CA1271715A - Monoclonal antibody - Google Patents
Monoclonal antibodyInfo
- Publication number
- CA1271715A CA1271715A CA000479685A CA479685A CA1271715A CA 1271715 A CA1271715 A CA 1271715A CA 000479685 A CA000479685 A CA 000479685A CA 479685 A CA479685 A CA 479685A CA 1271715 A CA1271715 A CA 1271715A
- Authority
- CA
- Canada
- Prior art keywords
- cells
- antibody
- human
- sample
- lysing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/821—Chemistry: analytical and immunological testing involving complement factors or complement systems
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/863—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving IgM
- Y10S530/864—Monoclonal
Abstract
Abstract of the Disclosure A monoclonal antibody capable of lysing human NK cells in vitro in the presence of complement.
Description
~171 ~
Back~ ound of the Invention This invention relates to monoclonal antibodies.
Human peripheral blood contains a sub-population of mononuclear cells called natural killer (NK) cells, which are defined by their ability to lyse tumor cells ln vitro in the absence of antibody of previous immunization.
Griffin et al. ~1983) J. Immunol. 130, 2947, describes a monoclonal antibody to NK cells (designated "N901"~ which is unreactive with non-NK human monocy~es, granulocytes, B
lymphocytes, ~ cells, erythrocytes, and platelets. The antibody is incapable of lysing NK cells and NK-like cells ln vitro in the presence of complement. (As used herein, the term "NK-like cells" refers to cells found elsewhere than in normal peripheral blood, e.g., cultured NK cells, or cells in other organs or in malignancies, and either having the lytic capability of NK cells, or bearing a surface antigenic determinant specific to NK cells).
Perussia et al., (1983) J. Immunol. 130, 2133, describes another monoclonal antibody reactive with NK cells.
Summarv of the Invention According to one aspect of the present invention there is provided a monoclonal antibody capable of lysing human NK
cells in vitro in the presence of complement, and said antibody recognizing an anti~Je~ determlnclnt on the surface of human NK
cells havlng a molecular weiyht of about 200-220 KD as determined by SDS-PAGE electrophoresis on a 10% polyacrylamide slab gel.
1a 60412-149 According to a further aspect of the present invention there is provided a method of detecting the presence of NK or NK-like cells in a human patient comprisiny contacting a cell-containing clinical sample from said patient with the antibody of claim 1 and detecting immune complexes as an indication of the presence of said cells.
In preferred embodiments, the antibody is unreactive with non-NK human granulocytes, non-NK human monocytes, human red blood cells, human platelets, human thymocytes, and non-NK human T3 lymphocytes~ the antibody .is of the IgM or IgG2 isotype; and it recognizes an approximately 200,000-220,000 dalton NK
~ .~ 7~
60412 14g6 cell-specific antigenic determinant on the surface of substantially all NK cells Ithat particular antigenic determinant is referred to herein as "NKHlA").
The antibody of the invention ("anti-NKHl~") can be labelled, e.g. with a radioactive or fluorescent label, and used to identify NK cells and to distinguish these cells from non-NK
cells. The antibody can also be used to destroy NK cells in tissue, e.g., bone marrow, to be infused in a patient, to inhibit graft-versus-host disease.
All monoclonal antibodies having the above specificity and characteristics are encompassed by the present invention.
These monoclonal antibodies are produced by hybrid cells made using conventional hybridization and screening techniques such as are described in Reinherz et al. (1979) J. Immunol. 123, 1312 and Ritz et al. Nature (1980) 283, 583. As is well-known in the monoclonal antibody field, each independently-produced hybrid cell line that produces a monoclonal antibody specific to the same particular antigenic determinant is nonetheless different from all others, as is each of the monoclonal antibodies so produced. Thus, while repetition of the procedure described below can result in the production of a hybrid cell line that produces a useful monoclonal antibody specific to and capable of lys~ng NI~ cclls, it is highly unlikely that it will produce a ccll llne that produces a monoclonal antibody which is chemically an exact copy of the monoclonal antibody described below.
Descr ption of the Preferred Embodiments We now turn to a description of the preferred embodiments of the invention.
, 7~
Hybr~doma Production and ~creeninq Six week old female Balb/c mice were immunized with cells of the human cloned NK cell line JT3, described in Heccend et al. (1983) Nature 301, 158. The 5 JT3 clone was derived from purified large granular lymphocytes (LG~) initially stimulated wi~h PHA and medium containing interleukin-2. For greater than 18 months, JT3 cells have been maintained in culture and have been found to display stable phenotype 10 (T3-T4-T8-Tll~N901+) and cytotoxic activity when tested against a large panel of in vitro established cell lines.
The JT3 clone was generated according to the limited dilution method described in Hercend et al.
