Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity.

Mechanisms of T cell-mediated cytotoxicity remain poorly defined at the molecular level. To investigate some of these mechanisms, we used as target cells, on the one hand, thymocytes from lpr and gld mouse mutants, and on the other hand, L1210 cells transfected or not with the apoptosis-inducing Fas molecule. These independent mutant or transfectant-based approaches both led to the conclusion that Fas was involved in the Ca(2+)-independent component of cytotoxicity mediated by at least two sources of T cells, namely nonantigen-specific in vitro activated hybridoma cells, and antigen-specific in vivo raised peritoneal exudate lymphocytes. Thus, in these cases, T cell-mediated cytotoxicity involved transduction via Fas of the target cell death signal.

T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.

Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

Regulation of Dopamine Uptake by Vasoactive Peptides in the Kidney.

Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na(+), K(+)-ATPase inhibition. Present results show that CNP did not affect either (3)H-dopamine uptake in renal tissue or Na(+), K(+)-ATPase activity; meanwhile, Ang-(1-7) was able to increase (3)H-dopamine uptake and decreased Na(+), K(+)-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na(+), K(+)-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on (3)H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on (3)H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on (3)H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na(+), K(+)-ATPase activity inhibition, contributing to its natriuretic and diuretic effects.

A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process.

PSA is an oncodevelopmental antigen usually expressed in human tumors with high metastatic potential. Here we set up a metastatic model in nude mice by using TE671 cells, which strongly express PSA-NCAM. We observed the formation of lung metastases when TE671 cells were injected intravenously, intramuscularly, and intraperitoneally, but not subcutaneously. Intraperitoneal injections also induced peritoneal carcinosis, ascites, and liver metastases. To evaluate the putative role of PSA in the metastatic process we used a specific cleavage of PSA on NCAM by endoneuraminidase-N on intraperitoneal primary tumors. Mice with primary intramuscular tumors were taken as control. Repeated injections of endoneuraminidase-N led to a decrease in PSA expression in primary intraperitoneal nodules and ascites but not in intramuscular primary tumors. Endoneuraminidase-N also increased the delay in ascitic formation and decreased the number of lung or liver metastases in the case of intraperitoneal tumors but not in the case of intramuscular tumors. When metastases occurred in endoneuraminidase-N injected animals, they strongly expressed PSA-NCAM. Therefore, we established a relationship between PSA expression on the surface of primary tumor cells and the metastatic process.

Immunohistology of carcinoembryonic antigen (CEA)-expressing tumors grafted in nude mice after radioimmunotherapy with 131I-labeled bivalent hapten and anti-CEA x antihapten bispecific antibody.

We have developed a pretargeting strategy, called the Affinity Enhancement System (AES), which uses bispecific antibodies (BsF(ab')2) to target radiolabeled bivalent haptens to tumor cells. We performed several radioimmunotherapy (RIT) experiments in nude mice grafted with LS174T colon carcinoma or TT medullary thyroid cancer. Mice were treated with 131I-labeled di-DTPA-indium-tyrosyl-lysine bivalent hapten (75-112 MBq) administered 15-48 h after anti-CEA x anti-DTPA-indium BsF(ab')2. Immunohistological studies were performed on tumors at their minimal relative volume (TT), on stabilized tumor nodules (LS174T), and on regrowing tumors (TT and LS174T). Untreated tumors were used as controls. On microscopic examination, regrowing tumors (2 months posttherapy) were similar to untreated tumors with cells showing their respective typical morphology (large cells with a high nucleocytoplasmic ratio for TT, small and very undifferentiated cells for LS174T). However, regrowing tumors showed larger necrotic areas and a higher mitotic index correlated with Ki-67 antigen staining. Immunostaining for CEA was as strong as for controls. By contrast, the immunohistology of TT tumors at their minimal relative volume (1 month posttherapy) or of LS174T residual nodules (8 months posttherapy) showed decreased mitotic indices correlated with poor Ki-67 antigen staining. Some clusters of LS174T presented with features of glandular lumen, which suggested a more differentiated and less aggressive status. In TT tumors, CEA expression remained unchanged (80-100% membrane and cytoplasmic staining), whereas only 70% of the LS174T tumors were stained, with 58% loss of the membrane expression. Repeated treatment early after the tumor has reached its minimal relative volume should thus be efficient and improve the overall efficacy of AES RIT.

Radioimmunotherapy of small cell lung carcinoma with the two-step method using a bispecific anti-carcinoembryonic antigen/anti-diethylenetriaminepentaacetic acid (DTPA) antibody and iodine-131 Di-DTPA hapten: results of a phase I/II trial.

