WO1992015698A1 - Anticorps monoclonaux murins reconnaissant des determinants polymorphes de l'antigene leucocytaire humain hla - Google Patents

Anticorps monoclonaux murins reconnaissant des determinants polymorphes de l'antigene leucocytaire humain hla Download PDF

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WO1992015698A1
WO1992015698A1 PCT/CA1992/000105 CA9200105W WO9215698A1 WO 1992015698 A1 WO1992015698 A1 WO 1992015698A1 CA 9200105 W CA9200105 W CA 9200105W WO 9215698 A1 WO9215698 A1 WO 9215698A1
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cells
hla
nfld
antibodies
antibody
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PCT/CA1992/000105
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Sheila Drover
William H. Marshall
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Sheila Drover
Marshall William H
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • This invention relates to the production and use of a set of monoclonal antibodies to HLA and its subtypes and to the set of monoclonal antibodies so produced.
  • monoclonal antibodies are specific for antigens coded by the HLA gene complex and therefore are useful in the typing of human tissue that is to be used in organ transplants.
  • the human system involves the production of serum proteins, known as antibodies, by the lymphoid cell series capable of reacting with antigenic determinants which trigger their production. Since the conventional response of the immune system to an antigen with many antigenic determinants is the production of antibodies to each determinant, the antiserum produced is heterologous in nature and polyclonal, or produced by many different cells each producing antibodies to a specific determinant. Antigenic determinants may be referred to as epitopes when more than one occurs on a single molecule and particularly when each elicits an antibody developing, immune response. A single antibody molecule is specific for a unique antigenic determinant or epitope.
  • the introduction of foreign material (antigenic material) into the body of a vertebrate animal provokes an immune reaction, the intent of which is to prevent the antigenic material causing damage to the body and to facilitate the removal of such material from the body.
  • the immune system achieves this by producing immunoglobulin molecules (hereafter referred to as antibodies) which have the property of selectively recognising and binding to characteristic sites on the antigenic material. These sites are known as determinants and an antigen may possess one or more such determinants.
  • Antibodies generated by the immune system each have specificity to only one determinant but a number of different antibodies may be produced if the antigenic material against which antibodies are raised possesses more than one determinant.
  • the primary function of antibodies is to protect the body from harmful foreign material, by agglutinating it, thereby assisting the normal body processes to remove the material.
  • Antibodies are proteins that have the ability to combine with and recognize other molecules, known as antigens. Monoclonal antibodies are no different from other antibodies and, except that they are very uniform in their properties, recognize only one antigen or a portion of an antigen known as a determinant.
  • the determinant recognized is an antigen on or in the cell which reacts with the antibody. It is through these cell antigens that a particular antibody recognizes, i.e. reacts with, a particular kind of cell. Thus the cell antigens are markers by which the cell is identified.
  • antigenic markers may be used to observe the normal process of cell differentiation and to locate abnormalities within a given cell system.
  • the process of differentiation is accompanied by changes in the cell surface antigenic phenotype, and antigens that distinguish cells belonging to distinct differentiation lineages or distinguish cells at different phases in the same differentiation lineage may be observed if the correct antibody is available.
  • Human antibodies have been used both for diagnostic and therapeutic purposes for a number of years.
  • Diagnostic techniques include blood typing for transfusion or transplantation. The oldest is isolation from immune serum. However, the small concentration of the antibody of desired specificity among those which are generally present in serum presents a serious drawback.
  • the preparation of hybridoma cell lines can be successful or not depending on such experimental factors as nature of the inoculant, cell growth conditions, hybridization conditions etc. Thus, it is not always possible to predict successful hybridoma preparation of one cell line although success may have been achieved with another cell line.
  • Monoclonal antibodies are uniform antibodies directed to a single determinant or epitope on the antigen molecule which may be repeated at several sites of the molecule. Obviously, to produce such monoclonal antibodies in vitro requires selecting a homogeneous antibody having the desired specifications from numerous antibodies elicited in a conventional polyclonal response.
