[Development of molecular targeting drugs for the treatment of cancer-therapeutic potential and issues to be addressed in global development].

A survey of cancer treatment in a sample of hospitals > 100 beds conducted in 1998 compared with experience in the US showed that good progress has been achieved in Japan in the screening and early treatment of gastric cancer, and that the prognosis for breast cancer is better than in the West. Although in the past, the cytotoxic therapies available to physicians in Japan vs the West have been different, recent acceleration of regulatory review will result in a convergence of treatment paradigms and some improvement in acute response in many tumour types. However, world wide there is a need for new improved therapies in all cancers evaluated. Particular needs are in the management of NSCLC, advanced disease and cancers which form micrometastases. The eventual hope is that cancer can be turned from a lethal disease into a chronic disease where patients maintain a good QOL. Apart from anti hormonal therapies, the usual approach has been to kill the cancerous cells. However, the new approaches to intervening in the growth and migration of cancerous cells or the host tissue response by molecular targeting offer the promise of achieving a step change in therapy. Although EGF tyrosine Kinase inhibitors such as ZD 1839 have been shown to cause a conventional tumour response in NSCLC, many of these new approaches are unlikely to show a short term response even if they have the capacity to affect tumour development and increase disease free survival. Some compounds will require combination therapy with a conventional cytotoxic or radiotherapy to show their full benefit. For conventional cytotoxics, the usual approach to development has been to select the maximum tolerated dose and then evaluate the efficacy in advanced disease. However, for the new approaches which will not have such severe dose limiting toxicities, it will be necessary to select a surrogate marker of the intended biological effect to select the optimal biological dose (OBD) and dose regimen in phase I/II studies for further evaluation in phase II or III studies which are designed to show the expected patient benefit. The tumour target, the stage of the disease and the possible need for concomitant therapy will also have to be considered according to the mechanism of action of the product.

Detection of abnormal peripheral blood mononuclear cell cytokine networks in human IgA nephropathy.

Dysregulated cytokine expression has been implicated in the pathogenesis of IgA nephropathy, but the mechanisms and selectivity of this response are poorly understood. In this study we have examined the expression of a range of immunoregulatory cytokine mRNAs by peripheral blood mononuclear cells (PBMNCs) from 45 patients with IgA nephropathy stratified empirically according to urinary red cell excretion: 10 in remission, and 35 with active disease (21 mild, 14 moderate), and 17 normal, and 15 disease, controls. We used a semi-quantitative polymerase chain reaction (PCR) technique. None of the patients had experienced recent episodes of macroscopic hematuria. Simultaneous analysis of monocyte class II antigen (DR) expression was also performed by two-color immunoflow cytometry. TGF-beta 1 mRNA was detected in 68% (24 of 35) of patients with active, and 70% (7 of 10) inactive IgA nephropathy, but in only 18% (3 of 17) normal (P < 0.005), and 27% (4 of 15) disease controls. IL-6 transcripts were identified in 37% (13 of 35) of patients with active IgA nephropathy, compared with 6% (1 of 17) normal controls (P = 0.015), with no significant increase in IgA remission, or disease control groups. TNF-alpha mRNA was detected in 29% (5 of 17) of normal and 53% (8 of 15) disease controls, but in only 7% (3 of 35) of patients with IgA nephropathy (P = 0.015). There was no significant change in TGF-beta 2, gamma-IFN, IL-2, IL-4, IL-1 alpha or IL-1 beta detection between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of lymphocyte anti-tumour responses in man: effector heterogeneity and the search for immunomodulators.

Data continues to accumulate on the immunological reaction against solid human cancers. The evidence at the present time supports the view that rather than being immunologically invisible, tumour cell antigens are recognised by at least three lymphocyte subsets. Helper T cells can be induced to proliferate upon exposure to cells of the autologous tumour and to secrete detectable levels of interleukin 2 (IL-2). Cultured T cell lines and clones can be shown to respond in primed lymphocyte tests not only to autologous tumour cells but also to allogeneic tumour cells of the same histology and anatomic location. Cytotoxic T cells manifest specific reactivity against cells of the autologous tumour which is distinguishable from natural killing (NK) on the basis of specificity and organ distribution. Natural killer cells can lyse freshly isolated autologous tumour cells after purification on Percoll gradients or when activated by IL-2. There is thus a demonstrable heterogeneity of response to human cancer in unseparated lymphocyte populations and at the clonal level. In limiting dilution assays lymphocytes at the tumour site respond more frequently to autologous tumour relative to NK targets. For at least some tumours there is evidence that the expression of auto-tumour reactivity but not NK correlates with the clinical course of the disease and is a favourable prognostic indicator. The finding of these auto-tumour reactivities has important implications for the search for immunomodulating drugs for cancer treatment. However, it must be recognised that the response is heterogeneous and that the immune system comprises multiple interactive elements that exhibit both positive and negative control. Any treatment modality must take this into account and seek to focus on specific activation of the tumour lytic populations or the inhibition of negative regulatory elements as opposed to seeking a more general augmentation of immune reactivity which may, by stimulating suppressor cells, have a counterproductive effect.