(1982) J . Immunol. 129. 129~ and Hercend et al. (19B3) 15 Nature 301, 158. The method involved cloning LGL at one cell/well on a ~eeder layer of autolo~ous irradiated (5000 rad) human peripheral blood mononuclear cells (PBMC) plus either P~ (2 ug/ml) or allogeneic or autologous irradiated (5000 rad) EBV transformed B
20 cells. Selected colonies were expanded by addition of culture medium containing LCM (10-15~ final dilution) every 3 days. Culture medium was RPMI 16~0 supplemented with 1~ penicillin-streptomycin, 1% sodium pyruvate, 20~
human ~B serum. The JT3 line was subsequently subcloned ' 25 2 times at 100 cells/well on a feeder layer of autologous irradiated PBMC plus icradiated EBV
transformed B cells. Following subcloning procedures, both phenotype and cytotoxic function have ren~airled stable.
3o I'lle immurlization sctle(lule consisted of an initial intraperitoneal in jection of ~ x 106 JT3 cells emulsified in complete Feeund's adjuvant (DifCo, Detroit, MI) followed by two weekly intraperitoneal ~7~
. -- 4 injections o~ ~ x 10 viable cells in phosphate bu~Lered saline (P~3S). Two weeks later. 2 x lO cells were injected intravenously, followed,by splenectomy 3 days later. Somatic cell- hybridization was carried out 5 by the method of Kohler and Milstein (1~75~ Nature 256, 495.
The initial screeninc3 of hybridoma supernatants was carried out as ~ollows: 10,000 51cr labeled JT3 cells were incubated with an aliquot of each hybridoma 10 supecnatant or 30 minutes at room temperature in V
bottom microtiter plates. Rabbit complement ~t an appropriate non-toxic dilution (usually 1:10) was ~hen added for 1 hour at 37 C. Subsequently, plates wc~re spun down and antibody reactivity was measured as 15 release of Cr into the supernatants using a gamma scintillation counter. Positive supecnatants wece then tested under ide~tical experimental conditions against ~n ~utolo~ous M~IC restricted cytoto~ic T cell clone as Wqll ~6 ~n ,lutolo~ous EBV tcansormed B cell line.
20 Cells wece selected which produced antibodies which, in the presence of complement, lysed JT3 cells but not the MHC restricted cytotoxic T cell clone or the EBV
tcansormed B cell line. Antibody producing hybridomas were subsequently recloned two times by limi~ing ' 25 dilution and immune ascites were produced.
AntibodY Characterization The reactivity pattern o tho antibocly was determined by testinq on puciiecl populatil)rls o~
lymphoid and nonlymphoicl colls by indirect 30 immunoL'luocescence assays and subseqllent analysis using either a FACS I~or Epics V low cytometer. To obtain ~- suitable cells for testing, PBMC were isolated from healthy volunteer donors by Ficoll-llypaque~(F/H) density 1~7~7~
.
~- -- 5 gcadient centrifugation. Monocyte enriched adherent _cells were obtained from PB~C by two step adherence on plastic culture dishes. Granulocytes were isolated from F/H gradient pellets using high molecular w~ight 5 dextran. Activated T cells were generated by stimulating E+ cells with phytohemagglutinin (P~lA) (Z ug/ml) and tested on day 7 following stimulation.
The antibody was found to be strongly reactive with JT3 immunizing cells. In addition, as shown in ~he 10 Table below, it was found to react with a small (12%) population of lymphocytes present in peripheral blood. Human monocytes, granulocytes, platelets, red blood cells, thymocytes~ T3 lymphocytes, and PHA
activated E+ cells were negative.
Tahle Peripheral blood lYmphocytcs % reactive cells ~ rcactive cclls Donor with anti-NKHlA Donor with NKHlA Mean + ~D
1 9 7 10 12.3 A further experiment demonstrated that anti-NKIIlA was unreactive with all the cells in a series o fourteen lymphohematopoietic cell lines derived either from vically tran~i~ocmcd cc115 or tumor c ~ I I s .
MorpholotJy of Nl<~llA~ cclls in unstimulated peripheral blood was determined following purification o~ these ceils by immunofluorescence cell sortintJ. For ~.~7~ S
. ~ .
.
this purpose, pecipheral blood lymphocytes ~rom ~wo individual donors were incubated with anti-NK~ plus GM-~TC. Small numbers (about 50,000);of NK~ +
cells were then purified using an Epics V cell sorter 5 and cytocentrifuge smears were prepared and analy~ed.
It was found that NK~IlA~ purified fractions consisted almost entirely (over 90%) of large gcanular lymphocytes. This finding was consistent with prev7Ous studies that have demonstrated that virtually the 10 entire NK activity present in peripheral blood was mediated by a small fraction of lymphocytes which can be identified morphologically because of their larger size and the presence of azurophilic granules in the cytoplasm.
