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Current diagnostic imaging procedures are commonly used to view a circumscribed part of the body such as head or thorax. Conventional nuclear medicine imaging procedures such as bone scans may be directed at an organ system, but only recently have single diagnostic imaging procedures become available to search the entire body and all tissues simultaneously for tumor deposits. Antibodies to tumor associated antigens, and ligand-receptor based imaging agents, such as the recently approved somatostatin receptor binding peptide,111 In pentetreotide, offer the opportunity to image tumors directly. Other radiolabeled imaging agents, such as 201 thallium, 99mTc-MIBI and positron emitters such as 18F-fluorodeoxyglucose (FDG) localize in areas of increased cellularity, vascularity or increased metabolism.

The detection of metastatic disease confined to the bone marrow compartment has in the past been technically limited. We have identified excellent imaging of bone marrow metastases during the evaluation of a Tc-99m labeled monoclonal antibody (NR-LU-10 Fab) (NeoRx Corp., Seattle, WA). This occurred during a study to assess the monoclonal antibody's ability to detect sites of small cell cancer (primary and metastatic). The study by design compares areas seen by the monoclonal antibody scan with those found by standard staging methods in patients with small cell lung cancer. Standard staging included chest x-rays, bone scans, CT studies of the abdomen, and histologic examination of the bone marrow. Fifteen patients have been evaluated, four on two occasions, for a total of 19 monoclonal imaging studies. Metastasis to the marrow compartment was identified by the monoclonal imaging in all patients whose bone marrow biopsies were positive for small cell carcinoma, and it was primarily responsible for the eventual detection of extensive disease (marrow involvement) in one patient. Thus it appears that compartmental bone marrow imaging for metastatic disease is possible with immunoscintigraphy.

Conjugates of monoclonal antibodies and Pseudomonas exotoxin A (PE) were formed with disulfide or thioether bonds. Thioether conjugates which formed with succinimidyl 4-( N -maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) modified PE and reduced antibody formed with an 80% yield of equimolar conjugate within 30 min with an offering of one to one (toxin:antibody). The efficiency and kinetics of thioether formation were much higher with SMCC than with other maleimide reagents as well as more efficient than disulfide linkers. Thioether linkage resulted in immunotoxin consistently more potent and more selective in vitro than disulfide bonded conjugate. Thioether bonded conjugates also proved to have other favorable in vivo properties compared to disulfide conjugates: (1) a longer half-life in serum; (2) increased tumor localization; and (3) reduced toxicity. Toxicity of thioether linked holotoxin conjugates was directed at the liver hepatocyte but was easily monitored by serum liver enzymes. The conjugates are currently undergoing clinical evaluation for treatment of ovarian cancer with intraperitoneal administration. Research is ongoing to further decrease residual toxicity without reducing the potency of the conjugate.

Trichothecenes are a class of mycotoxins that are potent inhibitors of protein synthesis (Alder et al., 1984). In fact, they are among the most active cytotoxic agents known when tested against cultured tumor cells. The manifestations of their toxicities against a living host are quite varied, ranging from necrotizing dermatitis to death.

We report the results of a trial of recombinant leukocyte A interferon in previously treated patients with non-Hodgkin's lymphoma who were no longer responsive to chemotherapy. Patients received recombinant leukocyte A interferon (50 X 10(6) U per square meter of body-surface area) by intramuscular injection three times weekly for three months or longer. Forty-five patients were enrolled in the study, and 37 were evaluated for a response. Thirteen of 24 (54 per cent) evaluable patients with low-histologic-grade non-Hodgkin's lymphoma had objective responses (nine partial responses and four histologically confirmed complete responses). Two of six (33 per cent) with intermediate-grade lymphoma responded (one partially and one completely), and one of seven (14 per cent) with high-grade lymphoma had a partial response. The median duration of responses was eight months. Four of the five complete responders have continued to receive maintenance interferon and have been in complete remission for 3, 7, 9, and 12 months, respectively; one had a recurrence at a site of previous disease seven months after interferon had been stopped. Side effects were noted in most patients. All 16 responders had been heavily pretreated with combination chemotherapy, including doxorubicin in 8 of the 16. These results suggest that recombinant leukocyte A interferon may be an effective new therapy for some patients with low- and intermediate-grade non-Hodgkin's lymphoma.

The need for improved specificity in cancer therapy is apparent. With the advent of monoclonal antibodies, the possibility of specifically targeted therapy is here. Early trials with monoclonal antibody in experimental animals and in man have demonstrated antibody can travel to specific tumor sites and localize on or around the tumor cells displaying antigens to which the antibody is directed. This evidence of specific targeting, along with early evidence of therapeutic efficacy for monoclonal antibodies and antibody immunoconjugates with drugs, toxins and isotopes, is encouraging. Some preliminary clinical observations from two monoclonal antibody trials are presented and the current status of clinical trials with monoclonal antibodies is reviewed.

