In vivo problems associated with RTAIT treatment

Immune response

The potential value of RTA-ITs in the treatment of human cancer may be limited by the development of host antibodies against the conjugate. Such antibodies could potentially alter Immunotoxin pharmacokinetics and pharmacodynamics as well as precipitate serum sickness or anaphylaxis. In fact, although in several instances no antibodies against either the antibody or the RTA portion of the conjugate could be observed (particularly in immunocompromised patients) (Hertler et al., 1988; 1989b; Amlot et al., 1993; LoRusso et al., 1995; Sausville et al., 1995) the development of anti-conjugate antibodies within a few weeks from the first IT infusion is a common finding in most subjects (Byers et al., 1989; Weiner et al., 1989; LeMaistre et al., 1991; Amlot et al., 1993; LoRusso et al., 1995; Sausville et al., 1995; Engert et al., 1997).

Hertler et al. (1987) measured serial anti-RTA and anti-murine immunoglobulin (anti-MIG) titers in 22 patients who received the anti-melanoma Immunotoxin XomaZymeR-Mel.

Significant titers of anti-RTA and/or anti-MIG were detected in 17 of 21 evaluable patients. Of the four patients not developing antibodies, two were likely immunosuppressed secondary to dexamethasone, and CCNU + dexamethasone respectively. Both patients who received ITs at a time when they had detectable anti-Immunotoxin antibodies experienced infusion reactions consistent with immune mediated allergic responses. In a further study using XomaZymeR-Mel (Mischak et al., 1990) human antibody responses to ITs components were evaluated in 21 melanoma patients. Twenty of the 21 melanoma patients produced antibodies against RTA, while 15 of 21 produced antibodies reactive with the murine monoclonal antibody component. Both IgM and IgG antibody responses were produced. Immunoglobulin responses were usually detected 1-2 weeks following initiation of therapy, with peak levels generally attained 2-4 weeks post-therapy. Titers of the anti-RTA antibodies were generally higher than those of the anti-murine monoclonal antibodies for the dose range tested. Anti-idiotype responses were demonstrated in eight of ten melanoma patients who had antimurine antibodies. Both the kinetics of appearance and the relative titers of the antiidiotype responses generally corresponded to the antimurine responses. Analogous results were obtained by measuring the humoral antibody response to XMMCO-791-RTA (anti-colorectal carcinoma RTA-IT) in colorectal cancer patients in a Phase I clinical trial (Durrant et al., 1989). All patients produced strong responses to the XMMCO-791 immunoglobulin and to RTA. The predominant response to the antibody was against the idiotypic determinant although anti-subclass and anti-mouse antibodies were also detected. A component of the anti-idiotypic immunoglobulin response in the colorectal cancer patients was directed against the combining site of XMMCO-791. These antibodies inhibited in vitro binding of XMMCO-791 to target 791 cells and so may be inhibitors of repeated ITs therapy.

Therefore, contrary to earlier hypothesis ITs do not abrogate the immune response to mouse immunoglobulin in vivo but instead are highly immunogenic. The development of anti-immunotoxin antibodies can reduce the therapeutic potential of ITs through several mechanisms and thus optimally effective, repeated courses of therapy may require some procedure for suppressing or abrogating the response against the Immunotoxin.

Strategies to prevent or reduce the titer of anti RTA-ITs antibodies were evaluated in animal models (Stoudemire et al., 1990; Byers et al., 1993) as well as in humans (Oratz et al., 1990). Stoudemire et al. (1990) conducted a study to determine if treatment with cyclophosphamide (CY) could suppress the formation of anti-murine and anti-RTA antibodies in rats treated with a murine mAb-RTA IT. Animals receiving IT alone developed significant titers of both anti-murine and anti-RTA antibodies. Compared with the response in the animals receiving single-course IT, the response to both of the components of the IT was significantly increased on days 28 and 35 in the animals receiving a second course of IT. The groups receiving a combination of either one or two courses of CY and IT demonstrated a significantly decreased antibody response to both the murine IgG and the RTA compared with the group receiving the IT alone (Stoudemire et al., 1990). Byers et al. (1993) developed instead anti-RTA mAb which recognize peptide epitopes and these were used to downregulate anti-RTA responses in RTA-IT treated mice. Of the five mAb produced two (608/7 and 596/134) inhibited anti-RTA responses by up to 73%. The down-regulation of anti-RTA responses appears to be effected by mAb interfering with Ag processing (Byers et al., 1993).

In a study in patients Oratz et al. (1990) treated 20 patients with the anti-melanoma immunoconjugate XMMME-001-RTA plus a single dose of intravenous cyclophosphamide. Although an overall response rate of 20% was observed-predominantly in pulmonary and soft tissue nodules there was no diminution in antibody responses against either the murine antibody or the ricin moiety.

Toxicity

A systematic study of in vivo lesions induced by RTA was first conducted by Jansen et al. (1982). They found that the LD50 of whole ricin after a single i.p inoculation corresponds to 0.32 g/mouse, that of pure native RTA to 466 g/mouse. Consequently RTA which is about 1500 times less toxic than ricin cannot be considered as a very toxic drug and if it becomes separated from the antibody of an IT in vivo its toxicity will be very limited. In an experiment of short-term cumulative toxicity over 5-10 days, doses corresponding to the LD50 were 4-5 times lower than a single injection. Histopathological alterations were also noticed by Jansen et al. (1982). After a lag period of about 10 h ricin at an i.v. dose corresponding to its LD50 causes lesions mainly in the reticuloendothelial system (RES) and the vascular system with disseminated intravascular coagulations and changes related to the shock syndrome. Such lesions were particularly apparent on the endothelium, the Kupffer cells of the liver and on the endothelium of myocardial capillaries. In comparison, RTA at a dose corresponding to its LD50 led to a quite different distribution of histopathological changes. Necrotic lesions were rapidly seen in the crypts of Lieberkuhn reaching maximum intensity 2-4 h after RTA injection. They tended to disappear 1 day later when lesions of the liver parenchyma became dominant. The liver lesions decreased after 2 days but at the same time tubular lesions of the kidney became apparent reaching a maximum on day 4. The necrotic lesions were paralleled by functional lesions. The different localization of ricin and RTA lesions could be explained by the immediate binding of ricin to every cell with which it is in contact (i.e. blood cells and endothelium) whereas RTA can penetrate deeper into the organs and attack mainly tissues with high mitotic index (e.g. crypts of Lieberkuhn), cells involved in detoxification (e.g. liver and tubular cells of the kidney), or cells bearing receptors for the sugars of RTA (e.g. liver cells, see above).