  • EBV transformation while successful in forming Mab-secreting cultures, will often fail to provide antigen specific EBV transformed cells which have sufficiently long life spans to provide reliable sources of the desired antibodies. This method fails to provide reliably for antibody production over extended periods.
  • Previously produced hybridomas between immunized human B cells and appropriately drug marked murine or human myeloma or human lymphoblastoid cell lines have suffered from low frequency of hybrid formation in the case of human-human hybridizations.
  • Murine-murine hybridomas are stable, but the antibodies produced are immunogenic if used in passive therapy.
  • An immunized experimental animal can sometimes serve as a source for specific antibody-secreting B cells to provide the immunized lymphoid member of the hybridoma.
  • This method cannot be used, however, to provide reagents for HLA or other blood type testing since when human antigens are injected, the plethora of antibodies elicited is mostly immunoreactive to antigens common to all humans, and the desired antigen-specific antibody is formed only as a very small percentage of the total response. Further, these non-human antibodies can themselves result in an adverse immune response if injected for human therapy.
  • lymphocytes There are two principal classes of lymphocytes involved in the immune system of humans and animals. The first of these (the thymus-derived cell or T cell) is differentiated in the thymus from haemopoietic stem cells. While within the thymus, the differentiating cells are termed "thymocytes". The mature T cells emerge from the thymus and circulate between the tissues, lymphatics, and the bloodstream. These T cells form a large proportion of the pool of recirculating small lymphocytes. They have immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are sometimes required for the secretion of these antibodies by the second class of lymphocytes discussed below. Some types of T cells play a regulating function in other aspects of the immune system. The mechanism of this process of cell cooperation is not yet completely understood.
  • the second class of lymphocytes are those which secrete antibody. They also develop from haemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they are differentiated in an organ analogous to the thymus, called the Bursa of Fabricius. In mammals, however, no equivalent organ has been discovered, and it is thought that these B cells differentiate within the bone marrow.
  • T cells are divided into at least several subtypes, termed “helper”, “suppressor”, and “killer” T cells, which have the function of (respectively) promoting a reaction, suppressing a reaction, or killing (lysing) foreign cells.
  • helper promoting a reaction
  • suppressing a reaction
  • kill a reaction that kill foreign cells.
  • These subclasses are well understood for murine systems, but they have only recently been described for human systems.
  • T cells The ability to identify or suppress classes or subclasses of T cells is important for diagnosis or treatment of various immunoregulatory disorders or conditions.
  • MHC human major histocompatibility complex
  • HLA antigens which include the HLA-A, HLA-B , and HLA-C antigens found on virtually every human cell and which have counterparts in other mammalian cells including the murine system; and Class II antigens including the HLD-, DR, DQ, and DP antigens found chiefly on the surface of immunocompetent cells including macrophages/monocytes, activated T-lymphocytes, and B lymphocytes. Class II antigens also have counterparts in other mammalian systems such as murine mammals. The presence of these Class I and Class II antigenic molecules plays a major role in the functional heterogeneity of peripheral T-cells.
  • T-cells The different regulatory and effector functions of T-cells are mediated by different subpopulation of cells which can be distinguished by differences in their phenotypes and antigenic determinants (identifiable by different monoclonal antibodies). This has led to the typing T cell functional subsets in accordance with the expression of specific surface molecules which are commonly designated by the letter "T” followed by a number.
  • T4 and T8 cells Based on functional differences between T4 and T8 cells, the peripheral blood T-cells can be broadly divided into two populations: one population constituting approximately 65% of peripheral blood T-cells is T4+; the other constituting approximately 35% of all peripheral blood T-cells is T8+.
  • the T8+ cell may be activated to become a cytolytic T lymphocyte (hereinafter termed "CTL cell”) which functions as a cytotoxic effector cell and plays an important role in the hosts' defense against foreign bodies.
  • CTL cell cytolytic T lymphocyte
  • NK cells natural killer cells
  • LAK cells lymphokine activated killer cells
  • the role of the T4+ cell has been traditionally viewed as an inducer cell for the activation of other T-cell subpopulation. This role is achieved in combination with an accessory cell or antigen presenting cell (hereinafter termed "APC”) which bears Class II MHC molecules on its surface and is able to take up and process an identifiable antigen.