15 Cell Ly!; i S
5ince anti-NKI-llA is an I~?V~ antibody, it was possible to investigate its capacity for lysing NK
cells in human peripheral blood in the presence o~
complernent. For this purpose, peripheral blood 20 mononuclear cells were incubated with either medium or anti-NKI~ for 30 minutes at room temperature.
Anti-N901 (discussed above), which is a nonlytic IgGl antibody directed at NK cells, was used as a control in these e~cperiments. Antibody excess was washed out and 25 complement was then added ~or ~5 minutes at 37C. This procedure was repeated two times. Subsequently, remaining viable cells were countc(l an(l 5lCr lat)clecl tarqet cells were added.
This proce(lllre WaS used on P~tJ target cells 30 from a clonor displaying a very hicJh level o spontaneous cytotoxicity against a 'I' cell leukemia line, indicating the presence o many NK cl?lls.
Treatment of these PBL with anti-NKHl~ plus 1~7~7~. j, complement was followed by a virtually complete loss o~
NK activity, indicating that anti-NK~-IlA Leacted with all NK cells in peripheral blood. Control-treated P~L
displayed a continued high level o cytotoxicity.
5 Characterization of NKHl~ Anti ~n Immunoprecipitation expe~iments were conducted to identify the molecular weight of t~le antigen defined by anti-NKHlA.
The irrlmunoprecipitations were done using 10 preformed complexes of rabbit anti-mouse Ig with anti-NKHlA. These preformed complexes were reacted with I surface labeled JT3 cell lysates which were precleared 3 times by incubation at 4 C for l hour with eit~er formalin fixed Staph A bacteria and/or preformed 15 complexes of rabbit anti-mouse Ig nonspecific monoclonal antibody. Aliquots of precleared lysate were then incubated with specific preformed complexes for 2 hours at 4 C. Immune precipitates were l~len washed ~ times with RIPA buffer and dissolved in sodium 20 dodecyl sulfate polyacrylamide gel electrophoresis (SVS-~AGE) sample buffer and loaded onto separate ;lots on a 10% polyacrylamide slab gel. Identical aliquots of immune precipitation were ~un in nonreduce~ and reduced conditions after addition of 2 mercaptoet~lanol 25 to SVS-P~GE sample buffer. Gels were drie~ and radiolabeled precipitates were visualized using standard methods. Anti-NK~ precipiL.Ite~(l a blo.ld band at appro~imately 200-220KD. ~dlli~. i 0~
exLeriments were performed irl which the precipi-at.es 30 were analyzed usin~ a 5-15~ polyacrylamide ~radienl and no additional specific bands were identified. When gradient gels were used, the specific band was seerl at a sli~htly lower molecular weight (lS0-200KI)) as compared to the lO~ polyacrylamide gt?ls.
~L~71~
.~ - 8 -It was demonstrated that anti-NK~lA and anti-N901 are probably directed at the same NK surface antigen molecule, in a se~ies of competitive binding exLeriments. Advantage was taken of the diferent isotypes of the two antibodies (anti-NK~ilA being an IyM and anti-N901 being an IgGl). Using iso~ype specific FITC in indirect immunofluorescence assays, it was ~ound that anti-N901 effectively blocked bindinq of anti-NKHlA whereas anti-NKHl~ did not signifi~ tly 10 af~ect binding of anti-N901. These results confirmed that both antibodies we~e specific for the same cell surface antigen and also suggested that the Ig~lantibody (anti-N901) had a higher affinity ~ an the IgM (anti-N~
15 De o~i L t Cells producing anti-NK~Il have been deposited in the ~merican Type Culture Collection, Rockville, Ma~yland, and are given ~TCC Accession No. ~I:~ ~6 20 Use Identification of NK cells and NK-like cells The monoclonal antibody of the invention can be used to identify NK and NK-like cells in perip~l(?ral blood samples and in samples fcom other tissue, e.g.
25 bone ma~row.
The antibody can be used to detect such cells in clinical samples using any eonvelltiotlal immulloassay technique, e.g., indict?et immunofLuorescerlce or a direct assay ellli?loyirlg antibody 1.3bc?1ecl, c.g., with a 30 radioactive label.
The antibody can also be used to detect NK or NK-like cells, or cell-~ree antigf?n, in plasma and other body fluids _ll vi~:!-7~
g Detecting and quantifying circulating NK cellscan permit correlations between various disease states and numbers of NK cells; such information can in turn further elucidate the role of NK cells in healthy and 5 sick individuals, a role which, evidence now suggests, may include the destruction of malignant cells.