As small cell lung carcinoma (SCLC) is frequently a widespread disease at diagnosis, highly radiosensitive and often only partially responsive to chemotherapy, radioimmunotherapy (RIT) would appear to be a promising technique for treatment. We report the preliminary results of a Phase I/II trial of RIT in SCLC using a two-step method and a myeloablative protocol with circulating stem cells transplantation. Fourteen patients with proved SCLC relapse after chemotherapy were treated with RIT. They were first injected i.v. with a bispecific (anti-carcinoembryonic antigen/anti-diethylenetriaminepentaacetic acid) monoclonal antibody (20-80 mg in 100 ml of saline solution) and then 4 days later with di-(In-diethylenetriaminepentaacetic acid)-tyrosyl-lysine hapten labeled with 1.48-6.66 GBq (40-180 mCi) of I-131 and diluted in 100 ml of saline solution. In patients receiving 150 mCi or more, circulating stem cells were harvested before treatment and reinfused 10-15 days later. Treatment response was evaluated by CT and biochemical data during the month before and 1, 3, 6, and 12 months after treatment. All patients received the scheduled dose without immediate adverse reactions to bispecific antibody or 1-131 hapten. Toxicity was mainly hematological, with two cases of grade 2 leukopenia and three cases of grade 3 or 4 thrombopenia. Body scanning 8 days after injection of the radiolabeled hapten generally showed good uptake at the tumor sites. Estimated tumor dose was 2.6-32.2 cGy/mCi. Among the 12 patients evaluated to date, we have observed 9 progressions, 2 partial responses (one almost complete for 3 months), and 1 stabilization of more than 24 months. Efficiency and toxicity were dose-related. The maximal tolerable dose without hematological rescue was 150 mCi. These preliminary results are encouraging, and dose escalation is currently continuing to reach 300 mCi. RIT should prove to be an interesting therapeutic method for SCLC, although repeated injections and hematological rescue will probably be required, as well as combination with other treatment modalities.

Successful surgical removal of occult metastases of medullary thyroid carcinoma recurrences with the help of immunoscintigraphy and radioimmunoguided surgery.

Patients with recurrent or metastatic medullary thyroid carcinoma (MTC) were referred for pretargeted immunoscintigraphy (Affinity Enhancement System; AES) and radioimmunoguided surgery (RIGS). Data collected from 13 patients establish that whole-body AES immunoscintigraphy revealed metastases < 360 mg and RIGS detected micrometastases (5-15 mg). All tissue samples removed by the surgeon were diagnosed by histology and immunohistochemistry of calcitonin to check the accuracy of IS and RIGS results. AES immunoscintigraphy is very sensitive. Of 34 metastases or recurrences detected, 22 had escaped physical examination or conventional imaging. The accuracy of RIGS was 86%, its sensitivity 75%, and its specificity was 90% (n = 208). IS and RIGS detected occult tumors that would have escaped surgery, clearly demonstrating clinical benefit. Serum calcitonin (normal, 10 pg/ml) and carcinoembryonic antigen (normal, 5 ng/ml) of two patients were restored to normal. In patients whose tumors were discovered, progression of their disease was slowed, as evidenced by the large decrease in serum calcitonin and carcinoembryonic antigen, an important prognostic factor. Surgery was canceled in one case where IS detected distant metastases out of surgical reach. Thus, AES immunoscintigraphy and RIGS might be of valuable help for the surgical management of medullary thyroid carcinoma.

Radioimmunotherapy in medullary thyroid cancer using bispecific antibody and iodine 131-labeled bivalent hapten: preliminary results of a phase I/II clinical trial.

