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Six human cell lines were compared with each other and with murine myeloma NS-1 as to their sensitivity to HAT medium and their ability to form hybrids with human lymphocytes, secret monoclonal immunoglobulin, clone, and maintain detectable levels of monoclonal immunoglobulin secretion for a period of time after fusion. Fusion efficiencies varied from 0 to 50%, and the incidence of immunoglobulin secretion ranged between 1 and 78% of the hybrids. Immunoglobulin secretion of cloned hybrids varied from 0.8 to 1.6 micrograms/ml/10(6) cells among the human-human hybrids and was 2.4 micrograms/ml/10(6) cells by the human-mouse hybrid. Among the lines tested, UC729-6 and HF2 appeared optimal for pursuing further studies with human-human hybridomas. In addition, although only a small percentage of hybrids were produced with HMy2, a very high percentage secreted immunoglobulin, so that this line also warrants further investigation to improve the efficiency of hybrid formation. Implications for specific monoclonal antibody production and for therapy of leukemias and lymphomas by anti-idiotype antibodies are discussed.

T gamma lymphocytes are those lymphocytes that express receptors for both the Fc portion of IgG and sheep erythrocytes. A very high proportion of normal T gamma lymphocytes are large granular lymphocytes (LGL), the cell responsible for most, if not all, natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) in humans, rats, and mice. In general, these cells are large lymphocytes with prominent azurophilic granules in the cytoplasm. Recently, a group of lymphoproliferative disorders made up predominantly of T gamma lymphocytes has been described. The most common and best studied of these disorders we refer to as “chronic T gamma-lymphoproliferative disease” (T gamma-LPD). In most cases, this disease represents the abnormal expansion of LGL, which is reflected by an increase in functionally active NK or ADCC effector cells. The chronic T gamma-LPD lymphocytes are generally characterized as E- and EA-rosette positive, acid-phosphatase, and beta-glucuronidase positive and express the pan-T antigens OKT3/Leu-4, OKT11/Leu-5, the suppressor-associated antigens OKT5,8/Leu-2, and the NK-associated antigens Leu-7/HNK-1. Typically, the patients are older, predominantly males and characteristically have a lymphocytosis of predominantly T gamma lymphocytes with lymphocyte infiltration of the bone marrow and often the spleen. While chronic T gamma-LPD is not usually an aggressive disease, the patients are often neutropenic and have recurrent bacterial infections requiring antibiotic therapy. Some patients have benefited from cytotoxic chemotherapy., but most patients have not required chemotherapy. An experimental LGL leukemia in F344 rats appears morphologically, functionally, and clinically similar to the human chronic T gamma-LPD and serves as an experimental model for further examining the ontogeny and function of LGL and may be applicable for exploring new and more effective means for the treatment of patients with chronic T gamma-LPD.

Human-murine hybridomas, generated by fusion of human CLL cells with non-Ig-secreting murine myeloma NS-1, and secreting the IgM kappa expressed on the CLL cell surface, were grown in the peritoneal cavity of doubly pristane-primed nude mice to produce large quantities of highly concentrated human IgM kappa for the development of anti-idiotype monoclonal antibodies. Although the hybridomas secreted only 0.8 to 1.5 micrograms/ml/10(6) cells in tissue culture, they produced 8 to 12 ml of ascites from two mice containing 1.7 mg/ml of human IgM. Partial purification was then easily accomplished by euglobulin precipitation. Two weekly injections of 100 micrograms of this material into BALB/c mice resulted in the development of both anti-idiotypic and anti-human IgM antibodies. This system overcomes the major hurdle of generating sufficient quantities of the human idiotypic Ig to immunize and screen for specific murine antiidiotypes, and provides the material to produce these therapeutically promising reagents rapidly for clinical trials. This is, to our knowledge, the first report of a human Ig produced in mouse ascites.

The sera of 78 patients with invasive amoebiasis were tested for antiamoebic antibodies by the techniques of enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination (IHA), indirect fluorescent antibody (IFA) and counterimmunoelectrophoresis (CIEP). Results showed that the ELISA compared favourably with IHA and IFA tests in terms of sensitivity and specificity. ELISA, IHA and IFA detected 97.4%, 96.2% and 98.7% of the patients respectively. CIEP was the least sensitive of the 4 serological methods with a sensitivity of 88.5%. The advantages and disadvantages of the 4 serodiagnostic procedures are discussed.

A 44-year-old man had mycosis fungoides and generalized plaque disease involving 80% of his skin surface with diffuse lymphadenopathy and alopecia of the scalp and groin. In addition, distal to the wrist, there were sclerodermatous changes involving the skin of the hands with associated sclerodactyly of all digits with loss of normal palmar creases. There were no subungual telangiectasis or digital ulcers. The changes in the hand that occurred in this case, no doubt arose as a result of the patient's neoplasm. Abnormalities of collagen biosynthesis and degradation probably occur with mycosis fungoides as a result of the extensive infiltration of the epidermis and dermis with malignant cells. To our knowledge, the association of sclerodactyly with mycosis fungoides has not been previously reported.

