Regulatory T cells (T REGs ) control the key aspects of tolerance and play a role in the lack of antitumor immune responses. Cyclophosphamide (CY) is a chemotherapeutic agent with a dose-dependent, bimodal effect on the immune system. Although a previous study demonstrated that CY reduces the number of T REGs , the mechanism involved in this process has yet to be defined. In this report, it is established that low-dose CY not only decreases cell number but leads to decreased functionality of T REGs . CY treatment enhances apoptosis and decreases homeostatic proliferation of these cells. Expression of GITR and FoxP3, which are involved in the suppressive activity of T REGs , is down-regulated after CY administration, though the level of expression varies depending on the time studied. This is the first report demonstrating that CY, in addition to decreasing cell number, inhibits the suppressive capability of T REGs . The relevance of the loss of suppressor functionality and the changes in gene expression are further discussed.

While several murine monoclonal antibodies (MAbs) directed against carcinoma associated antigens have shown excellent tumor targeting properties in clinical trials, the use of radiolabeled MAbs for both diagnostic and therapeutic applications has been hindered by two factors: (a) the induction of host anti-immunoglobulin (Ig) responses and (b) slow plasma clearance of unbound radiolabeled MAb, resulting in bone marrow toxicity for therapeutic application, and long intervals between MAb administration and tumor detection for diagnostic applications. This report describes the development of the first recombinant Ig with properties designed to reduce or eliminate both of the above problems: a complementarity determining region (CDR)-grafted humanized (Hu) MAb with a CH2 domain deletion (delta CH2). The MAb chosen for engineering was CC49, which is directed against a pancarcinoma antigen designated TAG-72 that is expressed on the majority of colorectal, gastric, breast, ovarian, prostate, pancreatic and lung carcinomas. When characterized for antigen binding in solid phase competition radioimmunoassays, the HuCC49 delta CH2 MAb completely inhibited the binding of murine (mu) CC49 and HuCC49 for TAG-72. The relative affinity constants (Ka) of MAbs HuCC49 delta CH2, HuCC49 and muCC49 were 5.1 x 10(-9), 2.1 x 10(-9) and 2.3 x 10(-9), respectively. The plasma clearance of 131I-HuCC49 delta CH2 was significantly faster than that of intact 125I-HuCC49 after either i.v. or i.p. administration in athymic mice (p(2)0.05). Biodistribution studies in athymic mice bearing human colon carcinoma xenografts after i.v. or i.p. administration of 131I-HuCC49 delta CH2 and 125I-HuCC49 demonstrated the efficient tumor localization and substantially lower percent of the injected dose (%ID/g) of the HuCC49 delta CH2 in normal tissues. This is reflected in the significantly higher radiolocalization indices (%ID/g in tumor divided by %ID/g in normal tissue) observed with the HuCC49 delta CH2 for most normal tissues tested (p(2)0.05). The differential between the rate of plasma clearance of HuCC49 delta CH2 and HuCC49 was even more pronounced in SCID mice, which have been shown to be an appropriate model to study the metabolism of human IgG. These studies thus describe the development of a recombinant Ig molecule which, for the first time, combines 1) the properties of more rapid blood clearance than an intact humanized Ig molecule--without loss of antigen binding affinity--and 2) reduced potential for eliciting a human anti-murine antibody (HAMA) response in patients. These studies also demonstrate the potential utility of HuCC49 delta CH2 for i.p. as well as i.v. radioimmunodiagnosis and radioimmunotherapy in patients with TAG-72 positive tumors.

Monoclonal antibody (MAb) CC49 reacts with tumor-associated glycoprotein (TAG)-72, a human pancarcinoma antigen. In clinical trials, radiolabeled CC49 has shown excellent tumor localization; however, many of the patients receiving MAb CC49 develop a human antimouse antibody response. In an attempt to prevent this antiimmunoglobulin response, we have developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto the variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28' CL, respectively, while retaining those murine framework residues that may be required for the integrity of the antigen combining-site structure. The HuCC49 MAb was compared with native murine CC49 (nCC49) and chimeric CC49 (cCC49), using a variety of assays. SDS-PAGE analysis under nonreducing conditions showed that the HuCC49 MAb has virtually identical mobility to that of cCC49. Under reducing conditions, the HuCC49 yielded two bands of approximately 25-28 and approximately 50-55 kDa, characteristic of heavy and light immunoglobulin chains. In competition radioimmunoassays, HuCC49 completely inhibited the binding of 125I-labeled nCC49 to TAG-72, although 23- to 30-fold more HuCC49 was required to achieve a level of competition similar to those of cCC49 and nCC49. The relative affinity of HuCC49 was 2- to 3-fold less than those of the cCC49 and nCC49 MAbs, respectively. The plasma clearance in mice of HuCC49 was virtually identical to that of cCC49. Biodistribution studies demonstrated equivalent tumor-targeting of HuCC49 and cCC49 to human colon carcinoma xenografts. These studies thus suggest that HuCC49 and genetically modified molecules, such as sFv and domain-deleted immunoglobulins developed by using the HuCC49 variable region as a cassette, may be potentially useful in both diagnostic and therapeutic clinical trials in patients with TAG-72-positive tumors.