Quantitative comparison of BCG strains and preparations in immunotherapy of a rat sarcoma.

Ten preparations of BCG, six clinical vaccines, and four experimental preparations were compared for suppression of tumor growth by regional application. The preparations differed widely in their proportions of viable bacterial units and in bacterial unit:dry weight ratios. As assessed by their ability to suppress tumor development following direct admixtures with cell inocula of a rat sarcoma, one of the six clinical vaccines (Connaught) was significantly superior to Glaxo on any parameter (dry weight, No. of total units, or No. of viable units), immuno BCG Pasteur F was superior to Glaxo on two parameters (dry weight and No. of viable units), and Pasteur scarification was superior to Glaxo only on a viable unit basis. The Tice and Rijks Institute vaccines were not significantly different from Glaxo on any basis. Experimental vaccines from the Trudeau Mycobacterial Collection, stored as frozen liquid suspensions, showed a less marked variation in physical properties; here too, the Pasteur strain was superior to two other Trudeau preparations examined (Tice and Phipps). Viable organisms were not essential for tumor suppression, gamma-radiation-sterilized vaccine being equally effective. Tests with pulmonary tumor deposits, treated by iv BCG, and tests with pleural deposits, treated by intrapleural BCG, indicated that agents identified as superior in the subcutaneous screening system were also superior in the treatment of thoracic deposits.

Comparison of biodistribution of 791T/36 monoclonal antibody and its Fab/c fragment in BALB/c mice and nude mice bearing human tumor xenografts.

Fab/c fragments, purified from pepsin digest of mouse IgG2b monoclonal antibody (MoAb) 791T/36, consist of one Fab arm and the intact Fc portion. Pharmacokinetic and biodistribution studies in BALB/c mice of radioiodine-labeled 791T/36 MoAb and its Fab/c fragments showed that, due to the presence of the Fc portion, the Fab/c fragment has the same catabolic rate as whole antibody (T1/2 = 64 h). Due to its lower molecular weight (105,000), the Fab/c fragment extravasated more quickly and to a greater extent than whole MoAb in organs in which the vascular endothelium was fenestrated or continuous. In organs in which the vascular endothelium is sinusoidal, such as in liver and spleen, their diffusion capacities were identical. Therefore, Fab/c fragments reconcile advantages of the intact antibody molecule (slow catabolic rate) and Fab or F(ab)2 fragments (increased extravascular diffusion), features required to improve targeting to solid tumors. Data from biodistribution studies in nude mice bearing subcutaneous 791T tumor (antigen positive) and Colo205 tumor (antigen negative) contralaterally showed important differences in the behavior of whole MoAb and Fab/c fragment: (a) Whole MoAb was cleared more rapidly from the body and from the blood than Fab/c fragment; (b) The MoAb was taken up by the spleen (tissue to blood ratio greater than 1 from 12 h after injection over the 3 days of the experiment) and the liver (0.6), whereas Fab/c fragment tissue to blood ratios were only slightly increased (0.34 and 0.35) compared to control nude mice (0.25 and 0.28) for the spleen and liver, respectively, 3 days after injection. Since both MoAb and Fab/c fragment bear the Fc portion, these data suggest that the reputed "nonspecific uptake" of antibodies due to the Fc portion could be an Fc-mediated specific uptake, e.g., uptake of immune complexes; (c) The tumor to blood ratios were 1.7 and 1.2 for MoAb and Fab/c fragment, respectively, from 24 h throughout the experiment, whereas the percentage of injected dose (% of ID) present/g of 791T tumor was at any time greater for Fab/c fragment (8% maximum of ID) than for MoAb (5% of ID). These results were not expected in view of the low immunoreactivity in vitro of Fab/c fragments compared to whole antibody. It is suggested that the distribution and the catabolic rate of whole antibody and its Fab/c fragment at the tumor level are modulated by their respective valency and immunoreactivity for the target cell.

Inhibition of growth of human tumor xenografts in athymic mice treated with ricin toxin A chain-monoclonal antibody 791T/36 conjugates.

Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 specifically inhibits growth of human tumor xenografts which express the gp72 antigen recognized by the antibody component. Dose schedule tests showed that the major response was obtained during the first 5 days of treatment and further prolonged treatment did not improve therapy. Expression of gp72 antigen on tumor cells derived from xenografts in immunotoxin-treated mice was not markedly altered indicating that treatment did not lead to the expansion of tumor antigen deficient tumor cells. The experiments indicate that treatment for short duration with immunotoxin may be the most effective protocol.