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B- derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.

Hairy cell leukemia is a lymphoproliferative disorder characterized clinically by cytopenias. Standard therapy following variable periods of disease stability consists of splenectomy that often restores normal hematologic parameters for periods ranging from weeks to years. Fifteen patients (five without prior splenectomy or chemotherapy) were treated with 3 X 10(6) units per day of recombinant leukocyte A interferon and 14 of 15 patients completed eight weeks of therapy and were evaluated for response. There was one complete and 12 partial responses for an overall response rate of 93 percent. All of these patients' conditions have remained in complete or partial remissions and they continue to receive interferon with a median follow-up of six months. Coincident with the normalization of peripheral blood counts was a return of natural killer activity and normalization of immunologic surface markers as determined by monoclonal antibodies. This study confirms and extends earlier observations with natural alpha-interferon and indicates that recombinant leukocyte A interferon in low daily doses is also very effective treatment for hairy cell leukemia. In fact, it may be the best single modality of therapy for inducing both hematologic and immunologic recovery of these patients and deserves consideration as initial therapy.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN- alpha A). The binding of iodinated recombinant interferon-alpha to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN- alpha A. HCL patients (8/10) responded to rIFN-alpha A therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-alpha A therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN- alpha A therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

Monoclonal antibodies to tumor-associated antigens (TAA) of human colorectal cancer were elicited using immunosorbents of lectins combined with peripheral protein extracts of xenografted colon adenocarcinoma. This method of immunization was compared with whole cells from surgical specimens and to crude membranes from xenografted tumors. The immunosorbent immunogens were superior to the other immunogens in three ways: (1) the number of hybrids reactive with colon tumor cells or extracts, but not with lymphoid cells or extracts, (2) the number of stable hybrids after cloning, and (3) the number of hybridoma clones reactive with tissue sections of colon tumors, but not normal colonic mucosa. In addition, lectin immunosorbents elicited primarily IgG antibodies, especially IgG3, with almost 50% of the clones of interest reacting to seemingly less immunogenic glycoproteins. The improved elicitation of monoclonal antibodies to TAA by the use of lectin immunosorbents and peripheral protein extracts has considerable potential for generating reagents useful in diagnosis and therapy of human tumors.

Eighty-one patients with a variety of refractory disseminated malignant neoplasms have been treated in the first multiple fixed-dose phase I trial of recombinant leukocyte A interferon (IFL-rA). Each patient received IFL-rA by intramuscular injection, three times weekly for 28 days. Dosages were escalated in different patients from 1 to 136×106units per injection. The toxic reactions seen with IFL-rA resembled those of nonrecombinant leukocyte interferon and included fever, chills, fatigue, anorexia, myalgia, headache, occasional nausea and vomiting, and dose-dependent reversible leukopenia and hepatic transaminase elevations. The pharmacokinetics of IFL-rA were also comparable with nonrecombinant leukocyte interferon. Objective evidence of antitumor activity was seen in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, breast cancer, and melanoma, indicating that IFL-rA, the first genetically engineered biological response modifier available for testing in cancer patients, is biologically active in vivo. (JAMA1982;248:2461-2466)

Recombinant leukocyte A Interferon Is a highly purified single molecular species of alpha-interferon prepared by recombinant DNA methods. In 1982, a phase II trial to evaluate the efficacy of recombinant leukocyte A Interferon for patients with previously treated chronic lymphocytic leukemia was begun, and 19 patients were entered In this study. Patients received one of two dose schedules depending on their pretreatment platelet counts. Those with platelet counts greater than 100,000/mm 3 received 50 × 10 6 units/m 2 intramuscularly three times weekly, with dose reductions to 25 × 10 6 units/m 2 and 5 × 10 6 units/m 2 for unacceptable toxicity. Those with platelet counts less than 100,000/mm 3 received 5 × 10 6 units/m 2 intramuscularly three times weekly. Toxicity was dose-dependent and included fever, chills, fatigue, anorexia, myalgias, headache, leukopenia, and thrombocytopenia. Response was evaluable in all but one of the patients entered In this study. Two of the 12 patients treated with 50 × 10 6 units/m 2 had a partial response, three had no response, and seven had progressive disease. Of the six patients starting at 5 × 10 6 units/m 2 in whom response was evaluable, two had no response and four had progressive disease. Five patients with progressive disease (three at 50 × 10 6 units/m 2 and two at 5 × 10 6 units/m 2 ) had an acceleration of disease while receiving recombinant leukocyte A Interferon. It Is concluded that the dose and schedule of recombinant leukocyte A interferon therapy tested in this study are not effective in previously treated patients with advanced chronic lymphocytic leukemia.