Therap~
Since NK cells may play a deleterious role in graft-versus-host disease ~GVHD) following organ or lO tissue transplants, the antibody of the invention can be useful in combatting GVHD by killing associated NK
cells. For example, in connection with a bone marrow transplant, the antibody plus complement can be used to treat the bone marrow prior to transplantation, 15 according to conventional techniques, to kill the NK
cells which could otherwise contribute to rejection of ~he nost by the bone marrow. The ability of the antibody to lyse NK cells in the presence of complement is a function of its being of the Igl~1 isotype;
20 generally, only Igt1 and IgG2 antibodies possess this prc~erty. The kill rate of NK cells in tissue can De increased by em?loying antibody to which has been cou~led, using conventional techniques, a cytoto~ic agent such as ricin or adiamycin.
l`he selective in vivo removal of NK cells may also prove usef~l in the treatment of autoimmune diseases such as 5LE which are in part mediated by NK
cells.
The antibody may al.so prove usc~lll in 3inhibiting the re-jection of al:LograLts such as bone marrow and kidl1eys by using anti-NKIIl~ to inactivate host NK cells which would otherwise attack the allograft.
60412~1496 Another use of the antibody is in the purification of NK
cells from peripheral blood or from a cell suspension of hematopoietic tissue, e.g. bone marrow, or lymphoid tissue, e.g.
thymus, spleen, or lymph node tissue. The antibody ean be used for such purification in connection with any conventional eell separation technique, e.g. cell sorting, immune rosetting, panning, or immune affinity column chromotography.
Such purification of NK cells can be useful where a particular medical disorder requires the infusion into the patient of NK cells, either from the patient or from a donor. Such infusion of NK cells can be used, e.g., to treat malignancies susceptible to attack of NK eells.
It may prove particularly useful to use a patient's own NK cells for such treatment. After anti-NK is used to purify NK
cells from the patient's blood or other tissue, a selected NK cell can be eultured and expanded and then reinfused into the patient;
appropriate culture methods are described in Hercend et al. (1982) J. Immunol. 129, 1299; and Hercend et al. (1983) Nature 301~158 Other embodiments are within the following claims. For example, the antibody can be labelled in a variety of ways, e.g.
with fluorescein-containing ligands, heavy metals, or 13C-containing compounds.
Back~ ound of the Invention This invention relates to monoclonal antibodies.
Human peripheral blood contains a sub-population of mononuclear cells called natural killer (NK) cells, which are defined by their ability to lyse tumor cells ln vitro in the absence of antibody of previous immunization.
Griffin et al. ~1983) J. Immunol. 130, 2947, describes a monoclonal antibody to NK cells (designated "N901"~ which is unreactive with non-NK human monocy~es, granulocytes, B
lymphocytes, ~ cells, erythrocytes, and platelets. The antibody is incapable of lysing NK cells and NK-like cells ln vitro in the presence of complement. (As used herein, the term "NK-like cells" refers to cells found elsewhere than in normal peripheral blood, e.g., cultured NK cells, or cells in other organs or in malignancies, and either having the lytic capability of NK cells, or bearing a surface antigenic determinant specific to NK cells).
Perussia et al., (1983) J. Immunol. 130, 2133, describes another monoclonal antibody reactive with NK cells.
Summarv of the Invention According to one aspect of the present invention there is provided a monoclonal antibody capable of lysing human NK
cells in vitro in the presence of complement, and said antibody recognizing an anti~Je~ determlnclnt on the surface of human NK
cells havlng a molecular weiyht of about 200-220 KD as determined by SDS-PAGE electrophoresis on a 10% polyacrylamide slab gel.
1a 60412-149 According to a further aspect of the present invention there is provided a method of detecting the presence of NK or NK-like cells in a human patient comprisiny contacting a cell-containing clinical sample from said patient with the antibody of claim 1 and detecting immune complexes as an indication of the presence of said cells.
In preferred embodiments, the antibody is unreactive with non-NK human granulocytes, non-NK human monocytes, human red blood cells, human platelets, human thymocytes, and non-NK human T3 lymphocytes~ the antibody .is of the IgM or IgG2 isotype; and it recognizes an approximately 200,000-220,000 dalton NK
~ .~ 7~
60412 14g6 cell-specific antigenic determinant on the surface of substantially all NK cells Ithat particular antigenic determinant is referred to herein as "NKHlA").
The antibody of the invention ("anti-NKHl~") can be labelled, e.g. with a radioactive or fluorescent label, and used to identify NK cells and to distinguish these cells from non-NK
cells. The antibody can also be used to destroy NK cells in tissue, e.g., bone marrow, to be infused in a patient, to inhibit graft-versus-host disease.
All monoclonal antibodies having the above specificity and characteristics are encompassed by the present invention.