T-cell control of IgA production. I. Distribution, activation conditions and culture of isotype-specific regulatory helper cells.

The capacity of T cells from different sites to augment IgA production by LPS-stimulated B cells has been investigated. Peyer's patch T lymphocytes activated with Con A induced up to a 20-fold increase in IgA production. The effect was isotype-specific, in that no consistent effect on IgG and a diminution of IgM synthesis were observed. Less activity was recorded in spleen and mesenteric lymph node T cells. Optimal activation of the Thy 1+ Lyt 1+2- helper cells required the addition of splenic adherent cells and the elimination of Thy 1+ Lyt 2+ suppressor cells prior to activation. T lymphocytes maintained regulatory activity for several months after expansion in medium supplemented with IL-2 and are now being cloned. We conclude that IgA production is under control of T cells sited preferentially, but not exclusively, in gut-associated lymphoid tissue, and that these T cells can augment IgA production by B lymphocytes from sites with low commitment to production of this isotype.

Human tumor-infiltrating lymphocytes: a marker of host response.

There is continuing interest in the possibility of immunologic intervention in the therapy of malignant disease. By employing a range of different techniques, it has been possible to show the presence of activated helper, suppressor, and cytotoxic T cells, B cells, NK precursors, and macrophages at the tumor site. The overwhelming impression from our data is that tumors may be subject to immunologic attack by heterogeneous effectors and that there is selective trapping of these effectors with corresponding depletion at the periphery. Like all inflammatory sites, however, the tumor contains both positive and negative regulatory mechanisms with the coexistence of cells with effector and suppressor functions, eg, T suppressors that modulate the proliferative response of T helpers and macrophages suppressing NK function contribute to the dynamic interplay in situ. Additional complexity is indicated by immunohistologic studies that clearly show that the stroma rather than foci of tumor cells are the site of infiltration, thereby further limiting effector function. We are now at the end of the descriptive stage of our investigations and further studies must approach the more difficult problem of modifying the host response in such a way as to alter the balance between effector and suppressor activity. A promising area of research would appear to be the use of cloned helper T cells or their products in the immunotherapy of cancer. The demonstration, by us, of selective trapping at tumor sites suggests that administration of the patients' own T cells with antitumor reactivity may serve as an efficient delivery vehicle to activate host effectors in situ. Studies in animal systems have shown the feasibility of this approach, although the failure of cultured T cells to undergo normal recirculation represents a considerable unresolved problem. Effector function by each of the tumor-infiltrating cell types described is under T cell control, and preliminary studies have already indicated the ability of helper T cells to accelerate allograft and tumor rejection. The increasing availability of gene-cloned materials with potent biologic activity opens new areas of research in cancer therapy. The lymphokines IL-2 and interferon are already undergoing clinical trials. Studies by Hersey demonstrate that administration of conditioned medium containing impure IL-2 results in the appearance of antitumor effectors in previously nonreactive melanoma patients, and Rosenberg, among others, has shown IL-2 to be a potent enhancer of alloimmune responses. Lymphokine-activating macrophages also augment antitumour responses.(ABSTRACT TRUNCATED AT 400 WORDS)

The human mixed lymphocyte-tumor cell interaction test. I. Positive autologous lymphocyte proliferative responses can be stimulated by tumor cells as well as by cells from normal tissues.

