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Twenty patients with metastatic malignant melanoma were studied with 99mTc-labeled monoclonal antibody (MoAb) Fab fragment (NR-Ml-05) reactive with a high molecular weight (Mr 240,000) melanoma associated antigen. Patients received 40 mg unlabeled irrelevant MoAb (NR-2AD-IgG) and 7.5 mg unlabeled NR-Ml-05 (whole IgG) prior to infusion of 10 mg 99mTc-labeled (10-25 mCi) NR-Ml-05 Fab. Unlabeled MoAb were given to block nonspecific and specific binding sites. Gamma camera scans and single photon emission computed tomography were performed at 8 and 24 h postadministration. Of 172 preexisting lesions, 136 were imaged for a sensitivity of detection of 79%. Imaging was site and size dependent with the greatest sensitivity for liver lesions (100%) and the least for bowel (0%). Six sites (2 skin, 1 lung, 3 liver) were detected by single photon emission computed tomography that were missed on routine planar images. Forty-one additional unconfirmed sites were seen. Of these, 7 (17%) have been confirmed as tumor after a median follow-up time of 6 months. False positive scans included scar tissue, areas of chronic inflammation, an infected femoral aneurysm, and septic emboli. Nonspecific uptake of radioactivity occurred in kidney, gallbladder, bowel, thyroid, and myocardium. Human anti-mouse antibodies were detected in up to 69% of patients. In summary, radioimaging with 99mTc-NR-Ml-05 is a sensitive test, especially for detecting liver lesions. It is safe, simple to administer, and convenient for the patient. Biodistribution and imaging sensitivity differ significantly from studies in which 111In-labeled anti-melanoma MoAb have been used.

We have previously reported that the addition of monocytes results in enhanced modulation of the T65 antigen when normal or leukemic lymphoid cells were cultured in vitro with the T101 monoclonal antibody. In the present investigation, we extend these findings to demonstrate that monocyte-enhanced modulation is a phenomenon that occurs with a variety of T and B lymphoid antigens identified by murine monoclonal antibodies. Two patterns of monocyte-enhanced modulation were observed: (1) augmentation by monocytes of existing antigen modulation by the T101 and anti-Leu-4 antibodies, and (2) induction by monocytes of previously unrecognized modulation with the anti-Leu-2 and anti-Leu-9 antibodies. Enhancement of modulation by monocytes was also detected with antibodies to surface IgM and HLA-DR antigens. Antigen modulation on lymphoid cell lines appeared to be more variable than on fresh cells, with or without monocytes. Monocyte-enhanced antigen modulation was not demonstrated with two monoclonal antibodies against solid tumors. Monocyte-enhanced modulation was shown to be dependent upon the Fc portion of the antibody, but independent of proteolytic or oxidative compounds released by monocytes. These findings indicate that the results obtained during in vitro studies of antigen modulation may vary with the source of cells and the extent to which monocytic cells are present. In addition, these findings suggest an enhanced role for Fc receptor-bearing cells of monocytic origin in antigen modulation following in vivo administration of monoclonal antibodies.

Antigenic modulation of the T65 cell surface antigen was assessed in chronic lymphocytic leukemia patients (CLL) receiving therapy with the murine monoclonal antibody T101 in a phase I clinical trial. A total of 12 patients received 1, 10, or 40 mg doses administered over 2 hr, or 50 or 100 mg doses administered over 50 hr. Decreases in T65 antigen expression (up to 90%) coincided with decreases in the circulating leukemic cell count in those patients who received T101 over a 2-hr period, indicating that clearance of circulating T65 antigen-positive cells could account for most of the observed decreases in T65 antigen expression. In contrast, analysis of bone marrow specimens from these patients indicated that decreases in T65 antigen density in this relatively stationary population resulted not from a cell decrement but rather from antigenic modulation. Pulmonary toxicity prevented administration of doses greater than 40 mg over a 2-hr period; therefore, higher doses (50 and 100 mg) were administered over a 50-hr period. This treatment schedule resulted in greater than 90% reduction in T65 antigen density of both circulating and bone marrow leukemic cells without dramatic drops in circulating leukemic cell counts, indicating that antigenic modulation accounted for most of the observed decreases in T65 antigen density under these conditions. Reexpression of T65 antigen by modulated cells was observed both in vitro and in vivo within 2 to 4 days. Immunoperoxidase staining of in vivo-modulated specimens and in vitro modulation studies with 125I-T101 suggested that T65 antigen-T101 antibody complexes were internalized during modulation. Although antigenic modulation inhibits the potential therapeutic effectiveness of unconjugated T101 antibody in CLL patients, treatment of CLL with T101 drug or toxin immunoconjugates under conditions that bring about rapid and extensive internalization of the T65 antigen may provide an effective means of therapy.