These monoclonal antibodies are produced by hybrid cells made using conventional hybridization and screening techniques such as are described in Reinherz et al. (1979) J. Immunol. 123, 1312 and Ritz et al. Nature (1980) 283, 583. As is well-known in the monoclonal antibody field, each independently-produced hybrid cell line that produces a monoclonal antibody specific to the same particular antigenic determinant is nonetheless different from all others, as is each of the monoclonal antibodies so produced. Thus, while repetition of the procedure described below can result in the production of a hybrid cell line that produces a useful monoclonal antibody specific to and capable of lys~ng NI~ cclls, it is highly unlikely that it will produce a ccll llne that produces a monoclonal antibody which is chemically an exact copy of the monoclonal antibody described below.
Descr ption of the Preferred Embodiments We now turn to a description of the preferred embodiments of the invention.
, 7~
Hybr~doma Production and ~creeninq Six week old female Balb/c mice were immunized with cells of the human cloned NK cell line JT3, described in Heccend et al. (1983) Nature 301, 158. The 5 JT3 clone was derived from purified large granular lymphocytes (LG~) initially stimulated wi~h PHA and medium containing interleukin-2. For greater than 18 months, JT3 cells have been maintained in culture and have been found to display stable phenotype 10 (T3-T4-T8-Tll~N901+) and cytotoxic activity when tested against a large panel of in vitro established cell lines.
The JT3 clone was generated according to the limited dilution method described in Hercend et al.
(1982) J . Immunol. 129. 129~ and Hercend et al. (19B3) 15 Nature 301, 158. The method involved cloning LGL at one cell/well on a ~eeder layer of autolo~ous irradiated (5000 rad) human peripheral blood mononuclear cells (PBMC) plus either P~ (2 ug/ml) or allogeneic or autologous irradiated (5000 rad) EBV transformed B
20 cells. Selected colonies were expanded by addition of culture medium containing LCM (10-15~ final dilution) every 3 days. Culture medium was RPMI 16~0 supplemented with 1~ penicillin-streptomycin, 1% sodium pyruvate, 20~
human ~B serum. The JT3 line was subsequently subcloned ' 25 2 times at 100 cells/well on a feeder layer of autologous irradiated PBMC plus icradiated EBV
transformed B cells. Following subcloning procedures, both phenotype and cytotoxic function have ren~airled stable.
3o I'lle immurlization sctle(lule consisted of an initial intraperitoneal in jection of ~ x 106 JT3 cells emulsified in complete Feeund's adjuvant (DifCo, Detroit, MI) followed by two weekly intraperitoneal ~7~
. -- 4 injections o~ ~ x 10 viable cells in phosphate bu~Lered saline (P~3S). Two weeks later. 2 x lO cells were injected intravenously, followed,by splenectomy 3 days later. Somatic cell- hybridization was carried out 5 by the method of Kohler and Milstein (1~75~ Nature 256, 495.
The initial screeninc3 of hybridoma supernatants was carried out as ~ollows: 10,000 51cr labeled JT3 cells were incubated with an aliquot of each hybridoma 10 supecnatant or 30 minutes at room temperature in V
bottom microtiter plates. Rabbit complement ~t an appropriate non-toxic dilution (usually 1:10) was ~hen added for 1 hour at 37 C. Subsequently, plates wc~re spun down and antibody reactivity was measured as 15 release of Cr into the supernatants using a gamma scintillation counter. Positive supecnatants wece then tested under ide~tical experimental conditions against ~n ~utolo~ous M~IC restricted cytoto~ic T cell clone as Wqll ~6 ~n ,lutolo~ous EBV tcansormed B cell line.
20 Cells wece selected which produced antibodies which, in the presence of complement, lysed JT3 cells but not the MHC restricted cytotoxic T cell clone or the EBV
tcansormed B cell line. Antibody producing hybridomas were subsequently recloned two times by limi~ing ' 25 dilution and immune ascites were produced.
AntibodY Characterization The reactivity pattern o tho antibocly was determined by testinq on puciiecl populatil)rls o~
lymphoid and nonlymphoicl colls by indirect 30 immunoL'luocescence assays and subseqllent analysis using either a FACS I~or Epics V low cytometer. To obtain ~- suitable cells for testing, PBMC were isolated from healthy volunteer donors by Ficoll-llypaque~(F/H) density 1~7~7~
.
~- -- 5 gcadient centrifugation. Monocyte enriched adherent _cells were obtained from PB~C by two step adherence on plastic culture dishes. Granulocytes were isolated from F/H gradient pellets using high molecular w~ight 5 dextran. Activated T cells were generated by stimulating E+ cells with phytohemagglutinin (P~lA) (Z ug/ml) and tested on day 7 following stimulation.
The antibody was found to be strongly reactive with JT3 immunizing cells. In addition, as shown in ~he 10 Table below, it was found to react with a small (12%) population of lymphocytes present in peripheral blood. Human monocytes, granulocytes, platelets, red blood cells, thymocytes~ T3 lymphocytes, and PHA
activated E+ cells were negative.