  • APC accessory cell or antigen presenting cell
  • T4+ cells The antigen presented by an APC bearing Class II molecules activates specific T4+ cells.
  • the activated T4+ cells in turn secrete a variety of lymphokines to initiate the effector and cytolytic functions of other T-cell lymphocytes.
  • all such immunotherapies utilize only those activated lymphocytes equipped with cytolytic effector function, e.g. CTL cells, NK cells, and LAK cells.
  • T-cells of the inducer phenotype are traditionally viewed as lacking the necessary cytolytic activity and therefore have not been considered useful for treatment of tumours as immunotherapeutic lymphocytes.
  • the human system of multiple T-cell subpopulation has a direct counterpart in the murine system.
  • T-lymphocytes There are two major functional subsets of T-lymphocytes in the murine system.
  • the L3T4+ subset of T-lymphocytes has inducer or helper functions and is generally activated by APCs that bear exogenous antigen and express Class II molecules (la) of the MHC.
  • This subset is equivalent to the T4+ lymphocyte subpopulation in humans.
  • the second major T-cell subset expresses Lyt-2 determinants and possesses either suppressor or cytolytic functions. These are equivalent to T8+ lymphocytes in humans. When activated, Lyt-2 cells become cytolytic T-lymphocytes (CTL cells) which generally lack the L3T4+ antigenic marker and which recognize Class I molecules of the MHC.
  • CTL cells cytolytic T-lymphocytes
  • L3T4+ inducer T cells help initiate the effector functions of other T-lymphocytes, but do not demonstrate any cytolytic effect themselves.
  • inducer T-cells generally in murine and human systems are able to express cytolytic effector function; whether all major types of antigen presenting cells are sensitive to such cytolytic activity; whether such cytolytic activity can be maximally expressed and, if so, under what conditions; and whether such inducer T-cell cytolytic activity can be utilized in-vivo for any therapeutic purpose.
  • MCT microcytotoxicity test
  • the donor organ is obtained from an identical twin since the antigens of the donor and recipient in such a case are identical and no histoincompatibility exists. Therefore, no immune response to the graft occurs in such a transfer, known as an isograft.
  • most transplants are between two less closely related individuals of the same species and histocompatibility differences in such an allograft may be strong or weak, depending on the individuals.
  • the fate of transplanted tissues and organs depends on a number of factors, but the recipient's immune response to graft antigens is the central event. Definition of antigenic systems which serve as strong barriers to transplantation has therefore become a major investigational interest, having both practical application in clinical transplantation and theoretical value in understanding the natural role of the histocompatibility antigens in immunobiology.
  • a single chromosomal gene complex codes for the major histocompatibility antigens in each vertebrate species investigated so far.
  • the histocompatibility antigens are produced by the HLA gene complex.
  • This complex occupies a portion of the short arm of the human C6 chromosome and consists of several series of paired alleles which are inherited from generation to generation in a dominant fashion, segregating randomly from other important antigens such as the ABH red blood cell type groups.
  • Antigens of the HLA system are divided into two classes. Each class I antigen consists of an 11.500-dalton ⁇ 2 -microglobulin sub-unit and a 44.000-dalton heavy chain which carries the antigenic specificity. Three gene loci (A, B and C) are recognized for the class I antigens. There are over sixty clearly defined A and B specificities while 8C locus specificities are known. Evidence that this gene complex plays the major role in the transplantation response comes from the fact that haplotype-matched sibling donor-recipient combinations show excellent results in kidney transplantation, in the vicinity of 85% to 90% long term survival.
  • D-locus antigens are not as yet clearly identifiable by serotyping techniques, serologically defined specificities closely related to the D-lo ⁇ us have been defined. These have the special property of that being expressed on platelets or unstimulated T lymphocytes. These specificities are termed class II having two glycoprotein chains of 29. (XX) ( ⁇ ) and 34. (XX) ( ⁇ ) daltons and lacking ⁇ 2 globulin. These antigens are also termed HLA-DR (D-related) and are important in tissue typing.