Biodistribution of ricin toxin A chain-monoclonal antibody 791T/36 immunotoxin and influence of hepatic blocking agents.

Immunotoxin constructed by conjugating ricin A chain to monoclonal antibody 791T/36 has a markedly altered biodistribution when compared to unconjugated antibody. This is principally manifest as hepatic uptake of immunotoxin which appears to be controlled by the ricin A chain (RTA) moiety. This was established by comparing the blood survival and organ distribution of immunotoxin with that of ricin A chain and free antibody using preparations in which either the RTA or antibody, alone or as components of the immunotoxin, was radiolabeled. Gel filtration chromatography of sera from immunotoxin treated animals demonstrated a preferential blood clearance of immunotoxin with high RTA-antibody ratio. Hepatic uptake is dependent upon Kupffer cell recognition of mannose-containing oligosaccharide structures on the RTA moiety of immunotoxin. Mannose-containing blocking agents given with immunotoxin were shown to prolong circulation time of the immunotoxin in blood including those species with higher RTA-monoclonal antibody ratios and reduce liver uptake. Effective blocking agents include ovalbumin, ovomucoid, and mannosyl-lysine (Man3Ly2). These studies demonstrate that agents specifically inhibiting hepatic uptake of immunotoxin significantly alter biodistribution and may improve their therapeutic efficacy.

Syngeneic anti-idiotypic antibody prevents localization of a murine monoclonal antibody in human tumour xenografts.

BALB/c mice were immunized against syngeneic mouse monoclonal antibody (Mab) 791T/36 to produce anti-idiotypic antibody. To examine the effect of this antibody on tumour localization of the Mab, serum from these mice was transferred to nude mice with human tumour xenografts and distribution was studied with I-125 labelled Mab. Serum containing anti-idiotypic antibody prevented tumour localization of the Mab. This finding has implications for the clinical use of human or humanized Mab since, if these evoke anti-idiotypic antibody, this alone may be sufficient to prevent tumour targeting.

The characteristics of blood-borne radiolabels and the effect of anti-mouse IgG antibodies on localization of radiolabeled monoclonal antibody in cancer patients.

Quantitative and qualitative aspects of the blood survival of 131I and 111In-labeled monoclonal antibody 791T/36 have been examined in patients with colo-rectal carcinoma, ovarian carcinoma and osteogenic sarcoma who were receiving labeled antibody in diagnostic immunoscintigraphy trials. The blood clearance of intact antibody radiolabeled with either 131I or 111In was similar. A bi-phasic decline of both radiolabeled preparations was measured with initial half-lives 0.62 and 0.42 days for 131I and 111In labels and then with 1.85 and 1.40 day half-lives, respectively. The Fab fragment of the antibody was lost more rapidly (initial half-life 0.20 days and then 0.78 days). Blood-borne radioactivity was associated predominantly with plasma rather than cellular elements. Radioactivity was still attached to undegraded, uncomplexed, and immunologically active antibody as demonstrated by molecular filtration, immune precipitation, and antigen binding assays. However, anti-mouse-IgG antibody detected within 7 days of administration of radiolabeled antibody was present for at least 10 mo and has implications for the efficiency of repeated image studies.

Drug-monoclonal antibody conjugates for cancer therapy: potentials and limitations.

The development of monoclonal antibodies against antigens associated preferentially with human tumors has reawakened interest in the use of antibodies as site-specific targeting agents for cancer therapy. There are now many reports of the construction of conjugates of drugs and toxins with antibodies which have in vitro toxic properties directed by the antibody moiety. Only recently, however, have limitations to the in vivo therapeutic application of these conjugates become appreciated. These include limited levels of antibodies accumulating in tumor deposits in patients, sometimes marked differences in the biodistribution of antibody-drug conjugates from those seen with free antibody, limited penetration of antibody and drug-antibody conjugates into tumor tissue together with heterogeneity of antigen expression within tumor, and limitation to the cytotoxicity of conjugates caused by the low drug to antibody molar ratios possible without denaturation. It is now becoming appreciated that effective development of this approach will require more than chemical conjugation of existing or even novel drugs or toxins to antibodies so that they are cytotoxic in vitro. Additional strategies will probably have to be employed, including the use of intermediate carriers to increase drug to antibody ratios in conjugates, the production of conjugates without extreme overall charge or conformation changes, techniques to increase antibody localization into tumors by, for example, regional perfusion, blood flow enhancement, or increase in antigenic expression, or the use of conjugates containing mixtures of antibodies or fragments directed against different tumor-associated antigens.