The toxicity and therapeutic efficacy of escalating doses of anti-carcinoembryonic antigen x anti-N alpha-(diethylenetriamine-N,N,N',N''-tetraacetic acid)-In bispecific monoclonal antibody (F6-734) and iodine 131-labeled bivalent hapten were determined in a Phase I/II trial. A total of 26 patients with recurrences of medullary thyroid cancer documented by imaging and a rise in serum thyrocalcitonin were enrolled. Twenty to 50 mg of F6-734 and 40-100 mCi of 131I-hapten were injected 4 days apart. Quantitative scintigraphy was performed after the second injection for dosimetry estimations in eight cases. Clinical, biological, and morphological follow-up was carried out for 1 year after treatment. The mean percentage of injected activity per gram of tumor at the time of maximum uptake was 0.08% (range, 0.003-0.26%). The tumor biological half-life ranged from 3 to 95 days, and tumor doses ranged from 2.91 to 184 cGy/mCi. The estimated tumor-to-nontumor dose ratios were 43.8 x 53.4, 29.6 x 35.3, 10.9 x 13.6, and 8.4 x 10.0 for total body, red marrow, liver, and kidney, respectively. Grade III/IV hematological toxicity was observed in seven patients, most of them with bone metastases. Among the 17 evaluable patients, 4 pain reliefs, 5 minor tumor responses, and 4 biological responses with decrease of thyrocalcitonin were observed. Nine patients developed human anti-mouse antibody. Dose-limiting toxicity was hematological, and maximum tolerated activity was 48 mCi/m2 in this group of patients, most of whom had suspected bone marrow involvement. The therapeutic responses observed in patients mainly with a small tumor burden are encouraging for the performance of a Phase II trial with minimal residual disease.

Delivery of therapeutic doses of radioiodine using bispecific antibody-targeted bivalent haptens.

UNLABELLED: Two-step pretargeting strategies have been designed to deliver radioisotopes to tumors more selectively than directly labeled antibodies or fragments. In this article, we compare quantitatively the potential of these strategies for the radioimmunotherapy of solid tumors. METHODS: Direct targeting was performed using iodine-labeled IgG and F(ab')2. As two-step strategies, we used the sequential injection of anti-CEA x anti-DTPA-In bispecific F(ab')2 (BsF(ab')2) and monovalent and bivalent DTPA derivatives labeled with iodine. The biodistribution of iodine in nude mice grafted with the LS174T human colorectal carcinoma was monitored in time and used for calculating radiation doses. RESULTS: In agreement with earlier studies, the IgG was more effective for delivering a radiation dose to the tumor than the F(ab')2 (7.8 versus 0.76 Gy/MBq, respectively) and both were moderately selective with respect to normal tissues (tumor:blood of 2.9 and 1.7, respectively). At their MTD, they should deliver 86 and 34 Gy, respectively, to the tumor. Using a nM-affinity DTPA-In bivalent hapten, the two-step protocol was optimized by varying the dosage of the BsF(ab')2, the stoichiometry of the reagents and the pretargeting time. The saturation of the tumor was obtained by injecting 5 nmol (500 microg) of BsF(ab')2. The pretargeted BsF(ab')2 was saturated by the injection of 0.5 mol of bivalent hapten per mole of antibody. With a 48-hr pretargeting time, the selectivity of the irradiation of the tumor was optimized (tumor:blood of 7.8) but only at the price of a lower efficiency (0.35 versus 0.86 Gy/MBq, 48-hr and 20-hr pretargeting time, respectively). Attempts to increase selectivity by using a microM-affinity DTPA-Y bivalent hapten or by chasing excess circulating radiolabeled hapten with an excess of unlabeled hapten also reduced tumor exposure. The use of a monovalent hapten resulted in both lower efficiency and selectivity. However, the two-step pretargeting of high-affinity bivalent hapten (Affinity Enhancement System, AES) should deliver 30-60 Gy to the tumor with less than 9 Gy to the blood in tumor-bearing mice. CONCLUSION: Radioimmunotherapy with AES is predicted to be as efficient and with lower hematological toxicity than direct targeting.

Two-step immunoscintigraphy for non-small-cell lung cancer staging using a bispecific anti-CEA/anti-indium-DTPA antibody and an indium-111-labeled DTPA dimer.

UNLABELLED: Immunoscintigraphy (IS) using anti-CEA F(ab')2 monoclonal antibody (MAb) is useful for improving mediastinal staging of nonsmall cell lung cancer (NSCLC), but the technique was limited because of an insufficient contrast between tumor and normal tissues. The aim of this study was to determine if the method could be improved by a two-step method which uses a bispecific anti-CEA/anti-di-DTPA antibody (Bs-MAb) and 111In-labeled di-DTPA-tyrosyl-lysine bivalent hapten. METHODS: Twelve patients were intravenously given a 30 min Bs-MAb infusion (0.1 mg/kg). Four days later, they were injected intravenously with 0.1 microgram/kg hapten labeled with 185 MBq 111In (5 mCi). Images were recorded immediately and 6 and 24 hr after hapten injection. A pharmacokinetic analysis was performed. Surgery was performed 3 days after 111In-hapten injection, and samples of tumor and normal tissues were collected for immunohistochemical and biodistribution studies. IS results were classified as true-positive (TP), false-positive (FP), true-negative (TN) or false-negative (FN) according to the surgical data. RESULTS: Primary tumors were visualized in nine patients. The contrast was excellent, generally higher than that obtained with direct labeling of anti-CEA. In the mediastinum, IS results were (after surgery) five TN, two TP and one FP. One case remains undetermined. The FP result was due to a Bs-MAb uptake in intrapulmonary lymph nodes. IS was in agreement with preoperative staging in six of these nine patients and discordant in three. CONCLUSION: Our study confirmed that the two-step method with a bispecific antibody could greatly improve the performances of IS for lung cancer staging.