  • Tissue typing is currently being carried out using sera obtained from multiparous women.
  • One specific anti-HLA serum appears to be a rabbit anti-A9 serum prepared by immunization with A9 antigen purified from human serum or urine.
  • A9 is actually a common determinate of A23 and A24, and thus possibly the allele-specific major epitope is not the only target with which this antibody reacts.
  • Antibodies have been produced by somatic cell hybrids between myeloma cells and spleen or lymph cells that are specific for malignant tumours. Continuous cell lines have been produced of genetically- stable fused-cell hybrids capable of producing large amounts of IgG antibodies against specific viruses.
  • Hybridomas have been provided which produce monoclonal IgG antibodies against tetanus toxin. Monoclonal antibodies have also been described against human tumour cells.
  • rat-mouse hybridomas have been disclosed which were reactive with determinants on cells from other species, e.g. humans.
  • HLA genes Matching for antigens determined by the HLA genes is an important component of the whole process of transplantation of organs and tissues. In the case of bone marrow transplantation this tissue typing and matching becomes critical; certain mismatches may lead to the death of the patient. Genes determine the production of at least six types of molecule of immunogenetic importance which fall into two classes, I and II. Class
  • HLA-A HLA-B and HLA-C while Class II
  • HLA-DR HLA-DR
  • HLA-DQ HLA-DP
  • HLA-DP HLA-DP
  • HLA-DQ is less well served with reagents and HLA-DP is for all practical purposes not typed by routine tissue typing laboratories.
  • the reason for the lack of DP typing is the unavailability of antibodies against the HLA-DP polymorphism.
  • E. L. Milford et al provided monoclonal antibodies for human tissue cross-matching. That patent provided an immortal, antibody-producing, hybridomally-produced clone and an antibody produced thereby.
  • the antibody was an immunoglobulin specific for an antigenic determinant encoded by an HLA gene complex in humans.
  • the clone was produced by an immortal cell line fused with a lymphocyte obtained from a first rat immunized against cells obtained from a second rat having a different histocompatibility antigen.
  • That patent therefore also provided novel hybridoma cell lines, novel monoclonal antibodies against a human HLA antigen, the antibody having been produced by a novel hybridoma cell line and a tissue-crossing assay kit, including a monoclonal antibody produced by a novel cell line and a dye.
  • U.S. Patent No. 4,634,666 patented January 6, 1987 by E. G. Engleman et al provided an ideal fusion partner for specific B-lymphoid cell lines, producing triomas that secreted specific antibodies of human character.
  • the immortalizing, non-secreting hybridoma having human characteristics was prepared by fusing mouse myeloma cells with human B lymphocytes and selecting the fusion product for stable immunoglobulin secretion and HLA surface antigen production, followed by treating the selected fusion product with mutagen and selecting the mutated product for non-secretion of immunoglobulin but retention of HLA antigen production. That invention also provided the products of fusing the immortalizing hybridomas with suitable human immunized lymphoid cells.
  • triomas are useful sources of desired Mab's.
  • That invention also provides human monoclonal antibodies which are produced by the triomas and their diagnostic and therapeutic compositions and uses.
  • the above patent provided a specifically recited immortalizing fusion partner for use in producing a trioma cell line capable of secreting a human monoclonal antibody specific against a selected antigen, when fused with a non-malignant b-lymphoid cell derived from a human donor exposed to such antigen. It also provided a trioma cell line capable of secreting a normal human monoclonal antibody specific against a selected antigen.
  • the cell line was the fusion product of a mouse myeloma/non- malignant human B-lymphocyte hybridoma fusion partner which expressed HLA surface antigens, did not secrete immunoglobulins, and was deficient in hypoxanthine phosphoribosyl transferase, as evidenced by the inability of the fusion partner to grow in hypoxanthine-aminopterin-thymidine or azaserin-hypoxanthine medium. and a non-malignant B-lymphoid cell derived from a human donor exposed to the selected antigen.