Co-culture of cancer patients' nonadherent peripheral blood lymphocytes with irradiated autologous fresh tumor cells, termed the mixed lymphocyte-tumor interaction (MLTI) test, resulted in significant stimulation of 3H-Tdr in corporation on day 6 in 19 of 37 autologous combinations. The MLTI test was performed in a microtiter wells (0.2 ml) and a variety of solid tumor cells (sarcomas and carcinomas) were used. Tumor cells were dissociated from the fresh biopsy tissue by nontrypsin enzymatic digestion (deoxyribonuclease, hyaluronidase, and collagenase) and the tumor cells enriched by depletion of macrophages using adherence procedures. Occasionally, further tumor cell purification was achieved by separation of cells on the basis of size on dis-continuous gradients. Positive MLTI resulted in stimulation as high as 20-fold over the backgrounds of PBL and tumor cells cultured alone. Mean positive MLTI was SI of 7.7. The negative MLTI were not a reflection of generalized immunosuppression, because tumor cell preparations that did not stimulate autologous PBL did stimulate allogeneic PBL. In an additional patient, PBL not responding in the autologous MLTI did respond to allogeneic tumors. MLTI using cryopreserved cells reproduced the MLTI results using fresh cells in 11 of 16 tests; the other five tests were all positive in the fresh MLTI and negative when using cryopreserved cells. Despite reports from many other groups it appears that positive MLTI were not tumor-specific. In 14 experiments we were able to simultaneously test the proliferative response to autologous tumor as well as to an autologous normal tissue (lung, liver, colon, and bowel). In eight of these experiments positive responses were obtained with tumor stimulators and in seven of these, positive proliferation was also obtained with normal tissue.

Effect of simple sugars on natural killing: evidence against the involvement of a lectin like mechanism in target recognition.

The spontaneous lysis of target cells sensitive to natural killer (NK) activity is accomplished in two distinct phases: (i) binding between target and effector cells and (ii) post-binding events leading to target cell destruction. To test the hypothesis that cell surface carbohydrate(s) might be involved in recognitive and/or lytic events, the binding and cytotoxicity of peripheral blood lymphocytes (PBL) towards NK sensitive K-562 targets was studied in the presence of simple sugars and after treatment of the targets with the antibiotic, tunicamycin. Lysis by peripheral blood lymphocytes was found to be inhibited by N-acetyl glucosamine, N-acetyl galactosamine and alpha-methyl mannoside in a dose-dependent manner under conditions where neither these sugars nor those (fucose, galactose) which had little effect on lysis inhibited the binding of effector cells to targets. Further, growth of K-562 in tunicamycin (which inhibits N-linked glycosylations occurring through the lipid intermediate pathway) with or without subsequent treatment with the enzyme neuraminidase, markedly reduced cell surface expression of sugars monitored by lectin binding. Treated cells showed no loss of NK susceptibility and were frequently more sensitive to lysis. Sugar inhibition profiles were the same as for untreated cells. These data suggest that carbohydrates are not the target sites of NK recognition but that simple sugars may have an inhibitory action at a later stage of the lytic process.

Limiting dilution analysis of the frequency of human T cells and large granular lymphocytes proliferating in response to interleukin 2. I. The effect of lectin on the proliferative frequency and cytotoxic activity of cultured lymphoid cells.

Human peripheral blood LGL that mediated NK and small T cells were isolated in high purity (98% by morphology) by density sedimentation on discontinuous Percoll gradients. The proliferative frequency of these subpopulations in the presence of lectin-free conditioned media containing IL 2 was determined by limiting dilution analysis. LGL showed a 20-fold greater frequency of proliferative cell precursors than small T cells (1/200 and 1/4970, respectively). The NK-like nature of cells expanded from LGL preparations in IL 2 was confirmed by parallel testing of the cytotoxicity against K562. Whereas T cell microcultures showed no lytic activity against K562 (cytotoxic precursor frequency less than 1/10,000), LGL cultures showed frequencies of cytotoxic precursors (1/170) comparable to those of proliferative precursors. Neither responder cell type gave rise to detectable lytic activity against NK-insusceptible mouse lymphoma RL male 1 or alloblasts. LGL proliferation was only minimally affected by the presence of PHA at the onset of culture (rise to 1/74 with 2 micrograms/ml PHA). By contrast, small T cells showed a dose-dependent increase of proliferative frequency, to reach 1/11 with 2 micrograms/ml PHA, provided accessory cells in the form of PBMC, monocytes, or LGL but not T cells were present. The cytotoxic activity of LGL and small T cells expanded in IL 2 was confirmed in bulk cultures. LGL-CLC showed high lytic activity against NK-susceptible cell lines and a majority of freshly isolated allogeneic human tumor targets. T cell-CLC showed little activity against cell line targets (K562, Raji, L1210, RL male 1) but were lytic for some fresh tumor cells. These data establish optimal conditions for the growth of human LGL in IL 2-dependent culture and suggest that a major contributor to lysis of allogeneic human tumors by CLC is likely to be NK cells. The data indicate that large numbers of activated T cells cannot be detected in vivo and that in vitro induction of IL 2 receptors by lectin/antigen is necessary for the establishment of antigen-reactive T cell lines. In contrast, a proportion of LGL appear to be spontaneously activated and susceptible to IL 2-dependent growth. Thus, in the absence of stimulation, culture of unfractionated lymphoid cells in the presence of IL 2 is likely to select for the growth of LGL with NK activity.