Toxicity associated with the formation and clearance of immune complexes between antitumour monoclonal antibodies and syngeneic anti-idiotypic antibodies in mice.

We have previously shown that Balb/c mice immunised against syngeneic monoclonal antibodies (mAbs), so that they have anti-idiotypic responses against those mAbs, will clear the mAbs from the circulation as a result of immune complex formation. We report here that with three separate mAbs, this clearance of complexes can result in toxicity to the animals. This was particularly severe with one mAb (NCRC-2), in some cases leading to death. It was not seen with Fab/c or Fab fragments of NCRC-2. This toxic response could be passively transferred with serum, indicating that it was due to formation of immune complexes or to their subsequent clearance. Treatment of mice with cobra venom factor, to reduce complement levels, reduced clearance of complexes but had no effect on toxicity. The anti-histamine pyrilamine did not reduce toxicity. This is one of only a few situations in which an anti-(mouse antibody) response has been reported to be potentially dangerous, and is particularly remarkable since it occurs as a result of such a limited anti-antibody response.

Biodistribution in tumour-bearing mice of polycationic, amphoteric and polyanionic branched polypeptides with a poly(L-lysine) backbone labelled with 125I and 111In: tumour accumulation less than that of labelled serum proteins.

The biodistribution has been studied in mice with subcutaneously transplanted solid tumours (mammary carcinoma and melanoma) of synthetic branched-chain polypeptides based on poly(L-lysine). The polypeptides were a poly(L-lysine) backbone with side-chains of three DL-alanine residues (AK, which is polycationic), AK with additional glutamic acid residues at the end of the side-chains (EAK, which is amphoteric) and EAK in which the terminal glutamic acid amino groups had been acetylated (AcEAK, which is polyanionic) or succinylated (SucEAK, which is highly polyanionic). Polypeptides were labelled with 125I by reaction with Bolton and Hunter reagent, or with 111In by chelation to diethylenetriaminepentaacetic acid previously conjugated to them. As controls, natural plasma proteins (immunoglobulin G, albumin and transferrin) were similarly labelled. Over a study period of up to 7 days, even with the polypeptides showing most prolonged blood survival (EAK and AcEAK) there was no particular uptake or retention in tumour tissue, over and above what was seen with control plasma proteins and/or in normal tissues. Overall these findings suggest that any enhanced permeability and retention in tumour tissue, reported by other workers with other synthetic macromolecules, operates poorly with the present polypeptides and/or tumours. Specific tumour targeting, for example with monoclonal antibodies, would seem a better option than non-specific accumulation of macromolecules.

Influence of syngeneic monoclonal anti-idiotypic antibodies to murine monoclonal antibodies against tumour-associated antigens on the biodistribution of their target antibodies and their fragments.

The effect of syngeneic anti-idiotypic (anti-id) antibodies on the biodistribution of three murine monoclonal antibodies (mAb) against human tumour-associated antigens, and also on that of their fragments, has been examined in mice using, as a model system, purified anti-id mAb against three different target mAb. With the IgG2b mAb NCRC-2, pretreatment of mice 24 h previously with its IgG1 anti-id mAb NCRC-60 caused clearance of subsequently administered NCRC-2. With the univalent Fab/c fragment of NCRC-2 there was little effect, even with anti-id to Fab/c pretreatment ratios of 20:1, although immune complexes were present in the circulation. With Fab of NCRC-2, anti-id mAb prolonged blood survival by reducing renal clearance, immune complexes surviving in the circulation. With the IgG1 mAb NCRC-23, immune complexes formed in vivo with the IgG2b anti-id mAb NCRC-59, but with only little hepatic clearance. With the Fab and F(ab')2 fragments of NCRC-23, blood survival was increased in mice pretreated with anti-id mAb, and with Fab this was clearly due to reduced renal clearance. The third mAb, the IgG3 NCRC-48, formed complexes with its IgG2a anti-id mAb NCRC-62, but these survived in the circulation with no accelerated clearance of the target mAb. These results are different from those previously seen with endogenous anti-id responses. They indicate the diversity of effects that anti-id mAb can have on the biodistribution of their target mAb, and emphasise the difficulty of using such anti-id mAb to modulate the pharmacokinetics of target mAb.

Capture of recombinant ricin A chain by a bispecific anti-RTA:anti-CEA monoclonal antibody pre-targeted to a human gastric carcinoma xenograft in nude mice.

