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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
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  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56972—White blood cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• This invention relates to methods for determining precursors of cytolytic T cells specific for antigens characteristic of or specific to tumors. More particularly, it relates to a non-obvious application and modification of the "limiting dilution" assay to determine the precursors discussed above.
• the applications include the ability to monitor therapeutic regimes and subject response, to particular treatments, including immune responses.
• T-cell response Where a T-cell response is involved, of course, measurement and study of T cells is of concern.
• T cells constitute a mixture of various types of lymphocytes, including cytolytic T cells, or "CTLs" as these will be referred to hereafter.
• CTLs interact with a molecule presented by their target cell via the T cell receptor. Following the interaction, the CTL causes the target cell to lyse.
• the actual target recognized by the CTL is frequently referred to as an antigen, and this term will be used hereafter.
• tumor rejection antigens the antigens expressed by these cells, now collectively referred to as "tumor rejection antigens", exist on murine tumors induced by viruses. chemicals, and ultra-violet radiation, as well as on spontaneous tumors. It has also been shown that, in some tvunor systems, these tumor rejection antigens induce cytolytic T cell responses and highly specific CTLs, directed against murine tvunor rejection antigens have been isolated. See Brunner et al., J. Immunol.
• the target antigen of these CTLs is relevant for recognition by a syngeneic host, since tumor cells which escape partial immune rejection in vivo have been found to have masked or to have lost the antigen CTLs recognize. See Uyttenhove et al., J. Exp. Med. 157: 1040-1052 (1983). Adoptive transfer with cloned CTLs can eradicate tvunor cells in animals bearing large tumors. See Kast et al., Cell 59: 603-614 (1989).
• the murine tumor P815 encodes a tumor rejection antigen, the gene for which has been identified and isolated, as per Van de Eynde et al., J. Exp. Med. 173: 1373-1384 (1991). It was found that this gene is identical to one that can be expressed by normal mouse cells, but has little or no expression in normal adult mouse tissues.
• MLTCs mixed lymphocyte tumor cell cultures
• MTLCs derived from PBMC generally contain responder cells which exert lysis on both tvunor cells and on NK targets, when analyzed after two weeks of culture. After an additional two or three weeks, lytic activity on tumor cells increases, and that on NK targets disappears. See Herin et al., supra. It has been possible to derive CTLs from MLTC responders which seem to be completely specific for tumors.
• the antigens recognized on tumor cells by these CTLs do not appear to be cultural artifacts, as they have been found to be present on fresh metastatic tumor tissue cells as well. See Mukherji, supra. Herin, supra; Knuth, supra. Panels of autologous CTLs permitted identification of four different stable antigens on human melanoma. See Van den Eynde et al., Int. J. Cancer 44: 634-640 (1989). Analysis of 70 additional CTLs from the patient on which the antigens were identified shows that all major stable antigens recognized by CTLs were in this set of four.
• CTL-Ps CTL precursors
• Figure 1 is a depiction of lytic patterns and criteria for their classification.
• Figure 2 presents levels of tumor cell lysis via limiting dilution microcultures and Poisson analysis of data.
• Figure 3 shows tumor cell lysis via limiting dilution, when competing K562 is added.
• Figure 4 shows results obtained for frequencies in anti-tumor lytic effectors, in presence and absence of K562.
• Figure 5 presents frequencies of lytic effectors among PBMCs
• CD4 + and CD8 + cells CD4 + and CD8 + cells.
• Human serum refers to pooled types A, B and O serum obtained from healthy donors.
• the HS had been decomplemented by treatment at 56°C for 30 minutes, partially delipified via centrifugation (45 minutes, 17,000g) filtration and sterilization.
• Fetal bovine serum is abbreviated as "FBS”.
• Interleukin-2 and Interleukin-4 are abbreviated as IL-2 and IL-4, respectively.
• IL-2 and IL-4 are abbreviated as IL-2 and IL-4, respectively.
• concentrations used were 30 U/ml (IL-2), and 5 U/ml (IL-4) in all experiments.
• IL-2 Interleukin-2
• IL-4 Interleukin-4
• IL-2 is defined as the concentration supporting half of maximal proliferation of CTLL-2 cell line.
• One unit/ml of IL- 4 is that concentration which yields 50% maximal proliferation of human T cells previously treated with phytohemagglutinin- A (“PHA").