A total of 11 patients were treated on an escalating, single dose trial of recombinant gamma interferon (rIFN-gamma), 6 patients by the i.m. and 5 patients by the i.v. route of administration. Dose ranges within each individual were from 0.05 mg/m2 of IFN (1 mg greater than or equal to 10 X 10(6) units of IFN) escalating to 10 mg/m2. All dosages were delivered twice weekly and the i.v. dose was infused over 5 min. The most common toxicities encountered included fever, chills, fatigue, anorexia, and granulocytopenia. The influenza-like symptoms were very similar to those encountered with IFN-alpha but were generally less severe. The granulocytopenia was dose-related and transient with recovery generally seen within 48-72 h following administration of rIFN-gamma. Absolute granulocyte counts only rarely dropped below 1000 mm3. Hepatotoxicity was not observed. IFN levels were determined by both a bioassay and an enzyme-linked immunosorbent assay. By the i.v. route, the peak level of IFN activity could usually be seen at completion of the infusion with a serum half-life of 30 min. By the i.m. route, the peak level of serum activity was generally detected between 4-8 h with a serum half-life of 4.5 h after the initial elimination phase. Peak IFN levels appeared to correlate with maximum toxicity. One patient with melanoma had a 25% reduction in a cutaneous lesion, but there were no other minimal, partial, or complete responses.

Biochemical and clinical signs of tumor lysis syndrome developed in a 57-year-old man with recurrent T cell lymphoma during therapy with recombinant leukocyte A interferon. When therapy was Interrupted due to thrombocytopenia and later resumed, biochemical changes compatible with tumor lysis recurred. This is the first case of tumor lysis syndrome observed during therapy with a biologic response modifier, a new class of agents entering cancer clinical trials. The atypical features of the clinical presentation and possible implications of these observations are discussed.

Nineteen patients with advanced refractory metastatic breast cancer no longer responsive to chemotherapy were treated in the first phase II efficacy trial of recombinant leukocyte A interferon (IFL-rA), a highly purified single molecular species of alpha interferon prepared by recombinant DNA methods. Patients received a previously determined maximum tolerated dose for this agent (50 X 10(6) U/m2 body surface area) by intramuscular injection three times weekly for up to 3 months. The symptoms of toxicity observed in this trial resemble those previously reported for alpha interferons and include fever, chills, fatigue, anorexia, and leukopenia. All patients required dose reductions, most often for reasons of severe fatigue. Of the 17 patients evaluable for tumor response, one patient had stable disease and 16 had evidence of tumor progression. We conclude that IFL-rA is not an active agent in the treatment of advanced, refractory breast cancer when used at a maximum tolerated dose on this treatment schedule.

Highly purified human lymphoblastoid interferon (HLBI) derived from virus-stimulated Namalwa cells was administered by 6-h IV infusion or IM injection to 40 patients with a variety of disseminated malignancies refractory to standard therapy. Each patient received doses escalating from 0.1 to 50 X 10(6) U for up to 5 weeks. Extensive monitoring for clinical effect, toxicity, and pharmacokinetics has revealed higher peak serum interferon levels and somewhat more pronounced systemic toxicity for the IV than for the IM route of administration. Objective evidence of tumor regression was observed in two patients receiving HLBI IV.

Sufficient preclinical and clinical data are now available on the use of monoclonal antibodies (MoAb) in the treatment of human solid tumors to describe their likely role in the treatment of cancer (1). The initial trials focused on the usual phase I considerations of toxicity and tolerance, but added the localization of the antibody in tumor deposits and the distribution of antibody in normal and neoplastic tissues as concurrent investigations (2–4). There is now a considerable body of evidence from the administration of 1 mg to several grams of a number of murine monoclonal antibodies that these agents are well tolerated by patients. Although clinical responses to unconjugated antibody have generally not been striking, there is unequivocal evidence that antibody binds to individual tumor cells after intravenous injection (5).