Pretargeted radioimmunotherapy of human colorectal xenografts with bispecific antibody and 131I-labeled bivalent hapten.

UNLABELLED: We have developed a pretargeting strategy, called the affinity enhancement system (AES), which uses bispecific antibodies to target radiolabeled bivalent haptens to tumor cells. The aim of this study was to evaluate the potential of the AES for the radioimmunotherapy (RIT) of LS174T colorectal xenografts in comparison with RIT with directly labeled F(ab')2 fragment. METHODS: A total of 6 groups of tumor-bearing mice were treated using anticarcinoembryonic antigen (CEA) x anti-diethylenetriamine pentaacetic acid (DTPA)-In bispecific antibody (BsF(ab')2) and 131I-labeled di-DTPA-In bivalent hapten. Three groups of mice were injected with various activities of 131I-labeled bivalent hapten (75, 96, and 112 MBq) 20 h after administration of BsF(ab)'2. Three other groups were injected with an almost constant activity of labeled hapten (102 MBq) at 3 time periods (15, 30, and 48 h) after BsF(ab')2 administration. For conventional RIT, mice were treated with 96 MBq 131-labeled anti-CEA F(ab')2. Control groups were left untreated. Toxicity and tumor growth were monitored at weekly intervals. RESULTS: Doses used for conventional RIT induced severe toxicity and resulted in death of several treated animals. Nevertheless, all surviving animals treated with 131I-labeled anti-CEA F(ab')2 relapsed shortly after treatment (tumor growth delay = 48+/-13 d). For animals treated with the AES reagents, toxicity varied with the pretargeting time interval and the administered activity. For 20-h pretargeting time, the maximum tolerated dose was 96 MBq. For all AES RIT except 1 (with 48-h pretargeting time interval and growth delay of 82+/-26 d), no tumor growth was observed over a period of 8 mo. Furthermore, based on clinical and histologic criteria, 33% of the treated mice were considered cured. CONCLUSION: High cure rates of LS174T colon carcinoma were achieved with the AES, and the flexibility of the pretargeting approach allowed the control of hematologic toxicity, which is the main limitation to dose escalation with conventional RIT.

Bispecific antibody and iodine-131-labeled bivalent hapten dosimetry in patients with medullary thyroid or small-cell lung cancer.

UNLABELLED: The purpose of this study was to estimate the dose delivered to tumor targets and normal tissues after two-step injection of an anti-CEA/anti-DTPA-In (F6-734) bispecific antibody and a 131I-labeled di-DTPA in-TL bivalent hapten in patients with medullary thyroid carcinoma (MTC) and small-cell lung cancer (SCLC). METHODS: Five patients with persistent disease or recurrences of MTC and five patients with primary SCLC or relapse were studied. In a first step, 0.1 to 0.3 mg/kg of F6-734 bispecific antibody was injected intravenously. Four days later, 6 nmole (5.8 to 9.8 mCi) of 131I-labeled di-DTPA in-TL bivalent hapten were injected. Quantitative imaging was performed during one week after the second injection. RESULTS: All 5 patients with MTC showed positive immunoscintigraphy (IS). In the smallest visualized and resected tumor (0.8 g), the fraction of injected activity per gram (% ID/g) was 0.1% at Day 3. IS was positive in 4 of the 5 patients with SCLC. The volume of the smallest visualized SCLC tumor was estimated at 11 +/- 2 ml, and tumor uptake was about 0.009% ID/g. Tumor dose estimates ranged from 4.2 to 174 cGy/mCi in patients with MTC and from 1.7 to 8 cGy/mCi in patients with SCLC. CONCLUSION: High absorbed dose values were calculated for small MTC recurrences. For SCLC recurrences the values were smaller but in the same range as those obtained by other investigators with the one-step technique in lymphoma.

Radioimmunodetection of medullary thyroid carcinoma using indium-111 bivalent hapten and anti-CEA x anti-DTPA-indium bispecific antibody.