A panel of monoclonal antibodies reactive with normal lymphocyte subsets was used to classify cases of lymphocytic leukemia on the basis of cell surface antigen expression. The antibodies employed were commercially available and included a common framework HLA-DR antibody, two pan-T antibodies (Leu-1 and OKT-3), and antibodies defining cytotoxic/suppressor (Leu-2 and OKT-8) and helper/inducer (Leu-3 and OKT-4) subpopulations of normal T lymphocytes. Cases of ALL could be subgrouped into non-T non-B, pre-T and T-ALL on the basis of reactivity with HLA-DR, Leu-1, and OKT-3 antibodies. Leukemic cells from patients with T-cell CLL could be divided into Leu-2/OKT-8 reactive and Leu- 3/OKT-4 reactive subpopulations, as well as a subgroup in which the majority of cells were unreactive with either of these antibodies. With the exception of one individual, all Sezary cell leukemias expressed a phenotypic pattern similar to that of the Leu-3 subgroup of T-CLL. Malignancies of B-cell lineage (B-CLL, prolymphocytic leukemia, and lymphosarcoma) that were examined were reactive with both the HLA-DR and Leu-1 antibodies. On the contrary, normal B lymphocytes and lymphoid cell lines of B-cell origin did not express surface antigens recognized by the Leu-1 antibody.

Immune reconstitution was studied serially by using T lymphocyte cell surface differentiation antigens in 37 individuals receiving bone marrow transplants. Antigen expression was assessed by immunofluorescence analysis using monoclonal antibodies to T lymphocytes including Leu-3, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation with suppressor or cytotoxic activity (L2+). These studies demonstrated that the L2+ subpopulation regenerated more rapidly after transplant than did the L3+ subpopulation. Imbalances between these two T lymphocyte subpopulations, indicated by a decreased L3/L2 ratio, persisted for periods up to 12 mo post-transplant. Expression of a cell surface antigen associated with immature lymphocytes (OKT-10), and of HLA-DR (Ia-like) antigens was markedly increased during the post-transplant period. HLA-DR antigen expression did not appear related to immune activation in that increased reactivity was not detected with a monoclonal antibody (anti-TAC) specific for activated T cells. These observations in bone marrow transplant recipients and other disorders characterized by lymphoid restoration or immaturity indicate that inversion of the normal L3/L2 ratio and increased expression of OKT-10 and HLA-DR antigens may be features of a regenerating immune system. Furthermore, serial observation of individual patients indicated that infection with cytomegalovirus was associated with a progressive decrease in the L3/L2 ratio.

Heterogeneity of leukemic cells was defined in a case of lymphoma. Four phenotypically distinct subpopulations of leukemic cells were identified. One subpopulation was observed to simultaneously express B- and T-cell characteristics. B-cell characteristics included monoclonal IgM (lambda) surface immunoglobulin, HLA-DR antigens, and expression of the B-cell antigen identified by the BA-1 monoclonal antibody. T-cell characteristics included E-rosette formation, expression of the pan-T- associated antigens recognized by the Leu-1 and OKT-11 monoclonal antibodies, and expression of the suppressor cytotoxic T-cell- associated antigen recognized by the Leu-2 and OKT-8 monoclonal antibodies. In addition to this subpopulation, three other phenotypically distinct subpopulations were identified, two of which expressed monoclonal IgM (lambda) surface immunoglobulin. The results of this investigation indicates that three phenotypically distinct lymphoid subpopulations bearing B-cell characteristics, and probably a fourth T-cell subgroup, were derived from a common lineage. These findings suggest that the malignancy involved a lymphoid progenitor cell that may possess diverse maturational capacity.