A bispecific antibody against carcinoembryonic antigen (CEA) and ricin A chain has been shown to localise in a CEA-producing human tumour xenograft. When labelled recombinant ricin A chain was subsequently injected there was uptake into tumours, indicating that this bispecific antibody can capture or carry toxin into tumour deposits.

Influence of recombinant tumour necrosis factor alpha on blood flow and antibody localisation in human tumour xenografts in nude mice.

Mice with s.c. grafts of gastric carcinoma MKN45 or osteosarcoma 788T were injected i.v. with recombinant tumour necrosis factor alpha (rTNF alpha) and tumour blood flow rates were determined 4 h later as a fraction of the cardiac output g tissue-1. With MKN45, the tumour blood flow rate was significantly reduced from a mean of 1.86% cardiac output g-1 to 0.84% and 0.65% with 50 and 200 micrograms kg-1 rTNF alpha respectively. With 788T, the tumour blood flow rate was reduced at 50 micrograms kg-1 rTNF alpha from 1.13% cardiac output g-1 to 0.56%. There were essentially no changes in blood flow rates in other organs. The effect of rTNF alpha on localisation of monoclonal antibodies into these xenografts were examined. When a single dose of rTNF alpha (50 micrograms kg-1) was given at the same time as labelled NCRC-2 antibody there was a significant reduction in localisation into 788T osteosarcoma xenografts. In other tests, mice were injected daily for 3 days with 50 micrograms kg-1 rTNF alpha. They were injected i.v. with monoclonal antibody 4 h after the first injection and dissected on the 4th day. With 788T there was a small but not statistically significant reduction in the absolute amount of NCRC-2 antibody localising in tumour, although this reduction was greater when results were expressed as tumour-to-blood ratios. With MKN45 xenografts, treatment with rTNF alpha had little effect on tumour localisation of an anti-(carcinoembryonic antigen) monoclonal antibody (NCRC-24). These studies show that TNF can be administered so as to reduce tumour blood flow and with little effect on tumour localisation of antibody, suggesting that combination therapy with TNF and antibodies or their immunoconjugates is feasible. Other studies have suggested that TNF can increase antibody localisation into tumours, but this was not seen here, and in some cases administration could reduce tumour localisation. It appears that this method of enhancing antibody localisation may be critically dependent on scheduling, and therefore it may not be extensively applicable.

Bacillus Calmette-Guérin contact immunotherapy of local and metastatic deposits of rat tumors.

Adjuvant contact therapy with BCG can be used to control tumor deposits at local subcutaneous sites and at other sites, particularly pulmonary metastases and pleural tumor growths. This treatment method is generally quantitatively more effective than general immunostimulation, or active immunotherapy that employs vaccines of adjuvant and tumor cells. Furthermore, this treatment is effective in hosts that lack full immunocompetence, making it still feasible in immunosuppressed patients. The current evidence indicates that local activation of host macrophages is probably the essential effector mechanism of adjuvant contact therapy.

Liposomal encapsulation augments delayed hypersensitivity reactions to tuberculin proteins.

Encapsulation of purified tuberculoproteins in liposomes augmented their ability to elicit delayed hypersensitivity reactions in BCG-immune rats. The effect was most marked with a low-molecular-weight tuberculopeptide that was relatively poor at eliciting reactions when in free form. These findings indicate that, in addition to the antigenic nature of the material, the physical form of presentation of mycobacterial test antigens can influence their ability to elicit hypersensitivity reactions.

Cellular interactions modulating host resistance to tumours.

Active specific immunotherapy of carcinogen-induced rat tumours can be effected using vaccines containing tumour cells admixed with bacterial vaccines such as BCG and C. parvum. The nature of the tumour antigen preparation is important, the most effective immunogen being viable tumour cells whose growth is controlled by responses generated by the bacterial agent in the vaccine. Soluble tumour antigen preparations are usually ineffective due to the preferential induction of suppressor lymphocyte responses in normal hosts. In comparison, removal of suppressor precursors by cyclophosphamide treatment of animals leads to the development of tumour immunity following immunization with soluble antigen preparations. One component of the immune rejection response involves the generation of non-specific effector cells. This type of response can also be induced by administering chemical hypersensitizing agents so as to localize tumour deposits. This approach has proved highly effective in treating the guinea pig line 10 hepatoma by intralesional injection of alkylcatechols. These compounds are highly potent hypersensitizers, being the active constituents of the poison ivy/oak (urushiol oil), and localize in tumour cell membranes.