• cytokine "gamma interferon" is abbreviated as "IFN- ⁇ ”.
• PBMCs Peripheral blood mononuclear cells
• the patient samples were subjected to density gradient centrifugation, and were cryopreserved prior to use in the limited dilution assays described infra.
• flow cytometric sorting was used, employing labeled anti-Leu 3 antibody (fluorescein), and anti-Leu 2 antibody (phycoerythrin).
• fluorescein labeled anti-Leu 3 antibody
• anti-Leu 2 antibody phycoerythrin
• Limiting dilution assays were set up using various quantities of either PBMCs or the subsets described supra. The numbers ranged from about 200 to 10,000.
• PBMCs/subset The particular amount, i.e., the defined number of PBMCs/subset chosen were seeded in 96 V-bottom microwells, together with 10 4 irradiated autologous tumor cells (i.e., tumor cells taken from the same patient as the PBMC source).
• the tumor cells had previously been used to establish cell lines, and the irradiated cells were taken therefrom. Irradiation was carried out using a Cs source (10000 rads).
• Irradiated tumor cells served as stimulator cells.
• the medium, as defined supra used to culture the mixture was supplemented with 10% HS and IL-4 as described supra.
• the wells were centrifuged for three minutes at 100g and incubated at 37°C in 8% CO 2 . On the third day of incubation, IL-2 as described supra was added.
• the rate of proliferation of the CTL precursors in the sample were measured using radiolabeled thymidine and conventional methods.
• four aliquots of 40 ul of the cells were transferred into microwells and lytic activity was assayed.
• the remaining samples were restimulated by adding 160 ul of medium, again containing the described IL-2, IL-4, and irradiated cells. At least 100 cultures were carried out for each number PBMC.
• Stimulation of the type described supra led to proliferation of sufficient cells to allow assay of lytic activity against tumor cell.
• the autologous tumor cells to be used were preincubated for 48 hours in medium containing 50 U/ml IFN- ⁇ . This enhances expression of histocompatibility molecules and adhesion molecules. When non-active natural killer cells ("K562", discussed infra) were added, these were not so treated. IFN- ⁇ does not modify susceptibility of these cells to lysis via NK-like effectors.
• the tumor cells were labeled via suspending them at 10 7 cells/ml in medium supplemented with 10% HS, and incubated at 37°C for 60 minutes, using 200m 51 Cr Ci/ml.
• the labeled cells were washed three times with medium and 2% HS, and were then suspended in medium at 10 4 cells/ml.
• 60 ul of medium augmented with 2% HS or with an additional 5x10 4 K562 cells/well were added to wells containing the previously stimulated cells.
• 1000 tvunor cells were added per well in 100 ul of medium.
• the mixtures were centrifuged (4 minutes, 200g) and then incubated for 4-5 hours at 37°C in 8% CO 2 atmosphere. Aliquots of supernatant (100 ul) were collected and 51 Cr specific release calculated.
• the following formula was used:
• ER experimental 51 Cr release
• SR spontaneous release (i.e., release by cells incubated in medium alone)
• Figure 2 shows results secured using samples taken from two patients (LB-33 & LB-30), using varied numbers of PBMCs and the percentage lysis of melanoma cells taken from the patient. High degrees of lysis were observed in many microcultures. In control cultures, i.e., those where no tumor cells, and hence no antigen was presented, there was no lysis.
• K562 is a human chronic myelogenous leukemia cell line, freely available from the American Type Culture Collection, e.g., under Accession Number ATCC CCL 243). This cell line has been described, e.g., at J. Nat. Cane. Inst. 59: 77-83 (1977), as a highly sensitive in vitro target in NK assays.
• the K562 specific lytic clones are referred to as "NK like", and it was important to determine if these were present.
• the clones considered to be anti-tumor CTLs were examined for specificity. This was done by testing lytic cultures on various targets. Table 2, which follows, presents one set of these data, where PBMCs of a patient (LB-33) were tested against autologous tumor cells, K562, autologous PHA blasts, and tumor cells from patients LB-30 and LB-34. These cells were taken from cell line cultures as discussed supra. The lysis percentage was determined 28 days after setting up a lytic assay as described supra. using 1333 PBMCs per well. In the case of PHA-blasts, lysis was checked using autologous PBMCs activated for 10 days with 1000 U/ml of
• This table shows activity against autologous tumor cells, both with and without non-viable K562.