Eleven patients with metastatic melanoma underwent serial gamma camera imaging and biodistribution measurements after i.v. injection of escalating doses of [111In]9.2.27, an antimelanoma murine monoclonal antibody. Patients received a fixed dose of 1 mg of [111In]9.2.27, with no additional 9.2.27 (five patients), or co-infused with 49 mg (five patients) or 99 mg (one patient) of unlabeled, unconjugated 9.2.27. Higher doses resulted in prolonged blood-pool retention, less uptake in spleen and bone marrow, and appeared to have a positive effect in improving tumor imaging. A dose of 1 mg of 9.2.27 permitted detection of tumors in two of five patients and two of ten lesions, while with greater than or equal to 50 mg, tumors were detected in all patients and in 24 of 32 lesions. Human gamma globulin injected prior to administration of [111In]9.2.27 failed to block the prominent liver, spleen, and bone marrow uptake. No toxicity was observed. These results indicate the feasibility of imaging metastatic melanoma with [111In]9.2.27 and suggest that antibody dose may be a critical determinant of biodistribution and tumor uptake.

Important insights into leukocyte differentiation and the cellular origins of leukemia and lymphoma have been gained through the use of monoclonal antibodies that define cell surface antigens and molecular probes that identify immunoglobulin and T cell receptor genes. Results of these studies have been combined with markers such as surface membrane and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte receptors on T lymphocytes, and cytochemical stains. Using all of the above markers, it is now clear that acute lymphoblastic leukemia (ALL) is heterogeneous. Furthermore, monoclonal antibodies that identify B cells, such as the anti-B1 and anti-B4 antibodies in combination with studies of immunoglobulin gene rearrangement, have demonstrated that virtually all cases of non-T-ALL are malignancies of B cell origin. At least six distinct subgroups of non-T-ALL can now be identified. T-ALL is subdivided by the anti-Leu-9, anti-Leu-1, and antibodies that separate T lymphocyte subsets into three primary subgroups. Monoclonal antibodies are also useful in the subclassification of non-Hodgkin's lymphoma, and certain distinct markers can be correlated with morphologic classification. The cellular origin of the malignant Reed-Sternberg cell in Hodgkin's disease remains uncertain. A substantial number of investigators favor a myelocyte/macrophage origin based on cytochemical staining; however, consistent reactivity with antimonocyte reagents has not been demonstrated. Although monoclonal antibodies are useful in distinguishing acute myeloid from acute lymphoid leukemias, they have less certain utility in the subclassification of acute myelogenous leukemia (AML). Attempts to subclassify AML by differentiation-associated antigens rather than by the French-American-British (FAB) classification are underway in order to document the potential prognostic utility of surface markers. Therapeutic trials using monoclonal antibodies in leukemia and lymphoma have been reported. Intravenous (IV) infusion of unlabeled antibodies is the most widely used method; transient responses have been demonstrated. Antibodies conjugated to radionuclides have been quite successful in localizing tumors of less than 1 cm in some studies. Therapy trials with antibodies conjugated to isotopes, toxins, and drugs are currently planned. Purging of autologous bone marrow with monoclonal antibodies and complement in vitro has been used in ALL and non-Hodgkin's lymphoma; preliminary data suggest that this approach may be an effective therapy and may circumvent many of the obstacles and toxicities associated with in vivo monoclonal antibody infusion.

The hyperviscosity syndrome refers to a group of symptoms and signs related to increased blood viscosity often produced by monoclonal immunoglobulins. It is most frequently associated with Waldenström's macroglobulinemia and, on occasion, with other immunoglobulins that are capable of forming highly polymerized molecules. This article is a report on the first case of pure light chain myeloma associated with the hyperviscosity syndrome. The hyperviscous plasma in this case is secondary to the unusual degree of aggregation of kappa light chain as demonstrated by high-resolution electrophoresis, serum immunofixation, and Sephadex g-200 (Pharmacia, Piscataway, NJ) column chromatography.