Circulating antigen: bad or good for immunoscintigraphy?

There is ample evidence to show that circulating antigen can restrict effective localization of radiolabelled murine monoclonal antibodies in human tumours growing as xenografts in Nude mice. This is the result of the formation of immune complexes in the circulation. Surprisingly this effect is not seen in patients with circulating antigen, although immune complexes are formed in the circulation, and immunoscintigraphy is not compromised. Moreover, at least in some situations, the presence and level of circulating antigen correlates positively with the sensitivity of tumour imaging, and circulating antigen can be used as a criterion of patient selection for immunoscintigraphy. The reason for the dichotomy between mouse and man is unclear, and seems to be the subject of little or no current research. The introduction of chimeric or fully human monoclonal antibodies in place of murine monoclonal antibodies means that clinical situations will now mimic more precisely the animal models. The species of antibody complexing with antigen will be homologous to the patients, and this could result in handling of those complexes in a manner different from the handling of complexes with foreign (i.e. murine) antibodies. Clearly this subject warrants further investigation.

Influence of a syngeneic anti-idiotypic antibody response to a mouse anti-tumour monoclonal antibody on biodistribution of its radiolabelled Fc-intact monovalent Fab/c fragment.

It has previously been shown that a syngeneic anti-idiotypic (anti-id) antibody response generated in Balb/c mice to the monoclonal antibody (Mab) NCRC-2 can cause its clearance from the circulation. However, there was a positive prolongation of the survival of the Fab fragment, which is monovalent but also lacks Fc. The present study was carried out to examine the effect against the Fab/c fragment of this Mab, since this is also monovalent but has intact Fc. There was accelerated clearance, rather than retention. This suggests that anti-id responses prolong survival of fragments because they lack Fc, rather than only because of their monovalency. Such monovalent, Fc deficient fragments, are the type of human antibody fragments most easily obtained by genetic engineering techniques for clinical use. The present findings have implication for the effects of patients' immune responses on the biodistribution of such fragments.

Evaluation of 153Sm-diethylenetriaminepentaacetic acid for radiolabelling of pharmaceutical dosage forms by neutron activation.

153Sm-DTPA provides a suitable alternative to 99mTc-DTPA and 111In-DTPA as a water soluble tracer for the evaluation of pharmaceutical preparations. The chelate was handled biologically in a similar way to 99mTc-DTPA and 111In-DTPA. The chelate can be incorporated into the formulation as a non-radioactive excipient and the intact dosage form can then be neutron activated to produce 153Sm.

Possible consequences of human antibody responses on the biodistribution of fragments of human, humanized or chimeric monoclonal antibodies: a note of caution.

The effect of anti-combining site antibody responses on the biodistribution of monoclonal antibodies intended for diagnostic or therapeutic purposes is considered. The new generation of chimeric, humanized or human antibodies may well still be immunogenic in man, due to reaction against their combining sites. The likely consequence of this immunogenicity is clearance of antibodies from the circulation. This is already appreciated, and can be modelled in animal studies. However the effect of such anti-combining site responses on the biodistribution of F(ab')2 or Fab fragments of chimeric or human antibodies, or the new generation of fragments such as Fv, is a yet unknown. Animal modelling studies suggest that anti-combining site responses may have totally unexpected consequences. They may not lead to clearance of fragments. Indeed survival of fragments can be greatly prolonged, but target recognition prevented.

Determination of the immunoreactivity of radiolabelled monoclonal antibody fragments by binding to immobilised anti-idiotypic antibodies.

The feasibility of using immobilised anti-combining site (anti-idiotypic) antibodies as targets for assessing the immunoreactivity of radiolabelled anti-tumour monoclonal antibodies has been assessed. With two anti-tumour monoclonal antibodies (anti-CEA and anti-gp72) it was possible to quantify binding of their 125I labelled Fab fragment preparations to their anti-idiotypic monoclonal antibodies immobilised on cyanogen bromide activated Sepharose. Binding was specific for immobilised anti-idiotypic antibodies reactive with the anti-tumour antibody fragments. Moreover binding was inhibited by unlabelled Fab or intact monoclonal antibody, but not by an irrelevant antibody or its Fab fragment. The use of anti-idiotypic antibodies for quantifying immunoreactions of radiolabelled antibodies has advantages over the use of initial target antigen, which may be available only in inconvenient forms, such as cultured tumour cells.