Tahle Peripheral blood lYmphocytcs % reactive cells ~ rcactive cclls Donor with anti-NKHlA Donor with NKHlA Mean + ~D
1 9 7 10 12.3 A further experiment demonstrated that anti-NKIIlA was unreactive with all the cells in a series o fourteen lymphohematopoietic cell lines derived either from vically tran~i~ocmcd cc115 or tumor c ~ I I s .
MorpholotJy of Nl<~llA~ cclls in unstimulated peripheral blood was determined following purification o~ these ceils by immunofluorescence cell sortintJ. For ~.~7~ S
. ~ .
.
this purpose, pecipheral blood lymphocytes ~rom ~wo individual donors were incubated with anti-NK~ plus GM-~TC. Small numbers (about 50,000);of NK~ +
cells were then purified using an Epics V cell sorter 5 and cytocentrifuge smears were prepared and analy~ed.
It was found that NK~IlA~ purified fractions consisted almost entirely (over 90%) of large gcanular lymphocytes. This finding was consistent with prev7Ous studies that have demonstrated that virtually the 10 entire NK activity present in peripheral blood was mediated by a small fraction of lymphocytes which can be identified morphologically because of their larger size and the presence of azurophilic granules in the cytoplasm.
15 Cell Ly!; i S
5ince anti-NKI-llA is an I~?V~ antibody, it was possible to investigate its capacity for lysing NK
cells in human peripheral blood in the presence o~
complernent. For this purpose, peripheral blood 20 mononuclear cells were incubated with either medium or anti-NKI~ for 30 minutes at room temperature.
Anti-N901 (discussed above), which is a nonlytic IgGl antibody directed at NK cells, was used as a control in these e~cperiments. Antibody excess was washed out and 25 complement was then added ~or ~5 minutes at 37C. This procedure was repeated two times. Subsequently, remaining viable cells were countc(l an(l 5lCr lat)clecl tarqet cells were added.
This proce(lllre WaS used on P~tJ target cells 30 from a clonor displaying a very hicJh level o spontaneous cytotoxicity against a 'I' cell leukemia line, indicating the presence o many NK cl?lls.
Treatment of these PBL with anti-NKHl~ plus 1~7~7~. j, complement was followed by a virtually complete loss o~
NK activity, indicating that anti-NK~-IlA Leacted with all NK cells in peripheral blood. Control-treated P~L
displayed a continued high level o cytotoxicity.
5 Characterization of NKHl~ Anti ~n Immunoprecipitation expe~iments were conducted to identify the molecular weight of t~le antigen defined by anti-NKHlA.
The irrlmunoprecipitations were done using 10 preformed complexes of rabbit anti-mouse Ig with anti-NKHlA. These preformed complexes were reacted with I surface labeled JT3 cell lysates which were precleared 3 times by incubation at 4 C for l hour with eit~er formalin fixed Staph A bacteria and/or preformed 15 complexes of rabbit anti-mouse Ig nonspecific monoclonal antibody. Aliquots of precleared lysate were then incubated with specific preformed complexes for 2 hours at 4 C. Immune precipitates were l~len washed ~ times with RIPA buffer and dissolved in sodium 20 dodecyl sulfate polyacrylamide gel electrophoresis (SVS-~AGE) sample buffer and loaded onto separate ;lots on a 10% polyacrylamide slab gel. Identical aliquots of immune precipitation were ~un in nonreduce~ and reduced conditions after addition of 2 mercaptoet~lanol 25 to SVS-P~GE sample buffer. Gels were drie~ and radiolabeled precipitates were visualized using standard methods. Anti-NK~ precipiL.Ite~(l a blo.ld band at appro~imately 200-220KD. ~dlli~. i 0~
exLeriments were performed irl which the precipi-at.es 30 were analyzed usin~ a 5-15~ polyacrylamide ~radienl and no additional specific bands were identified. When gradient gels were used, the specific band was seerl at a sli~htly lower molecular weight (lS0-200KI)) as compared to the lO~ polyacrylamide gt?ls.
~L~71~
.~ - 8 -It was demonstrated that anti-NK~lA and anti-N901 are probably directed at the same NK surface antigen molecule, in a se~ies of competitive binding exLeriments. Advantage was taken of the diferent isotypes of the two antibodies (anti-NK~ilA being an IyM and anti-N901 being an IgGl). Using iso~ype specific FITC in indirect immunofluorescence assays, it was ~ound that anti-N901 effectively blocked bindinq of anti-NKHlA whereas anti-NKHl~ did not signifi~ tly 10 af~ect binding of anti-N901. These results confirmed that both antibodies we~e specific for the same cell surface antigen and also suggested that the Ig~lantibody (anti-N901) had a higher affinity ~ an the IgM (anti-N~
15 De o~i L t Cells producing anti-NK~Il have been deposited in the ~merican Type Culture Collection, Rockville, Ma~yland, and are given ~TCC Accession No. ~I:~ ~6 20 Use Identification of NK cells and NK-like cells The monoclonal antibody of the invention can be used to identify NK and NK-like cells in perip~l(?ral blood samples and in samples fcom other tissue, e.g.