Limiting dilution analysis of the frequency of human T cells and large granular lymphocytes proliferating in response to interleukin 2. II. Regulatory role of interferon on proliferative and cytotoxic precursors.

Limiting dilution analysis has shown that large granular lymphocytes (LGL) and small T cells undergo proliferation in response to lectin-free IL 2. The latter is critically dependent on prior stimulation with lectin. Depending on conditions, alpha or beta interferons (IFN) were found to have either of two opposing effects on the frequency of proliferating cells. Pretreatment of responder cells with IFN resulted in dose-dependent augmentation of proliferative progenitors such that at 500 IU/ml, a fivefold increase of progenitor frequency was apparent. Under such conditions, approximately 5% of LGL could be expanded, and at least a proportion of the cultured cells killed the NK-sensitive K562 cell line. Similar results were apparent with T cell responders: whatever the initial frequency of proliferation (dependent on the PHA dose), augmentation was obtained with IFN pretreatment, although no killing of K562 was induced. In contrast, when IFN was present throughout the 7-day assay period, proliferative frequencies were reduced. This inhibitory effect was mediated through the presence of irradiated T cells in the feeder populations. With purified monocytes as feeder cells, little reduction was seen, whereas addition of T cells resulted in a 19-fold inhibition. Separation of OKT4+ and OKT8+ subsets demonstrated that both were essential for optimum induction of suppression. These data indicate that IFN has a powerful immunodulatory role on both NK and T cell proliferation, and that it may function both by induction of IL 2 receptors and activation of suppressive T cells. The interaction between different soluble mediators provides a complex immunoregulatory circuit in vitro.

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture. Clonal analysis of effector cells in cytotoxic and proliferative assays.

Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of 3H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members. MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols.

Modulation of IL 2-dependent growth of mouse NK cells by interferon and T lymphocytes.

The regulatory role of interferon (IFN) on the growth of mouse natural killer (NK) cells in the presence of interleukin 2 (IL 2) was analyzed by the limiting dilution assay. Pretreatment for 5 hr with IFN (600 U/ml) was able to augment the frequency of proliferating cells and NK effector cells when spleen cells of BALB/c nu/+ and BALB/c nu/nu were cultured for 7 days in the presence of IL 2. When IFN was present during the 7-day culture period, we again found an increase in proliferative and cytotoxic frequencies in cultures of spleen cells from nude mice, but in contrast, found a decrease in these frequencies in cultures of spleen cells from euthymic mice. Addition of irradiated (3000 R) spleen or thymus feeder cells from euthymic mice to the nu/nu cultures caused an inhibitory activity of IFN also on nu/nu cells. These data indicate that IFN can have both positive and negative regulatory effects on the in vitro growth and differentiation of mouse NK cells and that the inhibitory effects are mediated via T lymphocytes.

Flow cytometric analysis of lectin binding to human peripheral blood lymphocytes.

The binding of fluorescein-conjugated lentil lectin and concanavalin A to the surface membrane of human peripheral blood lymphocytes was studied by flow cytometry. The lymphocytes bound 3-fold more lentil lectin molecules compared with concanavalin A molecules and lentil lectin binding approached saturation at a much lower concentration than did that of concanavalin A. Lentil lectin identified two groups of lymphocytes: a low-binding T-cell fraction and a high-binding B-cell-enriched fraction. Concanavalin A did not discriminate between these populations in unseparated lymphocytes. Competition studies indicated that lentil lectin and concanavalin A were bound to different sites on the lymphocyte surface, although about 50% of lentil lectin sites were in close proximity to concanavalin A sites.

Quantitation of proliferative and cytotoxic precursor cells directed against human tumours: limiting dilution analysis in peripheral blood and at the tumour site.