A monoclonal antibody designated 'antibody 390' (Ab 390) with anti-human Thy-1 reactivity was prepared by the hybridoma technique from the splenocytes of BALB/c mice immunized with human fetal brain. This antibody was shown to have anti-human Thy-1 reactivity because (1) it precipitated a molecule with a molecular weight of about 24,000 daltons, (2) it had a pattern of reactivity similar to that of previously described anti-human Thy-1 antibodies and (3) purified human Thy-1 antigen specifically inhibited binding of Ab 390 to a known antigen-positive cell line. It was the intent of this study to investigate the distribution of Thy-1 on normal and malignant haematopoietic cells in humans and non-human primates. We show here that Ab 390 did not react with human peripheral blood leucocytes, bone marrow cells or splenocytes by immunofluorescence but did react with subcapsular and cortical fetal thymocytes by peroxidase-antiperoxidase immunohistology. A section of fetal spleen demonstrated staining of connective tissue and blood vessels and rare reactive lymphocytes. Adult spleen contained Thy-1-positive cells surrounding the white pulp and in the marginal zone, but single-cell suspensions of splenocytes did not react with Ab 390. Ab 390 was tested against a variety of fresh human leukaemia cells and human cell lines and was shown to react with only the acute lymphoblastic leukaemia T cell lines RPMI 8402 and HPB-MLT. Non-human primate studies revealed reactivity with a number of T cell lines from New World primates (cotton-topped and red-bellied marmosets) and peripheral blood granulocytes (owl monkey). Our studies support previous findings that suggest that human Thy-1 may be a marker for early T lymphocytes in man, and its distribution on non-human primate T cell lines suggests the same for certain species of non-human primates. Not consistent with the distribution on human cells was the demonstration of Ab 390 reactivity with owl monkey granulocytes.

We developed murine anti-idiotype monoclonal antibodies for each of four patients with B cell-derived leukemias and lymphomas. Idiotypic immunoglobulin was isolated from mouse X human tumor-cell hybridomas or from patients' serum and was used to immunize mice for the development of murine anti-idiotype monoclonal antibodies. Each patient's anti-idiotype antibodies demonstrated reactivity restricted to the immunizing immunoglobulin, thereby limiting their therapeutic utility to a single individual. In addition, we isolated isotype switch variants of hybridomas producing monoclonal anti-idiotypic antibody. The restricted specificity of these antibodies was found to be of value for the analysis of the extent of malignant B cell infiltration in a variety of tissues from several patients. Large populations of idiotype-bearing cells were detectable in biopsy specimens from patients K.T. and L.H. In contrast, although bone marrow specimens from patient G.D. were apparently devoid of morphologically abnormal cells, a small, highly fluorescent population of cells was demonstrable underscoring the potential utility of these antibodies for posttreatment evaluation as well as for therapy. In a fourth patient, H.M., anti-idiotype antibodies developed against the circulating macroglobulin isolated from his plasma failed to react with either his circulating or bone marrow hairy cell leukemia cells. However, examination of an enlarged inguinal lymph node revealed the presence of a large number of idiotype-bearing cells. Thus, the presence of two distinct malignant B cell clones were discovered in this individual through the use of anti-idiotype monoclonal antibodies. Anti-idiotype antibodies, therefore, represent a highly specific tool for the evaluation and potential therapy of B cell malignancies in individual patients.

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.

Five bone marrow transplant recipients with cytomegalovirus infections were treated with pure recombinant leukocyte A interferon produced by recombinant DNA technology from Escherichia coli. All five patients had documented interstitial pneumonia. The daily intramuscular dose of interferon ranged from 18 X 10(6) to 50 X 10(6) U; the mean duration of therapy was 11.0 days (range, 5 to 18 days). Two patients recovered, one improved, and two died. Clinical side effects (usually fever and chills) occurred in three patients. A 60% or greater reduction in the pretherapy peripheral granulocyte counts occurred in four patients, and four patients had a 37 to 80% reduction in their pretherapy platelet counts. Hematological toxicity was reversible, and there was no loss of marrow graft function. The toxicity of pure recombinant leukocyte A interferon in marrow transplants is similar to that of partially pure leukocyte interferon derived from human cells. Further controlled studies to establish the efficacy of recombinant leukocyte A interferon in marrow transplants are warranted but may be limited by hematological toxicity.