• the microcultures of pattern “1” or “2” were used to estimate CTL precursor (CTL- P) frequencies, and patterns “2", “3” or “4 " used to estimate NK-like effector cell frequencies.
• the microcultures did not use feeder cells in the cultures. For many patients, it is difficult to obtain the autologous PBMCs that are desirable feeder cells in sufficient amounts. It was also desired to present a generally applicable limiting dilution assay. In experiments performed by the inventors and not reported herein, when autologous, irradiated PBMCs were used as feeder cells, an increase in anti-tumor CTL frequency was observed, but this was coupled to higher NK-like frequency.
• NK inhibiting material i.e., non- viable K562 cells permits distinction between anti-tumor CTLs and NK like effectors which also lyse tumor cells.
• the NK like effectors are inhibited by binding to the K562 cells, which, since they are non viable, do not show 51 Cr release.
• Other methods to eliminate the effect of NK and/or NK like cells in the mixture, including other NK target cells, can also be used.
• CTL-Ps specific for antigens characteristic of tumor cells ranged from 1/33000 to 1/900 PBMCs.
• the latter number obtained with patient LB-33, may result from an autoimmune response against a tumor.
• the numbers observed should be compared to those secured by Borysiewicz et al., Eur. J. Immunol. 18: 269-275 (1988) of 1/5000-20,000 for CMV, by Schmid et al., J. Immunol. 140: 3610-3616 (1988) of 1/4000-8000 for HSV, or by Hickling et al., J. Virol. 61: 3463-3469 (1987), for varicella zoster of 1/1600-90000.
• Sharrock, Immunol Today 11: 281-286 (1990) discusses alloreactive frequencies of 1/500-500000.
• the invention thus teaches a method by which cytotoxic T cell precursors (CTL-Ps) specific for antigens characteristic of tumor cells can be determined via a limiting dilution assay.
• CTL-Ps cytotoxic T cell precursors
• a defined number of peripheral blood mononuclear cells, which contain the CTL-PS are contacted to an antigen which is characteristic of, charge for, i.e., is particularly associated with, a tumor type or types.
• the stimulation from the tumor antigen causes the CTL-Ps to develop into the actual cytolytic T cells. This development is monitored via monitoring the lysis of tumor cells either in or added to the mixture.
• the number of PBMCs used may vary, but preferably somewhere between about 200 and about 10000 are used per assay.
• the PBMCs may be added in a mixed sample, or as a pure culture of PBMCs.
• the antigen characteristic of the tumor is ideally added in the form of a tumor cell, generally non-viable.
• the tumor cell can be rendered non-viable via, e.g., irradiation.
• the tumor cells still present the antigen on their cell surfaces, and a proliferation linked reaction with CTL-Ps follows.
• the antigen can also be added, e.g., in pure form, but this is not the preferred mode.
• tumor cells i.e., tumor cells taken from the same subject as the PBMCs.
• the cell sample is as pure as possible to prevent reaction between CTL-Ps not directed against tumor cells and their targets. This is not always possible, however, and thus it is preferred to treat the mixture to eliminate natural killer ("NK") like cells contained therein.
• NK natural killer
• natural killer like includes natural killer cells, as well as cells which function in the same or an equivalent manner. One approach to doing this is by adding, e.g., an NK inhibitor.
• the lytic determination can be accomplished by means well known in the art, including the 51 Cr release method, described supra.
• CTL-Ps also enables one to monitor responses such as the immune response of a subject with respect to the subject's tumor. Changes in CTL-P levels are indicative of changes in the immune response, and serve an important diagnostic function in that changes over time can indicate a worsening or improvement in the subject's health.

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• 1993
  • 1993-01-07 KR KR1019940702508A patent/KR950700402A/ko not_active Application Discontinuation
  • 1993-01-07 JP JP5512545A patent/JPH07503134A/ja active Pending
  • 1993-01-07 EP EP93902961A patent/EP0624189A4/en not_active Withdrawn
  • 1993-01-07 CA CA002128424A patent/CA2128424A1/en not_active Abandoned
  • 1993-01-07 WO PCT/US1993/000083 patent/WO1993014189A1/en not_active Application Discontinuation
  • 1993-01-07 AU AU34352/93A patent/AU3435293A/en not_active Abandoned
• 1994
  • 1994-07-15 NO NO942660A patent/NO942660L/no unknown
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