25 bone ma~row.
The antibody can be used to detect such cells in clinical samples using any eonvelltiotlal immulloassay technique, e.g., indict?et immunofLuorescerlce or a direct assay ellli?loyirlg antibody 1.3bc?1ecl, c.g., with a 30 radioactive label.
The antibody can also be used to detect NK or NK-like cells, or cell-~ree antigf?n, in plasma and other body fluids _ll vi~:!-7~
g Detecting and quantifying circulating NK cellscan permit correlations between various disease states and numbers of NK cells; such information can in turn further elucidate the role of NK cells in healthy and 5 sick individuals, a role which, evidence now suggests, may include the destruction of malignant cells.
Therap~
Since NK cells may play a deleterious role in graft-versus-host disease ~GVHD) following organ or lO tissue transplants, the antibody of the invention can be useful in combatting GVHD by killing associated NK
cells. For example, in connection with a bone marrow transplant, the antibody plus complement can be used to treat the bone marrow prior to transplantation, 15 according to conventional techniques, to kill the NK
cells which could otherwise contribute to rejection of ~he nost by the bone marrow. The ability of the antibody to lyse NK cells in the presence of complement is a function of its being of the Igl~1 isotype;
20 generally, only Igt1 and IgG2 antibodies possess this prc~erty. The kill rate of NK cells in tissue can De increased by em?loying antibody to which has been cou~led, using conventional techniques, a cytoto~ic agent such as ricin or adiamycin.
l`he selective in vivo removal of NK cells may also prove usef~l in the treatment of autoimmune diseases such as 5LE which are in part mediated by NK
cells.
The antibody may al.so prove usc~lll in 3inhibiting the re-jection of al:LograLts such as bone marrow and kidl1eys by using anti-NKIIl~ to inactivate host NK cells which would otherwise attack the allograft.
60412~1496 Another use of the antibody is in the purification of NK
cells from peripheral blood or from a cell suspension of hematopoietic tissue, e.g. bone marrow, or lymphoid tissue, e.g.
thymus, spleen, or lymph node tissue. The antibody ean be used for such purification in connection with any conventional eell separation technique, e.g. cell sorting, immune rosetting, panning, or immune affinity column chromotography.
Such purification of NK cells can be useful where a particular medical disorder requires the infusion into the patient of NK cells, either from the patient or from a donor. Such infusion of NK cells can be used, e.g., to treat malignancies susceptible to attack of NK eells.
It may prove particularly useful to use a patient's own NK cells for such treatment. After anti-NK is used to purify NK
cells from the patient's blood or other tissue, a selected NK cell can be eultured and expanded and then reinfused into the patient;
appropriate culture methods are described in Hercend et al. (1982) J. Immunol. 129, 1299; and Hercend et al. (1983) Nature 301~158 Other embodiments are within the following claims. For example, the antibody can be labelled in a variety of ways, e.g.
with fluorescein-containing ligands, heavy metals, or 13C-containing compounds.
Claims (13)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A monoclonal antibody capable of lysing human NK cells in vitro in the presence of complement, and said antibody recognizing an antigenic determinant on the surface of human NK
cells having a molecular weight of about 200-220 KD as determined by SDS-PAGE electrophoresis on a 10% polyacrylamide slab gel.
cells having a molecular weight of about 200-220 KD as determined by SDS-PAGE electrophoresis on a 10% polyacrylamide slab gel.
2. The antibody of claim 1, said antibody being unreactive with non-NK human granulocytes, non-NK human monocytes, human red blood cells, human thymocytes, and non-NK human T3 lymphocytes.
3. The antibody of claim 1, said antibody being of the IgM
or IgG2 isotype.
or IgG2 isotype.
4. The antibody of claim 1, coupled to a cytotoxic agent.
5. The antibody of claim 1, labeled with a detectable label.
6. The antibody of claim 5, said antibody being radiolabeled.
7. The antibody of claim 5, said antibody being fluorescently labeled.
8. The monoclonal antibody produced by the hybridoma cell ATCC HB 8564 .
9. The hybridoma cell ATCC HB 8564 .
10. A method of detecting the presence of NK or NK-like cells in a human patient comprising contacting a cell-containing clinical sample from said patient with the antibody of claim 1 and detecting immune complexes as an indication of the presence of said cells.
11. A method of lysing NK cells or NK-like cells in a clinical sample comprising incubating said sample with the antibody of claim 1 in the presence of complement.