Antigenic modulation of the T65 cell surface antigen was assessed in chronic lymphocytic leukemia patients (CLL) receiving therapy with the murine monoclonal antibody T101 in a phase I clinical trial. A total of 12 patients received 1, 10, or 40 mg doses administered over 2 hr, or 50 or 100 mg doses administered over 50 hr. Decreases in T65 antigen expression (up to 90%) coincided with decreases in the circulating leukemic cell count in those patients who received T101 over a 2-hr period, indicating that clearance of circulating T65 antigen-positive cells could account for most of the observed decreases in T65 antigen expression. In contrast, analysis of bone marrow specimens from these patients indicated that decreases in T65 antigen density in this relatively stationary population resulted not from a cell decrement but rather from antigenic modulation. Pulmonary toxicity prevented administration of doses greater than 40 mg over a 2-hr period; therefore, higher doses (50 and 100 mg) were administered over a 50-hr period. This treatment schedule resulted in greater than 90% reduction in T65 antigen density of both circulating and bone marrow leukemic cells without dramatic drops in circulating leukemic cell counts, indicating that antigenic modulation accounted for most of the observed decreases in T65 antigen density under these conditions. Reexpression of T65 antigen by modulated cells was observed both in vitro and in vivo within 2 to 4 days. Immunoperoxidase staining of in vivo-modulated specimens and in vitro modulation studies with 125I-T101 suggested that T65 antigen-T101 antibody complexes were internalized during modulation. Although antigenic modulation inhibits the potential therapeutic effectiveness of unconjugated T101 antibody in CLL patients, treatment of CLL with T101 drug or toxin immunoconjugates under conditions that bring about rapid and extensive internalization of the T65 antigen may provide an effective means of therapy.

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.

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.

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.

The murine antimelanoma monoclonal antibody, 9.2.27, was administered intravenously to eight patients with metastatic malignant melanoma. Biopsies of metastatic nodules clearly demonstrate the selective localization of this antibody on the melanoma cell surface with a dose-response relationship to the quantity of administered antibody. The antibody infusions were clinically well tolerated and the pharmacokinetics of the antibody and the antiglobulin responses are described. This study indicates that murine monoclonal antibodies have potential as selective targeting agents in the design of future therapeutic trials using monoclonal antibodies or conjugates thereof in the treatment of cancer.

The relationship between dermal delayed hypersensitivity (DH) and granulomatous hypersensitivity was studied in rabbits sensitized with killed mycobacteria. Specific antigen challenge of sensitized animals resulted in extensive pulmonary granulomatous inflammation and induced suppression of both dermal DH and dermal granuloma formation. Whereas suppression of DH was concomitant with pulmonary granuloma formation, as is the case in a number of granulomatous diseases, a causal relationship between the two did not exist. Both DH and dermal granulomatous hypersensitivity were significantly suppressed whether or not the antigen challenge was of a granulomagenic (particulate) or nongranulomagenic (soluble) form. The data presented indicate that granulomatous hypersensitivity and DH are selectively suppressed with regard to different anatomical sites.

Immune reconstitution was studied serially by using T lymphocyte cell surface differentiation antigens in 37 individuals receiving bone marrow transplants. Antigen expression was assessed by immunofluorescence analysis using monoclonal antibodies to T lymphocytes including Leu-3, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation with suppressor or cytotoxic activity (L2+). These studies demonstrated that the L2+ subpopulation regenerated more rapidly after transplant than did the L3+ subpopulation. Imbalances between these two T lymphocyte subpopulations, indicated by a decreased L3/L2 ratio, persisted for periods up to 12 mo post-transplant. Expression of a cell surface antigen associated with immature lymphocytes (OKT-10), and of HLA-DR (Ia-like) antigens was markedly increased during the post-transplant period. HLA-DR antigen expression did not appear related to immune activation in that increased reactivity was not detected with a monoclonal antibody (anti-TAC) specific for activated T cells. These observations in bone marrow transplant recipients and other disorders characterized by lymphoid restoration or immaturity indicate that inversion of the normal L3/L2 ratio and increased expression of OKT-10 and HLA-DR antigens may be features of a regenerating immune system. Furthermore, serial observation of individual patients indicated that infection with cytomegalovirus was associated with a progressive decrease in the L3/L2 ratio.

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.

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.

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.

A murine monoclonal antibody to a human melanoma-associated antigen has been evaluated at the molecular, cellular, preclinical, and clinical levels. Our systematic assessment of antibody 9.2.27 and its conjugates ricin A, abrin A, and gelonin has indicated that the antibody forms highly potent and specific immunotoxins which can inhibit growth of established human melanoma in nude mice. Clinical evaluation has indicated that unconjugated 9.2.27 can be given in cumulative doses up to one gram, with dose-dependent localization in cutaneous lesions eliciting only weak, antiglobulin responses. Both preclinical and clinical evaluation of 9.2.27 is directed to optimizing immunoconjugate trials in the near future.