Blood and tumour-infiltrating lymphocytes (TIL) from 16 cancer patients have been examined under limiting dilution conditions to determine the frequency of cells responding in mixed tumour-lymphocyte cultures (MLTC) to autologous tumour and Interleukin-2 (IL-2). Tumour-derived lymphocytes showed a high spontaneous response to IL-2 alone 1/1,900 in TIL; 1/6,000 in PBL suggesting the presence of "activated" T cells in situ. Proliferative frequencies were increased in MLTC in both blood (1/3,779) and TIL (1/1,084). Phenotypic analyses showed that total T-cell contents of the responder populations were comparable but TIL were enriched for the OKT8+ subset with a corresponding reduction in OKT4+. TIL showed increased numbers of OKMI+ and Tac+ lymphocytes. The major cytotoxic precursor expanding under these conditions was reactive against autologous tumour. K562 (NK) were present at a lesser frequency--particularly in TIL. The data show a concentration and activation of reactive lymphocytes at the tumour site and establish conditions for the clonal expansion of specifically cytotoxic T cells.

Lectin-binding characteristics of human natural killer cells.

Human natural killer (NK) cells separated initially by density centrifugation of lymphocytes (E+) forming rosettes with sheep red blood cells (SRBC), were further fractionated on gradients of bovine serum albumin (BSA). Low density fractions contained effector cells which displayed high cytotoxicity against the NK-sensitive erythroleukaemic cell line, K562. These low density cells, which expressed receptors for Fc and the monoclonal antibody OKMI, showed enhanced cytotoxicity when treated with lymphoblastoid interferon (IFN-alpha). They also showed an increased response to phytomitogen in comparison with unseparated cells or those recovered from high density fractions. Two lymphocyte subsets one of high and one of low lectin binding capacity were identified in the E+ populations by their reactivity with Lens culinaris agglutinin (LCA). High LCA binding was observed only in low density fractions and was associated with a marked enrichment of NK activity. This property was used to separate the NK active population in E+ cells by fluorescence-activated cell sorting (FACS). These data add a new dimension to the cell surface properties of human NK cells and suggest the presence of LCA-reactive glycoproteins which are either enriched in, or uniquely associated with, cells of the NK subset. The experiments indicate that lectins can serve as useful probes of lymphocyte function and provide the basis for effective cell sorting.

Role of autologous lymphocyte cytotoxicity in colonic neoplasia.

The T-lymphocyte mediated killing of autologous carcinoma colon cells was investigated. There was no change in the incidence of activity with advanced disease, age, or nutritional status of the patient and no difference could be demonstrated in lymphocytes extracted from blood, draining lymph nodes, or the tumour itself. Nevertheless. T-lymphocyte activity did appear to be specific for the patients's own tumour, as it was rarely observed with allogeneic tumours. There was also no correlation with lymphocyte natural killer activity. The in vitro studies demonstrated patient specific T-lymphocyte activity in 23 of 47 patients with carcinoma of the colon, but the results do not correlate with clinical and pathological findings.

Specific cytotoxicity against autologous tumour and proliferative responses of human lymphocytes grown in interleukin 2.

Peripheral blood lymphocytes of cancer patients were sensitized in vitro to autologous tumour cells in mixed lymphocyte-tumour culture (MLTC). Blast cells were isolated on discontinuous Percoll gradients from MLTC which showed significant stimulation of [3H]-thymidine incorporation. Cultured T cells (CTC) were derived from these blasts by growth in conditioned medium containing interleukin-2 (IL-2) and maintained for up to 51 days by repeated feeding with IL-2 and in some cases by addition of irradiated allogeneic blood mononuclear cells as "fillers". These cultures showed specific cytotoxic reactivity against autologous tumour and in only a few cases was natural killing (NK) of K562 cells apparent. Restimulation of CTC with tumour was measured in primed lymphocyte test (PLT). Increased uptake of [3H]-thymidine was found upon stimulation by autologous tumour and allogeneic tumour of the same site and histology but there was no response to non-related tumours or to a panel of allogeneic lymphocytes. No sensitization to autologous HLA-D/DR could be detected by restimulation or cytotoxicity against monocytes in the majority of cases. These data suggest that, by careful selection of sensitised blasts from MLTC, it is possible to obtain CTC with both helper (proliferative) and cytotoxic T cells and that such CTC have specific reactivity against tumour cells. These cellular reagents will be useful in defining the antigenicity of human neoplasms and possibly in therapy.