George J. Schreiber

Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue

SummaryMeasurements in cancer patients showed that the pH of tumors averages 0.8 unit lower than that of the surrounding normal tissues, confirming published work. Based on this, the anti-carcinoma monoclonal antibody (mAb) L6 was used to prepare immunoconjugates with daunomycin (DM), the drug being released at the acidic pH of the tumor. A direct linking of the aconitic derivative of DM (AcoDM) to mAb L6 led to conjugates that either had a low drug/antibody ratio (<5:1) or precipitated in vitro. In order to increase the drug load and avoid precipitation, several biopolymers were tested as spacers between the drug and the L6. To attach the polymer derivative to the mAb, the former was maleimidized and the mAb was thiolated. The AcoM/mAb ratio obtained was 20, and the mAb retained its highly specific binding to tumor cells. At pH 6 the AcoDM-L6 conjugate was toxic to cultured C-3347 carcinoma cells with an inhibitory concentration (IC50) of 5 µg/ml. The conjugate was less effective than the free DM with an IC50 of 0.2 µg/ml. The L6 alone was not toxic. At a tumor pH of 6.5, 15% of the AcoDM was released. The amount of released drug reached a maximum 24–48 h after exposure to the acidic medium.In vivo localization studies demonstrated a similar tumor uptake of the conjugate and mAb L6 with 18% of the injected dose/g tumor and a maximum uptake in tumor 48 h after injection. Our data indicate that it is possible to construct conjugates based on a pH-sensitive linker that can be targeted successfully to a tumor with release of a portion of the drug at the tumor site, but testing is needed to establish whether such release has anti-tumor activity in vivo and offers an advantage over treatment with unconjugated drug.

An affinity column method for determination of the immunoreactivity of 131I-chimeric L6 monoclonal antibody and comparison to in vivo tumor localization

An affinity column method was developed to determine the immunoreactivity of 131I-ChiL6 (chimeric L6 monoclonal antibody), a candidate for radioimmunotherapy. This method involved assessing the binding of the radiolabeled antibody to antigen containing membranes bound to a Reacti-gel agarose matrix. The immunoreactivity determined by the affinity column method correlated to other in vitro binding assays including the Lindmo infinite antigen excess method. In tumor-bearing mice which had been injected with 131I-ChiL6, which possessed high immunoreactivities (90-82%), a high tumor uptake (13.5-10.5% ID/g) was observed. A decrease in tumor uptake (5.2-4.8% ID/g) was observed with 131I-ChiL6 samples of low immunoreactivity (42% and 31%, respectively). While a moderate loss of immunoreactivity (4-18%) of the 131I-ChiL6 samples could be detected by the affinity column method, the loss of tumor uptake in vivo observed was not as significant. This method was found to be an efficient and sensitive method for detecting damage to the antibody during radiolabeling and applicable as a quality control method for clinical trials. This rapid method, compared to the other in vitro binding assays (including the Lindmo infinite antigen excess method) has distinct advantages as a quality control method since it requires less manipulation and can be semi-automated.