Maximally tolerated doses of interferon alfa-2a, 50 X 10(6) U/m2 administered intramuscularly (IM) 3 times weekly, were given to 20 patients with advanced stages of cutaneous T-cell lymphoma (CTCL) to determine the efficacy and toxicity of this therapy. All patients were heavily pretreated and had failed standard therapies. Objective remissions were noted in 45% of the patients, including two patients who achieved complete remissions and seven patients who had partial remissions. The median duration of response was 5.5 months, with responses lasting a minimum of 3 months and a maximum of more than 3 years. Responses in excess of 2 years occurred in three of the nine responding patients. These results were achieved with moderate toxicities. The dose-limiting toxicity was a flu-like syndrome consisting of malaise, anorexia, weight loss, and falling performance status. Toxicity was observed in all patients but was always alleviated by dose reduction. Patients with indolent B-cell non-Hodgkin's lymphoma who received the same therapy had a similar objective response rate (54%) and showed the same toxicities. These trials were followed by an ongoing trial using the same dose of interferon in a different schedule given for 12 weeks followed by a dose escalation to 100 X 10(6) U/m2. Three partial responses were observed in the first 13 patients on this trial treatment. Other studies examining lower dose interferon compared to the 50 X 10(6) U/m2 are in progress. This study establishes interferon alfa-2a as a treatment of choice for patients with advanced cutaneous T-cell lymphomas refractory to chemotherapy and other standard therapies. Trials combining interferon with other standard treatments and the use of interferon in earlier stages of disease are needed.

We have reported that [111In]T101 is highly effective in the detection of cutaneous T-cell lymphoma (CTCL) in nodal and cutaneous (erythroderma and tumor) sites. This study compares the biodistribution of [131I]T101 (1 to 7.1 mg, 2 mCi) in four patients with CTCL; two of these patients also received [111In]T101 (1 mg, 5 mCi). There was rapid clearance of [131I]T101 from whole-body, spleen, liver, and bone marrow, with evidence of loss of 131I tracer from the T101. Lymph node uptake was minimal in three of four patients, and there was no localization in skin lesions. This contrasted with [111In]T101 where there was prolonged retention of activity in these organs and excellent uptake in skin tumors, erythroderma, and lymph nodes. The study showed that [131I]T101 was suboptimal for imaging CTCL patients and demonstrates that the isotope or labeling method can dramatically alter the apparent biodistribution and tumor targeting of a given monoclonal antibody.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease. Some patients require no therapy and have prolonged survival. Others have brief survival despite intensive treatment. Several staging systems have been developed. These are useful in predicting outcome and need of therapy, but controversies exist. Additional prognostic factors have been identified such as cytogenetics, lymphocyte levels or doubling time, bone marrow morphology, and others. Patients with low-stage CLL have not been shown to benefit from treatment. In patients with intermediate disease, combination chemotherapy with cyclophosphamide, vincristine, and prednisone (CVP) has not been shown to be superior to chlorambucil with or without prednisone. The therapy of high stage CLL is controversial; therapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) may be superior to CVP. Future directions of therapeutic research include the treatment of patients with low stage disease, more precise identification of patients requiring therapy, and more precise definition of therapeutic response.

Acute myelogenous leukemia (AML) is a neoplastic disease characterized by the proliferation of malignant myeloblasts and other immature myeloid cells. Infiltration of the bone marrow by these leukemia cells results in the impairment of normal hematopoiesis, with resultant anemia, granulocytopenia, and thrombocytopenia. If untreated the disease is rapidly fatal, with a median survival of less than 2 months. Death frequently results from infection, hemorrhage or both [1].

In 1981, the National Cancer Institute undertook Phase II trials of interferon alfa-2a in patients with non-Hodgkin's lymphoma (including cutaneous T-cell lymphoma [CTCL]) and chronic lymphocytic leukemia (CLL). A dose of 50 X 10(6) U/m2, three times per week, was used initially, then adjusted downward as dictated by toxic effects. A 54% response rate was achieved among 24 patients with low-grade non-Hodgkin's lymphomas, and the median duration of response was 8 months. Less encouraging results emerged from studies in patients with intermediate- or high-grade disease. Responses were noted in only two of six patients in the former group, and only one of seven in the latter group. Results have likewise been disappointing in patients with CLL. Of 18 individuals treated, only two exhibited brief, partial responses. In CTCL, on the other hand, alpha interferon may be the most effective single agent. Among 20 patients with advanced disease who had failed previous therapies, 45% responded. The primary dose-limiting toxicity in all these trials has been flu-like symptoms, particularly fever and fatigue. Fever has generally resolved as treatment has been continued, but dosage reductions are usually necessary to alleviate fatigue. Future studies are likely to focus on the use of alpha interferon in combination with chemotherapeutic agents or other biologic response modifiers, such as monoclonal antibodies.

The HK-9 strain of Entamoeba histolytica was cultured axenically. A crude extract of the trophozoites was used as antigen in the enzyme-linked immunosorbent assay (ELISA) for the determination of antiamoebic IgG, IgM, IgA and IgG subclasses in sera of patients with E. histolytica infection. Sera from amoebiasis patients had significantly higher mean ELISA values for all classes and subclasses of immunoglobulins examined compared with healthy controls. The ELISA for IgG, IgM and IgA was positive for 76 (97.4%), 34 (43.6%) and 62 (79.5%) respectively of 78 amoebiasis sera. Analysis of IgG subclasses by ELISA showed that 76.7%, 25%, 33.3% and 66.6% of the patients were positive for antiamoebic IgG1, IgG2, IgG3 and IgG4 respectively. IgG1 and IgG4 were the dominant IgG subclasses involved in amoebiasis. The role of antiamoebic antibodies in amoebiasis is discussed.