UNLABELLED: Pretargeting labeled bivalent hapten with bispecific antibodies has proven feasible in the clinic, and our earlier results have suggested the technique may be very sensitive for detecting small recurrences and metastases. Medullary thyroid carcinoma (MTC) is an example where this technique may be the most useful since local recurrences and isolated metastases are removed surgically when detected, and thyrocalcitonin provides a specific and sensitive tumor marker. In our current study, we evaluated pretargeted immunoscintigraphy in a larger number of MTC patients. METHODS: Anti-carcinoembryonic antigen (CEA) x anti-diethylenetriaminepentaacetic acid (DTPA) indium bispecific antibody and 111In-labeled bivalent DTPA hapten were administered sequentially (4-5 days apart) to 44 patients with elevated circulating calcitonin after resection of primary MTC. Immunoscintigraphy was performed 2, 5 and 24 hr after hapten injection and, when necessary, at longer time intervals. When available, a handheld gamma probe was used during surgery. RESULTS: Fifteen patients had known tumor sites before immunoscintigraphy. Tumors were imaged in 12 (80%) of these patients, including 3 with liver metastases. Five unknown tumor sites were detected. For the 29 patients with occult disease, immunoscintigraphy detected high-activity uptake sites in 21 patients (72%), including 5 in the liver. Twelve were confirmed by surgery, 1 by guided morphologic imaging and 1 by venous catheterization. There were 2 false-positive patients. The other 5 patients have not yet been confirmed. All detected liver metastases were high-activity uptake areas. Radioimmunoguided surgery was used in 14 patients. It was considered helpful by the surgeon in 12 patients, including 4 patients where it determined the resection of small, not palpable nor visible, tumor-involved lymph nodes. Surgical resection resulted in a significant decrease (8 patients) or normalization (1 patient) of circulating calcitonin and CEA. CONCLUSION: This technique affords high sensitivity and specificity for detecting small tumor lesions including liver metastases. Its use for immunoscintigraphy and guided surgery should improve the therapeutic management of recurrent MTC.

Pretargeting with the affinity enhancement system for radioimmunotherapy.

The pretargeting technique referred to as the Affinity Enhancement System (AES) uses bispecific antibodies and radiolabeled bivalent haptens that bind cooperatively to target cells in vivo. Experimental and clinical data demonstrate that AES can deliver large radiation doses to tumor cells with high tumor to normal tissue contrast ratios and long activity residence time in tumors. Preliminary clinical results of radioimmunotherapy of medullary thyroid carcinomas and lung cancers look promising.

Pretargetted imaging of colorectal cancer recurrences using an 111In-labelled bivalent hapten and a bispecific antibody conjugate.

In 11 patients recurrence of colorectal cancer was suspected by a rise in serum carcinoembryonic antigen (CEA) (nine cases), by a subocclusive clinical situation (one case) or by endoscopy (on an anastomosis, one case). Two-step tumour targetting was performed by a first injection of 0.1 mg kg-1 of unlabelled bispecific antibody conjugate (an anti-CEA Fab' fragment chemically coupled to an anti-diethylene triamine pentaacetate (DTPA)-indium fragment) followed 4 to 5 days later by injection of the bivalent DTPA hapten labelled with 5 to 8 mCi 111In. Planar scintigraphy, single photon emission computed tomographic (SPECT) 360 degrees acquisitions and whole-body scans were obtained 4.5 and 24 h after injection of the radiolabelled hapten. Biodistribution was determined for eight patients at 48 h. The final diagnosis was confirmed histologically in nine patients (eight by second-look surgery, one by laparotomy). Overall, results were one true negative (1-year follow-up) and 10 true positive; however, for the three large liver metastases (3 to 6 cm), only the periphery of the metastasis had high uptake compared to normal liver. For pelvic recurrences, immunoscintigraphic (IS) contrast was better for small tumours. The highest tumour uptake was found for a 1 cm diameter pelvic recurrence (7.2% i.d. kg-1). Mean tumour-to-blood ratios were 6.4. Thus, this two-step tumour targetting technique, which uses a bispecific antibody conjugate and an 111In-labelled bivalent hapten injected sequentially without chasing the excess bispecific antibody, provided satisfactory results in this preliminary clinical trial for detection of recurrent colorectal cancers.

Targeting medullary thyroid carcinomas with bispecific antibodies and bivalent haptens. Results and clinical perspectives.