12. A method of lysing NK cells or NK-like cells in a clinical sample comprising contacting said sample with the cytotoxic agent-coupled antibody of claim 4.
13. The method of claim 11 or claim 12 wherein said clinical sample is a bone marrow sample.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US603,181 | 1984-04-23 | ||
| US06/603,181 US4772552A (en) | 1984-04-23 | 1984-04-23 | Monoclonal antibody which recognizes a 200-220 KD antigen on natural killer cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1271715A true CA1271715A (en) | 1990-07-17 |
Family
ID=24414404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000479685A Expired - Fee Related CA1271715A (en) | 1984-04-23 | 1985-04-22 | Monoclonal antibody |
Country Status (6)
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|---|---|
| US (1) | US4772552A (en) |
| EP (1) | EP0160486B1 (en) |
| JP (2) | JPS6128000A (en) |
| AT (1) | ATE71149T1 (en) |
| CA (1) | CA1271715A (en) |
| DE (1) | DE3585049D1 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5028424A (en) * | 1987-10-16 | 1991-07-02 | University Of Georgia Research Foundation | Antibodies to receptor and antigen for natural killer and non-specific cytotoxic cells |
| US4895796A (en) * | 1988-01-06 | 1990-01-23 | Becton Dickinson And Company | Identification of NK cells and cytotoxic T lymphocytes |
| DK172110B1 (en) | 1988-04-15 | 1997-10-27 | Gen Hospital Corp | Method for Isolating Mutants of DNA Sequences and Their Use in Identifying Cell Surface Proteins |
| US5089384A (en) * | 1988-11-04 | 1992-02-18 | Amoco Corporation | Method and apparatus for selective cell destruction using amplified immunofluorescence |
| AU679437B2 (en) | 1992-01-08 | 1997-07-03 | General Hospital Corporation, The | Induced tolerance to xenografts |
| US5658564A (en) * | 1992-02-19 | 1997-08-19 | The General Hospital Corporation | Xenograft thymus |
| US5663481A (en) * | 1993-08-06 | 1997-09-02 | Mount Sinai Hospital Corporation | Animal model of the human immune system |
| DE69430846T2 (en) * | 1993-08-27 | 2003-01-16 | Dana Farber Cancer Inst Inc | FOR NATURAL KILLER CELLS SPECIFIC ANTIGENS AND IDENTIFYING ANTIBODIES |
| JP4294286B2 (en) | 2002-09-24 | 2009-07-08 | 富士重工業株式会社 | Vehicle differential limiting control device |
| CA2564246A1 (en) * | 2004-04-30 | 2005-11-10 | Innate Pharma | Compositions and methods for treating immunoproliferatifs disorders such as nk-type ldgl |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4364936A (en) * | 1980-01-08 | 1982-12-21 | Ortho Pharmaceutical Corporation | Monoclonal antibody to a human monocyte antigen and methods of preparing same |
| US4607007A (en) * | 1983-04-07 | 1986-08-19 | Becton, Dickinson And Company | Differentiation of natural killer cell subpopulations of cells |
| US4599307A (en) * | 1983-07-18 | 1986-07-08 | Becton, Dickinson And Company | Method for elimination of selected cell populations in analytic cytology |
| US4599304A (en) * | 1983-10-07 | 1986-07-08 | Becton, Dickinson And Company | Method for monitoring activated cell subpopulations |
| US4572834A (en) * | 1984-04-10 | 1986-02-25 | Clinical Reference Laboratory, Inc. | Biologic and method of preparing same |
-
1984
- 1984-04-23 US US06/603,181 patent/US4772552A/en not_active Expired - Lifetime
-
1985
- 1985-04-22 CA CA000479685A patent/CA1271715A/en not_active Expired - Fee Related
- 1985-04-22 EP EP85302806A patent/EP0160486B1/en not_active Expired - Lifetime
- 1985-04-22 AT AT85302806T patent/ATE71149T1/en not_active IP Right Cessation
- 1985-04-22 DE DE8585302806T patent/DE3585049D1/en not_active Expired - Lifetime
- 1985-04-23 JP JP8735485A patent/JPS6128000A/en active Granted
-
1992
- 1992-12-02 JP JP4323131A patent/JPH0734742B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0588115B2 (en) | 1993-12-21 |
| JPH0734742B2 (en) | 1995-04-19 |
| EP0160486A2 (en) | 1985-11-06 |
| JPS6128000A (en) | 1986-02-07 |
| US4772552A (en) | 1988-09-20 |
| EP0160486A3 (en) | 1987-06-16 |
| DE3585049D1 (en) | 1992-02-13 |
| EP0160486B1 (en) | 1992-01-02 |
| JPH0662845A (en) | 1994-03-08 |
| ATE71149T1 (en) | 1992-01-15 |
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