Human anti-murine immunoglobulin responses were assessed in serum from three groups of patients receiving murine monoclonal antibody therapy. Each of the three patient groups responded differently. Chronic lymphocytic leukemia patients demonstrated little or no preexisting murine immunoglobulin G-reactive antiglobulin prior to treatment, while the cutaneous T-cell lymphoma and melanoma patients demonstrated preexisting antiglobulin levels in the same range as those demonstrated in healthy controls. None of 11 chronic lymphocytic leukemia patients receiving the T101 monoclonal antibody demonstrated an antiglobulin response, whereas all four of the cutaneous T-cell lymphoma patients receiving the same antibody developed increased levels of antiglobulins. Three of nine malignant melanoma patients receiving the 9.2.27 monoclonal antibody showed an increase in antiglobulin titers. In patients developing antiglobulin responses, the response was rapid, typically being detectable within 2 weeks. The antiglobulins were primarily immunoglobulin G and, with the exception of a single melanoma patient in whom the response appeared to have a substantial 9.2.27-specific component (i.e., antiidiotype), were cross-reactive with most murine immunoglobulin G preparations tested. This pattern of results suggested that the antiglobulin was a secondary immune reaction with elevation of the levels of preexisting antiglobulin which was cross-reactive with the mouse antibody administered. While the presence of serum antiglobulin would be expected to present major complications to monoclonal antibody therapy, no clinical toxicity related to antiglobulin responses was observed in these patients, and no inhibition of antibody localization on tumor cells was seen.

Antibody localization at the tumor site was assessed in melanoma patients who received the murine monoclonal antibody 9.2.27. Antibody was administered twice weekly in escalating doses from 1 to 500 mg. Localization was assessed by biopsies of cutaneous and lymph node lesions obtained 24-96 hours following therapy. The percentage of tumor cells that bound the antibody in vivo was dose dependent, with similar findings obtained by either flow cytometry or immunoperoxidase staining techniques. Little or no in vivo binding of the 9.2.27 antibody to tumor cells was found following 1- and 10-mg doses, whereas all specimens demonstrated in vivo binding of the antibody following 200- and 500-mg doses. Fluorescence staining intensity, as quantitated by flow cytometry, was employed to determine the degree of in vivo saturation of antibody binding sites following therapy. The degree of saturation was found to vary substantially among patients: Some patients demonstrated nearly 100% saturation after 200-mg doses of 9.2.27 antibody, whereas others demonstrated only half maximal saturation after doses of 500 mg. Although immunoperoxidase staining provided important qualitative information regarding the distribution of antigen and antibody within the tumor, these studies demonstrated the usefulness of immunofluorescent flow cytometry for quantitative assessment of antibody localization in solid tumors and provided information necessary for the design of further trials of monoclonal antibodies and immunoconjugates.

Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

The interferons represent an important first member of a family of biologic response modifiers used in treating human malignancies. Activities associated with the interferons include inhibition of viral replication, influence on cellular protein production, direct antiproliferative effects, and a variety of modulatory effects on the immune response. These regulatory functions of interferon underlie the interest in its use as an anticancer agent. Interferon alpha is the most extensively studied interferon species. Although antitumor activity has been seen both in vivo and in vitro in some solid malignancies, the most impressive responses have occurred in the hematologic malignancies. For the low-grade non-Hodgkin's lymphomas, response rates of 50%, with 10% to 15% complete responses, have been reported. A response rate of 15% has been reported for chronic lymphocytic leukemia in studies outside of the National Cancer Institute (NCI); in our phase II trials at the NCI, only two (11%) of 18 patients had brief partial responses to recombinant interferon alpha. For patients with cutaneous T-cell lymphomas (mycosis fungoides and the Sézary syndrome), a response rate of 45%, with 10% complete responses, was seen in patients treated with recombinant interferon alpha. Based on such findings, interferon appears to be one of the most effective single agents for cutaneous T-cell lymphomas. Further phase II trials are being conducted to determine whether lower doses of interferon alpha are as effective as the high doses used in the previously reported studies. Phase III trials will involve the use of interferons in combination with chemotherapeutic agents as well as in the adjuvant setting.

Factors in the queous humor capable of increasing the random migration and chemotaxis of monocytes were demonstrated during experimental uveitis in the rabbit. Injection of endotoxin-free human serum albumin into the vitreous produced a severe inflammation detectable after 7 days and maximal between 12 to 21 days. Aqueous humor was measured for chemotactic activity with a modified Boyden chamber, which permitted the use of volumes of less than 0.025 ml. Chematactic activity was measurable during the primary inflammation and remained elevated for a short period of time after clinical inflammation and anterior chamber cellular reaction had decreased. A severe reinduction uveitis could be initiated by the intravenous injection of human serum albumin after day 30. Chemotactic factors could be measured at high titers by 8 h after this intravenous challenge.