The present article reviews the clinical trials that have been performed in recurrent medullary thyroid carcinoma patients with the Affinity Enhancement System. This technique uses bispecific antibodies to target radiolabelled bivalent haptens to tumour cells. Its sensitivity in the detection of known tumour sites is high (90%) and this technique also achieves good sensitivity (61%) in the detection of occult disease as revealed by abnormal thyrocalcitonin blood levels. Due to its high targeting capacity, this technique is now considered for use as a therapeutic agent in medullary thyroid carcinoma patients.

CTLA-4 and CD28: similar proteins, neighbouring genes.

Subtractive cloning and screening yielded a cDNA clone corresponding to a molecule expressed in activated T cells, called CTLA-4. At the protein level, CTLA-4, a single-V-domain member of the immunoglobulin superfamily, was found very homologous to the lymphocyte activation molecule CD28. In particular, the hinge region included the hexamer MYPPPY, completely conserved for both molecules and in mice and humans. By immunizing mice with a human CTLA-4 peptide, an anti-CTLA-4 monoclonal antibody (MAb) was obtained, which enabled to establish the MW of the protein (26 and 40 kDa under reduced and non-reduced conditions respectively) and its preliminary tissue distribution. Also, CTLA-4 and CD28 were very similar at the message and at the gene structure level. The corresponding genes had previously been found to co-map on mouse chromosome IC and on human chromosome 2q33. We show that they can be found on the same yeast artificial chromosomes bearing human genomic DNA, and that they are 25 to 150 kb apart. These marked homologies and gene proximity strongly suggest that CTLA-4 and CD28 are the direct products of a duplication event, and raise the question of the function of CTLA-4.

CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.

To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location.

CD28, initially detected on human T lymphocytes with the help of antibodies, and CTLA-4, obtained by reverse genetics through its preferential expression in mouse activated T cells, are both single-V domain members of the Ig superfamily. Early work showed a relationship between these two molecules, which we wished to further document, in particular because of the growing realization of the functional importance of CD28 in some T cell activation pathways. Isolation and analysis of the mouse CTLA-4 gene and further analysis of the human CTLA-4 gene showed that both of these and the human CD28 gene share the same overall intron/exon organization. The nucleic acid sequence homology of the exons was found to extend across both molecules and species, whereas the 5' and 3' flanking regions exhibited homology across species but not between molecules. Message expression of human CTLA-4 was only detected in activated T cells and, thus, shares with that of mouse CTLA-4 and of mouse and human CD28 a lymphoid tissue distribution, although apparently broader for the latter. Two main human CTLA-4 transcripts of about 1.8 and 0.8 kb were detected, the smaller of which may derive, as reported for human CD28, from the use of an alternate degenerated polyadenylation signal sequence. The nucleic acid sequence data allowed a direct comparison of the four putative complete protein sequences of CD28 and CTLA-4 in the mouse and the human, showing striking homologies, especially in some stretches (such as a MYPPPY hexamer in the hinge region) conserved across molecules and across species. The mouse CD28 gene was localized to chromosome 1 band C by in situ hybridization with three different radioactive probes, indicating, together with previous data, that the CD28 and CTLA-4 genes map to the same chromosomal region in both the mouse and the human. Thus, CD28 and CTLA-4 were found to be strikingly similar in most respects, in terms of structure, sequence, expression, and gene location, furthermore in two species, strongly suggesting that their genes are the direct products of a duplication event and raising the possibility of functional homologies between the corresponding proteins.

Intracellular uptake and catabolism of anti-IgM antibodies and bi-specific antibody-targeted hapten by B-lymphoma cells.

The efficiency of radioimmunotherapy with iodine-labelled antibodies is often limited by intracellular internalisation and catabolism after initial binding to the cellular targets. We have developed a technique called affinity enhancement system (AES) which uses bi-specific antibodies to target radiolabelled bivalent haptens to cells. This targeting method has been applied successfully to tumour imaging in colorectal cancer patients and is now considered for therapy. We have investigated the potential of this technique to target iodine radioisotopes by comparing it to targeting with covalently iodine-labelled antibodies in a rapidly internalising antigenic system, the surface IgM of a B-lymphoma cell line. A 5-fold increase in the intracellular retention time of activity as compared to 125I-labelled F(ab')2 or IgG was observed. The radiolabelled hapten did not undergo any catabolism after internalisation. Resistance to cellular proteases and failure of recognition of the hapten by amino acid transporter systems may be potential explanations for these observations. This should make non-covalent targeting, particularly the AES, a method of choice to target modulating antigens for the therapy of malignant hemopathies.