F(ab')2 and Fab fragments of murine monoclonal antibody 9.2.27, that recognizes the 250 kD melanoma-associated antigen, were labeled with 99mTc using the bifunctional chelate method of Fritzberg et al. Twenty-seven (27) patients received, intravenously, 10 mg of either F(ab')2 (8), or the Fab (27), labeled with up to 30 mCi of 99mTc. These doses were preceded by an infusion of cold irrelevant antibody. The average serum T1/2 of the F(ab')2 and the Fab were 11 hr and 2 hr, respectively. Twenty-two percent (22%) of the total injected F(ab')2 dose was excreted in the urine in 20 hr, compared to 55% for the Fab group. Imaging was optimal 6-9 hr postinjection for the Fab patients. No nonspecific uptake in liver, spleen, bone marrow, or lung was observed for either antibody form. Overall, (43/53) 81% of known metastases were seen with visualization of tumors as small as 250 mg and tumor localization as high as 0.03% injected dose/g. Immunoperoxidase staining of freshly-frozen tumor nodules removed 24 hr postinjection confirmed antibody deposition in the tumor. Thirty-six previously unknown ("occult") metastatic sites were detected. To date, 12/36 of these sites have been confirmed. We conclude that 99mTc-labeled antibody to melanoma produces high resolution images with a high sensitivity of detecting metastatic melanoma. The detection of previously unknown sites of disease has proven helpful in directing additional diagnostic studies (i.e., CT) as well as planning of therapeutic options.

We developed murine anti-idiotype monoclonal antibodies for each of four patients with B cell-derived leukemias and lymphomas. Idiotypic immunoglobulin was isolated from mouse X human tumor-cell hybridomas or from patients' serum and was used to immunize mice for the development of murine anti-idiotype monoclonal antibodies. Each patient's anti-idiotype antibodies demonstrated reactivity restricted to the immunizing immunoglobulin, thereby limiting their therapeutic utility to a single individual. In addition, we isolated isotype switch variants of hybridomas producing monoclonal anti-idiotypic antibody. The restricted specificity of these antibodies was found to be of value for the analysis of the extent of malignant B cell infiltration in a variety of tissues from several patients. Large populations of idiotype-bearing cells were detectable in biopsy specimens from patients K.T. and L.H. In contrast, although bone marrow specimens from patient G.D. were apparently devoid of morphologically abnormal cells, a small, highly fluorescent population of cells was demonstrable underscoring the potential utility of these antibodies for posttreatment evaluation as well as for therapy. In a fourth patient, H.M., anti-idiotype antibodies developed against the circulating macroglobulin isolated from his plasma failed to react with either his circulating or bone marrow hairy cell leukemia cells. However, examination of an enlarged inguinal lymph node revealed the presence of a large number of idiotype-bearing cells. Thus, the presence of two distinct malignant B cell clones were discovered in this individual through the use of anti-idiotype monoclonal antibodies. Anti-idiotype antibodies, therefore, represent a highly specific tool for the evaluation and potential therapy of B cell malignancies in individual patients.

Extensive immunologic studies were done in 97 patients with severe aplastic anemia between 1973 and 1979. Sixteen young male patients with hepatitis-associated aplastic anemia appeared to constitute a unique subset. Compared with most patients with aplastic anemia from other causes, these 16 patients had significant reductions in the mean values of circulating T lymphocytes, serum IgG and IgM, mitogen reactivity, and decreased cutaneous hypersensitivity. The ratio of peripheral blood helper to suppressor T lymphocytes identified by monoclonal antibodies was within normal limits in 3 patients studied with hepatitis-associated aplastic anemia. Interestingly, the ratio was low (less than 1) in 3 of 7 patients studied with aplastic anemia from other causes, although the mean for these 7 patients was normal. These data suggest that patients in this subset with hepatitis-associated severe aplastic anemia have a severe immunodeficiency. Whether this immunodeficiency is the cause or result of the hepatitis or aplastic anemia, or both, is unknown.

The dermal response to purified protein derivative (PPD) in rabbits sensitized with Mycobacterium bovis strain Bacille Calmette Guèrin (BCG) is composed of at least two distinct phases. The early phase occurs within 24 h after injection of tuberculin and is characterized by accumulation of polymorphonuclear leukocytes. The second phase is delayed in onset, depending on mononuclear cell accumulation, and constitutes the classic delayed hypersensitivity reaction. The early component of the reaction of PPD is not immunologically specific, in that it can be elicited in either BCG-sensitized or nonsensitized rabbits and appears to be induced by pyrogenic components present in tuberculin preparations. Rabbit fever index assays indicated the presence of sufficient pyrogen in tuberculin PPD to account for the observed reactions; however, Limulus amebocyte pyrogen assays were negative. These findings suggest that tuberculin contains pyrogenic components other than endotoxin. Radiometric assays for mononuclear cell accumulation as well as desensitization experiments indicated that the early phase was independent of the delayed mononuclear phase.

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.