The toxic A chain of abrin was isolated by affinity chromatography and was demonstrated to be a potent inhibitor of protein synthesis in a cell-free rabbit reticulocyte system with a complete inhibitory dose at 1 X 10(-9) M. This A chain was coupled by a disulfide linkage to a purified monoclonal antibody directed against a tumor-associated antigen found on the line 10 hepatocarcinoma tumor in strain two guinea pigs. The immunoconjugate retained the functions of the individual components, i.e., antigen binding to the intact cell in vitro and inhibition of its protein synthesis. This conjugate was a selective antineoplastic agent with a cytocidal dose at 5 X 10(-9) M toward antigen-bearing cells in vitro. Several antigen-negative cells were much less susceptible to its cytotoxic effect. The cytotoxicity of the conjugate appeared to be by antibody-mediated delivery of toxic A chain into the target cell. When cells were pretreated with excess free antibody followed by a brief exposure to conjugate, there was a reversal of the cytotoxicity to antigen-positive cells but not to the antigen-negative cells. The therapeutic efficacy of the conjugate was assayed by injecting a single dose s.c. or i.v. into syngeneic guinea pigs bearing established line 10 tumors. These in vivo studies showed that (a) the conjugate was not toxic at a dosage of 60 to 1120 micrograms/guinea pig, (b) the conjugate decreased or abolished the growth of established solid tumors, (c) the conjugate delayed or inhibited tumor metastases to lymph nodes, and (d) 20 to 40% of the animals in selective groups had a long-term complete regression.

Biologicals and biological response modifiers (BRMs) represent a new class of agents for cancer therapy. Historically, there have been many attempts to stimulate the immune response with nonspecific immunomodulators in the form of bacterial extracts, viruses, and chemicals. Although these approaches have occasionally proven useful under defined conditions in experimental models, their extension to the clinic has been largely unsuccessful. Recent advances in molecular biology and hybridoma technology have made available genetically engineered lymphokines and cytokines, as well as monoclonal antibodies, as highly purified biologicals for cancer treatment. These agents may act directly on tumor cells and/or may act on the patient's own biological responses to induce an antitumor response. Selective defects in T-cell function have recently been identified in cancer patients and in patients with acquired immunodeficiency syndrome (AIDS). Simultaneously, the availability of gamma interferon (gamma-IF) and interleukin-2 (IL-2) may allow for the selective correction of these T-cell deficits, leading to restoration of the patient's immune responses and perhaps correction of the clinical syndromes. Preliminary data suggest that gamma-IF and IL-2 have in vitro activity on these T-cell defects, and the preliminary evidence that these agents have activity in vivo will be reviewed. Extensive trials are being conducted at the National Cancer Institute with monoclonal antibodies as anticancer agents. Animal model experiments have demonstrated considerable antitumor activity of immunoconjugates using monoclonal antibodies tied to toxins. Preliminary clinical results suggest that T-101 in leukemia and lymphoma and 9.2.27 in malignant melanoma may prove useful as specific reagents in the treatment of these disorders. While the antitumor effects with these antibodies have not been dramatic, our preliminary data in approximately 30 patients with leukemia, lymphoma, and melanoma clearly demonstrate the ability of intravenous monoclonal antibody to locate and specifically label tumor cells bearing the target antigens. It has been possible to localize antibody on the tumor cells in melanoma deposits that are barely visible in the skin. These data and radioimaging data suggest a future role for immunoconjugates as anticancer agents.

One-hundred and seven patients with acute myelogenous leukemia (AML) ranging in age from 15 to 82 yr who were previously untreated, received a 70 day high-dose remission induction regimen consisting of daunorubicin, cytarabine, and thioguanine (TAD). Identical complete remission rates of 65% were observed for 33 patients 60 yr of age and older and for 74 patients age 15-59 yr. Median remission duration and survival were 14 mo and 22 mo for patients 60 yr and older, and 16 mo and 22 mo for patients 15-59 yr. These differences are not significant. These data indicate that older patients respond to intensive chemotherapy in a similar manner to younger patients with this disease.

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 x 10(6) units 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.