A variety of interferons have now been tested in clinical trials. Most of these trials have utilized leukocyte (α) interferon preparations. Early trials were conducted with partially purified material derived from the supernatants of virus-stimulated leukocytes that were of low purity and inconsistent pharmaceutical quality. More recently, trials have been conducted with a lymphoblastoid cell line interferon (α) of high purity and good reproducibility (Knost et al., 1983). Several recent studies have utilized recombinant α-interferon derived by cloning a gene for α-interferon in an E. coli expression system (Sherwin et al., 1982, 1983). Despite the major differences between these α-interferon preparations, many of the toxicities, immunological modulating effects, and therapeutic effects have been similar. Fever, chills, headache, fatigue, and anorexia have been rather constant side effects of these interferon preparations. At higher doses, mild hematological depression and transient hepatic enzyme abnormalities have been seen. Occasional cardiac effects including arrhythmias and ischemic effects have been observed in the context of these trials. Some central nervous toxicity including confusion, decreased ability to concentrate, and rarely seizures at very high doses have been seen in these studies. It is unclear whether all these effects are due to the direct action of the interferon preparation since the induction of fever, tachycardia, and fatigue may have secondary effects (Oldham, 1983a). The phase I trials for the α-interferons are virtually complete.

Inhibitors of human interferon action that might be relevant to tumour resistance or escape mechanisms were investigated in a macrophage system. The effects of IFN on macrophage Fc gamma receptor expression were inhibited by three preparations: (1) a low-molecular-weight component of normal autologous serum; (2) a low-molecular-weight component of carcinoma supernatant; and (3) physiological concentrations of retinol and retinoic acid. Since human carcinoma tissue contains abnormally high levels of retinoic acid-binding protein, the possibility that a tumour-associated retinoid contributes to tumour-induced inhibition in vitro was investigated. Inhibition of IFN action in vitro by retinoic acid (vitamin A acid) was found to be reversed by beta-carotene (pro-vitamin A). When tested in the tumour system beta-carotene also reversed inhibition by the human-carcinoma-derived signal. These data are consistent with the view that at least one of the tumour-derived signals inhibitory towards IFN is a tumour-associated retinoid, although firm evidence for this must await further physicochemical characterization of the inhibitory signal(s). The present data clearly show, nevertheless, that human tumour-induced inhibition of IFN in vitro can be reversed by the pro-vitamin beta-carotene.

Human monoclonal antibodies are desirable, especially as therapeutic agents, but the best means of producing them is still a matter of investigation. It is clear that human antibodies of predicted specificity from patients with autoimmune disease can be derived, and this may help unlock some of the mysteries of these illnesses. Human monoclonal antibodies against tumor-specific antigens for use in in vivo diagnosis and therapy remain desirable goals. Problems involved in their routine development include the lack of available, adequately immunized, and differentiated lymphocytes and the nature and paucity of the available human “myeloma” cell lines. These lines have been compared now by a number of authors who have reached similar conclusions. Our study directly compared the greatest number of cell lines and found UC729-6 and HF2 to be the best; on the other hand, our success in developing IgG-secreting hybridomas from U-266, using hyperimmunized lymphocytes, suggests that this line may only be capable of secretion with the more differentiated cell, the human equivalent of those hyperimmunized murine spleens. Hence both sides of the fusion equation must be made optimal. Two new approaches to circumvent this problem involve the use of either a human-murine myeloma chimera as the parental myeloma line or, more recently, genetic engineering techniques to substitute human constant regions for the murine while retaining the murine hypervariable region, preserving the binding specificity of the murine antibody.

We report the successful generation of human T-cell hybridomas that constitutively secrete lymphokines. An acute lymphoblastic leukemia T-cell line, CCRF-H-SB2, free of reverse transcriptase and mycoplasma, was sensitized to hypoxanthine, aminopterin, and thymidine (HAT) by selecting out a mutant deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT) in 8-azaguanine. Peripheral blood T lymphocytes from normal donors were incubated in vitro with 10 micrograms/ml of concanavalin A for 48 h and subsequently fused with the CCRF-H-SB2 HAT-sensitive cell line. Following 5 weeks in culture, 38 of 440 wells (8.6%) demonstrated hybridoma growth. Supernatants of these cultures were screened for interleukin-2 (IL-2), chemotactic factor, interferon, migration inhibition factor, and macrophage-activating factor activities. Twelve (of 38) hybrids exhibited IL-2 activity, and eight of these were successfully cloned. The highest secreting clone was demonstrated to have mRNA to IL-2 while the parent CCRF-H-SB2 had no detectable mRNA to IL-2. Three hybrid cultures produced chemotactic factor; one was successfully cloned and grown in serum-free medium, where it continued to constitutively produce chemotactic factor as well as IL-2 activity. The chemotactic factor was determined to have the same molecular weight (12,500 daltons) as leukocyte-derived chemotactic factor. Constitutive IL-2 production remained stable for over 12 months. None of the hybridomas tested produced detectable levels of gamma interferon, migration inhibition factor, or macrophage activation factor. Because these T-cell hybridomas produce lymphokines constitutively and this phenotype is stable, they can be an important source of highly purified human lymphokines for clinical and laboratory investigations.