Results of the first set of clinical trials with IT have shown several problem areas that must be addressed in order for IT to become broadly effective therapeutics. Among these toxicity is a particularly relevant issue in human treatment. Toxicities have been of two types: general and specific. General toxicities that are toxin dependent have been seen with both RTA and PEA (Pseudomonas Exotoxin A) ITs. RTA ITs produce a VLS with low serum albumin, edema and weight gain and, rarely, pulmonary edema. The etiology of the VLS is unclear. According to Weiner et al. (1989) it may result from IT Fc interaction with monocytes. Others (Soler-Rodriguez et al., 1992; 1993) have found that endothelial cells show great sensitivity to RTA and may be directly damaged by RTA or RTA-ITs. Similar capillary leak phenomena have been observed with other recombinant drugs, including IL-2 and GM-CSF (Rosenberg et al., 1987; Brandt et al., 1988). If the syndrome represents a physiological opening of endothelial gap junctions, shorter treatment schedules and the use of steroids may perhaps reduce toxicity. Nevertheless, multiple courses of IT therapy have been tolerated in patients, indicating that toxicity is not cumulative.

Recently a series of investigations specifically devoted to studying in great detail the toxic effect of RTA-based ITs have been reported. Muraszko et al. (1993) examined the pharmacokinetics, stability and toxicity of ITs injected into the intrathecal space in rats and rhesus monkeys. mAb specific for the human (454a12 and J1) and rat (OX26) TfnR were coupled to rRTA. In monkeys the MTD of the anti-human IT was a dose that yielded a nominal CSF concentration of approximately 1.2 X 10~7 M. In rats the LD10

of the anti-human IT was a dose yielding a nominal CSF concentration of 8.8 X 10~7 M whereas the LD10 of the anti-rat IT was a dose yielding a nominal CSF concentration of 1.2 X 10~7 M. Thus the species-relevant IT resulted in toxicity at a concentration one-seventh that of the IT with the irrelevant mAb. Dose-limiting toxicity corresponded with the selective elimination of Purkinje cells in both rats and monkeys and was manifested clinically as ataxia and lack of coordination. There was evidence of only minimal inflammation within the CSF, and there was no signs of systemic toxicity (Muraszko et al., 1993).

A series of acute and multiple dose toxicology studies were performed by Kung et al. (1995) to support the clinical dose and to evaluate the systemic toxicity of an anti CD5 IT (mAb H65 linked to RTA). The LD50 was 60-62.5 mg/kg in the rat. H65-RTA was administered to the rat and the monkey as a bolus injection at doses of 0.1, 0.5 and 2 mg/kg and over 1-h infusion at 0.2 and 2 mg/kg, respectively. Following repeated doses of H65-RTA the following was found: peripheral edema, decreased body weight, in addition to a general inflammatory reaction evidenced by changes in hematology, clinical chemistry and urinalysis parameters. Histopathologically, chronic inflammation in the nonarticular soft tissue was found in the rat at doses of 0.1 mg/kg and higher and monkeys developed much more severe toxicity when compared to the rat at the same doses. Inflammation, hemorrhage and/or edema were evident in a variety of tissues. Myeloid hyperplasia was also evident. All toxicity was reversible. The toxicity observed in this study was not related to T lymphocytes and was probably due to a series of acute to subacute inflammatory reactions caused largely by the RTA moiety of H56-RTA. During safety evaluation studies in rats using the RTA-IT ZD0490 a number of reversible inflammatory changes were seen (Westwood et al., 1996). The synovial membranes of articular joints showed a marked degeneration and necrosis with an associated inflammation. Some nonspecific skeletal muscle toxicity occurred. However, tongues from the intravenously (tail) injected rats consistently showed inflammation specifically located in the ventral subepithelial area with myocyte degeneration and necrosis. Also, hepatic peliosis primarily located in the subcapsular areas was induced. Studies with rRTA alone demonstrated that rRTA is responsible for these findings. It is likely that cells of a macrophage type with the ability to specifically bind rRTA may at least in part determine the location and nature of the lesions observed. This is also supported by earlier observations (Weiner et al., 1989) that binding of mAb-RTA conjugates to monocytes/macrophages may result in the pathologic phenomena associated with VLS. Further observations that may help clarify pathogenetic phenomena related to VLS were gathered by Baluna et al. (1996) who examined the effect of dgRTA on HUVECs in the presence of fibronectin (Fn), an extracellular matrix protein which plays a role in the maintenance of vascular integrity. The addition of exogenous Fn greatly inhibited dgRTA-mediated morphological changes in HUVEC6 monolayers, dgRTA-mediated inhibition of [3H]-Thymidine incorporation and the binding of [125I]-dgRTA to HUVECs. Should the same phenomenon occur with RTA-based IT in vivo, this might shed light on the development of RTA-mediated VLS during IT therapy.

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