Thirteen patients with metastatic malignant melanoma received intravenous therapy with the murine antimelanoma monoclonal antibody 9.2.27. Five patients were entered on a dose escalation protocol with twice weekly escalating doses of 10-500 mg, in an extension of a previously reported trial. These patients demonstrated near saturation of available antibody binding sites in vivo following the 500 mg dose, with minimal toxicity. The remaining patients were entered onto a dose schedule comparison study, with a 500 mg dose administered either in a single 2 h infusion or as five daily 2 h infusions of 100 mg to examine the effects of different dose schedules and of an interrupted schedule on subsequent therapy with the same antibody. Intratumor localization of the monoclonal antibody did not appear to vary with respect to the dose schedule; however, interruption in therapy for 4 weeks was accompanied by somewhat poorer localization of antibody. This effect appeared to be primarily attributable to development of human antimurine antiglobulin in 25-30% of patients with resultant decrease in intratumor localization of antibody and more rapid clearance of the 9.2.27 antibody from the circulation. Earlier reports with other antibodies notwithstanding, initial infusions of 500 mg of 9.2.27 did not induce tolerance to the murine immunoglobulin. This study confirms and extends the findings of our initial trial of the 9.2.27 antibody by demonstrating that, although clinical responses were not observed, the antibody can be safely administered at doses up to 500 mg, with good intratumor localization of antibody. The diminished localization of antibody associated with antiglobulin responses indicates the importance of monitoring antiglobulin levels during therapy, and the necessity of controlling or preventing this phenomenon when monoclonal antibodies are administered in multiple doses as drug, toxin, or radionuclide immunoconjugates.

Interferon alfa-2a (Roferon-A) was given to 20 patients with advanced cutaneous T cell lymphomas, all of whom were heavily pretreated. Objective responses were seen in nine of the 20 patients, including seven partial and two complete responses. The median response duration was 5.5 months, with three of the responses lasting 2 or more years. Toxicity was moderate but was alleviated by dose reductions. These results demonstrate that Roferon-A is highly effective in the treatment of advanced refractory cutaneous T cell lymphomas. The role of this treatment in earlier stages of disease and in combination with other standard forms of therapy needs to be evaluated.

Two patients with advanced lymphoma involving the lungs that was resistant to chemotherapy had an initial excellent response to interferon therapy. After two months of stable disease, alpha interferon A therapy was discontinued, with relapse of a few months later. Both patients had extensive pulmonary involvement with relapse of the lymphoma, and both showed a dramatic response to retreatment with alpha interferon A. Unusual interferon-related pulmonary complications were observed in one patient, and their management is detailed.

Poly(I,C)-LC was administered in low (1 mg/m2) and intermediate (4 mg/m2) doses to cancer patients by intramuscular injection or intravenous infusion to evaluate the immunomodulatory effects. Natural killer cell (NK) activity was elevated slightly at the low dose and remained unchanged overall, but some depression was observed at the 4 mg/m2 intravenous dose. Monocyte function was elevated in all groups of patients, as was the interferon-induced enzyme 2'5'-oligo-A synthetase. These increases were observed at the 1 mg/m2 intramuscular dose, despite a lack of detectable circulating serum interferon (IFN). In regard to cell surface markers, poly(I,C)-LC induced an increase in OKT10-positive cells and a small but consistent trend toward increases in the ratio of Leu-3/Leu-2-positive cells. Lymphocyte proliferation in response to concanavalin A was depressed by poly(I,C)-LC administration. Although an optimum immunomodulatory dose and schedule was not determined, the data indicate that low doses produce significant changes in immune function and that induction of detectable levels of circulating interferon is not required for poly(I,C)-LC to have biological effects.

Using data from 12 patients, we have analyzed the pharmacokinetics of 111In-9.2.27, an antimelanoma monoclonal antibody, following i.v. infusion. Plasma data and scintillation camera images obtained from patients receiving either 1, 50, or 100 mg of monoclonal antibody indicated dose-dependent (i.e., saturable) kinetics. Based on these observations and known immunoglobulin kinetics, we developed a nonlinear compartmental model to describe the biodistribution of 111In-9.2.27 and the other coinjected 111In-associated compounds. The model included (a) three compartments representing intact 111In-9.2.27 ("plasma," "nonsaturable," and "saturable binding" compartments), (b) four compartments representing 111In-diethylenetriaminepentaacetic acid, and (c) one compartment representing 111In in an undetermined chemical form ("extravascular delay" compartment). Analysis of the rate of urinary excretion relative to plasma concentration indicated that the saturable binding compartment was a site for catabolism of monoclonal antibody. Further examination of the urinary data, together with previous studies of the site(s) of immunoglobulin catabolism, suggested that additional elimination took place from either the plasma or the nonsaturable compartment. The model indicated that to fill the saturable sites would require a dose of approximately 0.5 mg and suggested that greater than 3.5 mg would maintain saturation for 200 h. Computer integration of gamma camera counts over the spleen revealed a clear saturable component of uptake, whereas integration over the liver showed no such pattern. The proposed model was fitted to the liver and spleen imaging data by summing fractions of model simulations of each compartment. That analysis confirmed the suspected saturable uptake by the spleen (21% of the saturable binding compartment) and revealed a quantitatively important component of saturation in the liver (35% of the saturable binding compartment) that was not obvious from initial examination of the images. When the results were expressed on a concentration basis, the spleen accounted for 247% of the saturable compartment per kg, whereas the liver accounted for 25%/kg. The bone marrow also showed saturable uptake; hence, the saturable uptake may relate to the sinusoidal blood supply characteristic of liver, spleen, and marrow. The model predicts the dose levels required to overcome saturable background, suggests appropriate doses and schedules for cold loading strategies, and provides a format for explicit inclusion of tumor antigen.

