentran a mayor concentración en las fases iniciales del desarrollo. Nigrina b es 103-104 veces menos tóxica que la ricina, una proteína inactivadora de ribosomas relacionada estructuralmente con la nigrina b y extremadamente tóxica presente en Ricinus communis L., que se utiliza para la construcción de inmunotoxinas para la terapia del cáncer. Nigrina b se internaliza en las células superiores por una vía intracelular distinta a la de la ricina independiente de temperatura y de brefeldina A. La administración de dosis letales de nigrina b produce lesiones intestinales irreversibles específicas por destrucción de las criptas y desaparición del epitelio intestinal lo que ocasiona hemorragias intestinales letales. Los datos sobre estructura primaria de las nigrinas indican que la diferencia de toxicidad con la ricina se basa en cambios de aminoácidos clave en los dominios de fijación de galactosa en las cadenas B de ambas proteínas. Esta característica de la nigrina b es de enorme utilidad en la construcción de los denominados "proyectiles mágicos" o fármacos inteligentes capaces de interaccionar y destruir blancos específicos. Como ejemplos de dichos proyectiles mágicos se han construido conjugados transferrina- nigrina b/ebulina l que han mostrado ser efectivos contra células cancerosas que sobreexpresan el receptor de transferrina y una inmunotoxina antitumoral contra el CD105 de ratón que identifica a la células CD105+ y las destruye de manera selectiva tanto in vitro como in vivo. Ello convierte a la nigrina b en una herramienta útil en la inmunotoxiterapia del cáncer (AU)

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker.

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.

A new screening model for safety evaluation of superantigen-antibody recombinant fusion proteins (mAb Fab-SEA/E) using telemetric monitoring in conscious rabbits.

INTRODUCTION: Carcinoma recognising monoclonal antibodies (mAb) and mutated forms of the T-cell-activating bacterial staphylococcal enterotoxin A/E (SEA/E) have been combined in single hybrid constructs (mAb Fab-SEA/E). By introducing substitutions in an MHC class II binding site, these harmful toxins can be converted into tolerable immunotoxins. Rabbits and humans are sensitive to SE toxins, and cardiovascular effects in rabbits are similar to those seen in septic shock in man. A new screening model using telemetry in conscious rabbits was applied in the safety evaluation of different mAb Fab-SEA/E constructs administered intravenously. METHODS: Telemetry transmitters were implanted in the peritoneal cavity of animals with a pressure catheter in the aorta and electrodes for ECG recording subcutaneously following administration of mAb Fab-SEA/E constructs intravenously. RESULTS: The responses in body temperature, heart rate, and blood pressure varied depending on the treatment regimen and the mutations of the drug given. For example, 25 micro g/kg of C215 Fab-SEAmut9 were given as a first treatment cycle on days 1, 5, and 7 and as a second treatment cycle on days 13-15. The first dose induced high fever, whereas the second and third doses induced fever responses more rapidly and were of lower and shorter duration. The second treatment cycle, starting on day 13, did not induce any responses probably due to anti-SEA antibodies formed because of the treatment. Another construct, 5T4 Fab-SEA/E-11 at 50 micro g/kg, induced a similar response as C215 Fab-SEAmut9 on days 1, 5, and 7. In this case, the pharmacologic response was still present on days 13-15, though no clinical signs developed or no formation of anti-SEA antibodies occurred. When 50 micro g/kg of 5T4 Fab-SEA/E-11 was administered once daily for 4 days, body temperature after the first dose increased slowly during the first 24 h, whereas the second to fourth doses induced more rapid and higher responses. The fourth dose of another compound, K305 Fab-SEA/E-11 (50 micro g/kg), induced an even more pronounced response both in magnitude and in duration as well as in adverse clinical signs. DISCUSSION: By using continuous telemetric registration in the rabbit as a tool in superantigen-antibody (mAb Fab-SEA/E) drug selection, it has been possible to evaluate the dynamics of drug-induced immune effects (fever) and concomitant engagement of the cardiovascular system, conditions that are essential before clinical trials can be initiated.

Eradication of colorectal xenografts by combined radioimmunotherapy and combretastatin a-4 3-O-phosphate.

Solid tumors have a heterogeneous pathophysiology, which has a major impact on therapy. Using SW1222 colorectal xenografts grown in nude mice, we have shown that antibody-targeted radioimmunotherapy (RIT) effectively treated the well-perfused tumor rim, producing regressions for approximately 35 days, but was less effective at the more hypoxic center. By 72 h after RIT, the number of apoptotic cells rose from an overall value of 1% in untreated tumors to 35% at the tumor periphery and 10% at the center. The antivascular agent disodium combretastatin A-4 3-O-phosphate (CA4-P) rapidly reduced tumor blood flow to 62% of control values by 1 h, 23% by 3 h, and between 32-36% from 6 to 24 h after administration. This created central hemorrhagic necrosis, but a peripheral rim of cells continued to grow, and survival was unaffected. Changes in the pattern of perfusion across the tumor over time were zonal. Untreated mice showed perfusion throughout the tumor, with greatest activity at the rim. There was an overall reduction at 1 h, and total cessation of central perfusion from 3 h onward. A narrow peripheral rim of perfusion was always present, which increased in intensity and extent between 6 and 24 h, either through reperfusion or new vessel growth. Combining these two complementary therapies (7.4 MBq (131)I-labeled anti-carcinoembryonic antigen IgG i.v. plus a single 200 mg/kg dose of CA4-P i.p.) produced complete cures in five of six mice for >9 months. Allowing maximal tumor localization of antibody (48 h) before blood flow inhibition by CA4-P increased tumor retention by two to three times control levels by 96 h without altering normal tissue levels, as confirmed by gamma counting and phosphor image analysis. The success of this combined, synergistic therapy was probably the result of several factors: (a) the killing of tumor cells in the outer, radiosensitive region by targeted radiotherapy; (b) enhancement of RIT by entrapment of additional radioantibody after combretastatin-induced vessel collapse; and (c) destruction of the central, more hypoxic and radioresistant region by CA4-P. This work demonstrates the need to consider cancer treatment in a biologically heterogeneous setting, if results are to be effectively translated to the clinic.

The effects of local hyperthermia on the catabolism of a radioiodinated chimeric monoclonal antibody.

Local hyperthermia has been shown to increase the tumor uptake and tumor:normal tissue ratios of radiolabeled monoclonal antibodies (mAbs) in athymic mouse xenograft models. The current study was undertaken to determine whether this behavior was related in part to alterations in mAb catabolism by local hyperthermia. Human/mouse chimeric 81C6 mAb reactive with tenascin and a nonspecific control mAb were labeled with 125I using Iodo-Gen and given to separate groups of athymic mice bearing s.c. D-54 MG human glioma xenografts. Half of the animals were then subjected to 4-h tumor-localized hyperthermia at 41.8 degrees C, a protocol previously shown to enhance the specific tumor uptake of the mAb in this xenograft model. The tumor, serum, liver, kidney, and urine were collected from heated as well as control animals 4 and 24 h after injection of the mAb and analyzed by SDS-PAGE and trichloroacetic acid precipitation. At 24 h, a significantly higher percentage of 81C6 was present as intact mAb in the tumors harvested from heated animals compared with those from controls. Unexpectedly, intact mAb was found in the urine of mice immediately after hyperthermia, but not in unheated control animals. We conclude that local hyperthermia decreases the catabolism of the mAb in the tumor and increases the urinary excretion of the mAb through a transient increase in glomerular permeability.