Serial gamma camera imaging was performed in 11 patients with cutaneous T-cell lymphoma after s.c. injection between the toes of 111In-labeled T101, a pan T-cell monoclonal antibody. Two of the patients also received 111In-labeled 9.2.27, an isotype-matched control monoclonal antibody. Three doses of T101 (0.1, 0.5, and 1.0 mg), coupled with 0.5 mCi 111In, were used to measure lymph node uptake and dose response. A single 0.5-mg (0.5 mCi) dose of 111In-9.2.27 was used as control antibody to assess nonspecific uptake. Low-dose (0.1 mg) T101 administration produced rapid, intense uptake in inguinal-femoral and iliac nodes with minimal uptake in the paraaortic nodes and liver. The 0.5-mg dose and higher produced consistent uptake in all subdiaphragmatic nodes with greatest accumulation of liver activity at the 1.0-mg dose. In the two control studies, the ratio of specific:nonspecific antibody uptake in the inguinal-femoral, iliac, and paraaortic nodes averaged 7.7,5.9, and 1.9 at 48 h, respectively. In another patient, inguinal-femoral node biopsy contained over 2% of the injected dose of 111In-T101 per gram at 7 days. These findings suggest efficient and specific antibody binding in nodes nearest to the injection site, with progressive uptake of remaining antibody in more distant nodes as proximal sites approach saturation. Higher doses increase overflow of unbound antibody into the systemic circulation. There appears to be an optimal s.c. dose of approximately 0.5 mg (0.25 mg/foot) for T101 immunolymphoscintigraphy of subdiaphragmatic lymph nodes in patients with cutaneous T-cell lymphoma.

Recombinant and natural forms of interferon alpha have been shown to bring about tumor regressions in patients with low-grade non-Hodgkin's lymphoma. Complete and partial response rates in up to 50% of patients have been documented. Although these studies are encouraging, interferon alpha's role in the current management of patients with low-grade non-Hodgkin's lymphoma is not apparent. The high doses of interferon that were used in some of the recent studies were not well tolerated by these patients. Current studies using interferon in patients with low-grade non-Hodgkin's lymphomas are evaluating lower, and perhaps better tolerated doses of interferon. In addition, in vitro studies have documented additive and sometimes synergistic antitumor effects of interferon in combination with cytotoxic drugs. This approach is being extended to the clinic, and trials are now underway evaluating combinations of interferon and various cytotoxic drugs known from previous studies to be active in patients with low-grade non-Hodgkin's lymphomas. This report reviews studies using interferon alone in these patients, and discusses ongoing and proposed studies for the future use of interferon in treating this disease.

Serotonin and histamine are released during the early phase of inflammatory reactions. To determine if these vasoactive amines might play a role in the cell-mediated phase of inflammation, studies were undertaken to assess their influence on some immunologic functions of human peripheral blood leukocytes (PBL). PBL were cultured with serotonin or histamine, and assayed for a proliferative response and their cell-free supernatants were assayed for the presence of a mediator with chemotactic activity for monocytes. Neither serotonin nor histamine were directly chemotactic for monocytes. When serotonin was cultured with PBL factors chemotactic for monocytes as well as polymorphonuclear leukocytes were produced in the absence of a proliferative response. Production of the factor chemotactic for monocytes was specifically blocked by methysergide, a serotonin antagonist and nonspecifically blocked by cycloheximide which interferes with leukocyte protein synthesis. Some dearboxylated precursors of serotonin also induced the monocyte chemotactic factor. This serotonin-induced chemotactic factor was demonstrated to be a product of mononuclear cells, and the majority of chemotactic activity was physiochemically similar to the previously characterized lymphocyte-derived chemotactic factor, suggesting that this factor is a lymphocyte product. In contrast to serotonin, histamine did not stimulate the production of monocyte chemotactic factor, nor did it induce proliferation. However, when 10(-5) M histamine was added to a substimulatory concentration of serotonin (10(-5) M), chemotactic activity comparable to or greater than the peak activity of serotonin alone was measured. This synergistic effect between serotonin and histamine suggests that the combination of these two amines may contribute to the induction of monocyte exudation in inflammatory reactions.

Important insights into leukocyte differentiation and the cellular origins of leukemia have been gained through the use of monoclonal antibodies that define cell surface antigens and molecular probes that identify immunoglobulin and T cell receptor genes. Results of these studies have been combined with markers such as surface membrane and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte receptors on T lymphocytes, and cytochemical stains. Using all of the above markers, it is now clear that acute lymphoblastic leukemia (ALL) is heterogeneous. Although monoclonal antibodies are useful in distinguishing acute myeloid from acute lymphoid leukemias, they have less certain usefulness in the subclassification of acute myelogenous leukemia (AML). Attempts to subclassify AML by differentiation-associated antigens rather than by the French-American-British (FAB) classification are underway in order to document the potential prognostic usefulness of surface markers.

Thirty-three patients were treated in an escalating single-dose trial of partially purified nonrecombinant interferon-gamma (IFN-gamma). The first seven patients received intramuscular injections of IFN-gamma in doses up to 20 X 10(6) units/m2. When it became clear that these patients had no detectable antiviral activity in their serum, subsequent patients were treated by the intravenous route of administration, generally with 2-h infusions. A total of 26 patients received the agent intravenously in single escalating doses ranging from 0.2 to 60 X 10(6) units/m2, on a twice-weekly schedule for 4-6 weeks. The most common toxicities encountered included fever, chills, fatigue, anorexia, and occasional nausea and vomiting. No myelosuppression or hepatic toxicity was observed. A maximum tolerated dose for single-dose intravenous administration was defined as 50 X 10(6) units/m2 on the basis of unacceptable fatigue and prolonged systolic hypotension. Antiviral activity was detected in the serum following doses greater than 2 X 10(6) units/m2 when the IFN-gamma was administered intravenously. No evidence of antitumor activity was seen in this Phase I trial, although the treatment regimen employed did not lead to high or prolonged levels of serum IFN activity in the majority of patients. An accurate assessment of the antitumor activity of this particular IFN-gamma preparation will require Phase II trials employing multiple-treatment regimens.

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.

The interferons represent an important first member of a family of biological response modifiers used in treating human malignancies. Activities associated with the interferons include inhibition of viral replication, influence on cellular protein production, direct antiproliferative effects, and a variety of modulatory effects on the immune response. These regulatory functions of interferon underlie the interest in its use as an anticancer agent. alpha-interferon is the most extensively studied interferon species. While antitumor activity has been seen both in vivo and in vitro in some solid malignancies, the most impressive responses have occurred in the hematologic malignancies. For patients with cutaneous T cell lymphoma (CTCL) (mycosis fungoides and the Sezary syndrome), a response rate of 45%, with 10% complete responses, was seen in patients treated with recombinant alpha-interferon at the National Cancer Institute (NCI). Interferon appears to be one of the most effective single agents for cutaneous T cell lymphoma. Current trials include further phase II trials to determine whether lower doses of alpha-interferon will be as effective as the high doses used in the previously reported studies. Additional trials include phase III trials where interferons are included in combination with chemotherapy agents, as well as using interferon in the adjuvant setting. Results for patients with chronic lymphocytic leukemia (CLL) were not as encouraging. A response rate of 15% was reported for crude and recombinant alpha-interferon in studies outside the NCI, and in our trial, only two of 18 patients (11%) had brief partial responses to recombinant alpha-interferon.

Immunologic approaches to the classification of acute lymphocytic leukemia (ALL) have led to a new awareness of the heterogeneity of this disease. Surface membrane markers including surface membrane immunoglobulin, complement receptors, and sheep erythrocyte (E) receptors have demonstrated at least three subtypes of ALL, which include non-B, non-T, ALL, T-ALL, and B-ALL. In addition, hetero-antisera to Ia-like antigens and ALL-associated antigens have been used to positively identify non-B, non-T ALL, which was previously a diagnosis of exclusion. This paper reports 17 cases of childhood ALL whose lymphoblasts were studied for surface membrane immunoglobulin, sheep erythrocyte receptors, and the presence of four antigens detected by well-characterized heteroantisera. Every non-B, non-T lymphoblast was positively identified by the Ia-like antiserum and/or the ALL antiserum. One T lymphoblast was identified by E receptors and the T antiserum, whereas two did not have E receptors but did react to the T antiserum. None of these three T lymphoblasts reacted with the Ia-like antiserum.

A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either 125I or 111In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

Chronic lymphocytic leukemia (CLL) is usually stable over months to years however a small proportion of cases may transform to more aggressive forms. These transformations include diffuse lymphoma (Richter syndrome), prolymphocytic transformation, acute lymphoblastic leukemia and multiple myeloma. There are a variety of techniques to determine whether the transformation represents a clonal evolution of the original CLL or an independent disease. These include cytogenetic analysis, immunoglobulin gene rearrangement by Southern blot analysis, and anti-idiotypic antibodies. While identity can be determined by gene rearrangement and anti-idiotypic antibodies, lack of identity can occur because of somatic mutation within a single clone. Thus, even with optimal techniques it is difficult to definitively exclude lack of identity in some instances. In most cases where transformation has taken place, definitive studies were not performed. In instances where detailed data are available, most do not suggest clonal evolution. Further studies are needed to allow definite conclusions.