  • P.C. Kung provided methods and compositions using monoclonal antibodies to human T cells.
  • This patentee provided a novel hybridoma which was capable of producing a monoclonal antibody against an antigen found on essentially all normal human peripheral T cells. The antibody so produced was monospecific for a single determinant on normal human T cells and contained essentially no other anti-human immuneglobulin.
  • the patentee also provided a novel hybridoma producing antibody to an antigen found on essentially all normal human T cells, the antibody itself, and diagnostic and therapeutic methods employing the antibody.
  • U.S. Patent No. 4,681,760 patented July 21, 1987 provided a method of conferring immunotolerance to a specific antigen. That patent provided a method for suppressing undesired immune responses, e.g. allergic reactions, to antigens whose administration to the subject was either desired or inevitable but otherwise harmless. It also provided a method for inducing tolerance to tissue transplants. The patented method involved the co-administration of the antigen for which immunotolerance is sought and an antibody which is specific for the "L3T4-equivalent" differentiation antigen on T cells, thus preventing these helper T cells from participating in the immune response otherwise concurrently mounted against the particular co-injected or co-administered antigen.
  • U.S. Patent No. 4,692,405 patented September 8, 1987 by A. Freedman et al provided monoclonal antibodies to antigen on activated human B-cells and assays therefor, protein antigenic determinants therefor and methods of making same.
  • That invention provided a monoclonal antibody recognizing an antigenic determinant on activated human B-cells.
  • That invention also provided a substantially pure protein having an antigenic determinant or determinants substantially identical to determinants of a single-chain polypeptide having an apparent molecular weight of approximately 75,000 daltons under reducing conditions and 67,000 daltons under non-reducing conditions, the single-chain polypeptide being a protein on the surface of activated human B-cells.
  • That invention also provided a specifically recited process for preparing the antigenic protein.
  • That invention also provided kits useful for assaying a biological sample for the presence of cells expressing the antigen of the invention and for assaying a biological sample for the presence of antibody to the cells expressing the antigen of the invention. These kits contained one or more containers, each holding separately detectably labelled or unlabelled antibody or antigen of the invention, and in another compartment, a means for detecting the formation of immunocomplexes.
  • OKT11 (designated OKT11) which was capable of producing monoclonal antibodies against an antigen found on essentially all normal human peripheral T cells and on approximately 95% of normal human thymocytes, but not on normal human B cells or null cells.
  • the antibody so produced was monospecific for a single determinant on essentially all normal human peripheral T cells and contained essentially no other anti-human immune globulin.
  • U.S. Patent No. 4,843,004 patented June 27, 1989 by C. Platsoucas provided a specifically recited method for the production of human T-T cell hybrids and production suppressor factor by human T-T cell hybrids.
  • the patented method was developed for the production of human haematopoietic cell hybrids especially T-T cell hybrids as determined by HLA typing.
  • Some of these T-T cell hybrids produce factors useful for biotherapy or exhibiting specific-immunological functions. This is accomplished by fusing cells from human T cell lines with appropriately sensitized or induced human T cells exhibiting specific immunological function or producing the desired factors.
  • U.S. Patent No. 4,861,589 patented August 29, 1989 by S. T. Ju provided a method for therapeutically treating abnormal cells expressing a major histocompatibility complex class II antigen using cytolytic inducer T4 cells. That patent provided a specifically recited method for treating a subject afflicted with tumour cells expressing a major histocompatibility complex Class II antigen either constitutively or inductively.
  • U.S. Patent No. 5,009,995 patented April 23, 1991 by A. Albino provided monoclonal antibodies to melanoma cells.
  • the patent related to monoclonal antibodies recognizing the gp130 antigen of human cells.
  • Monoclonal antibodies which recognize distinct determinants on this antigen and methods of detecting the determinants by immunoassay with the monoclonal antibodies which recognize them are also disclosed.
  • Hybridoma cell lines which produced such monoclonal antibodies were also disclosed.
  • the monoclonal antibodies are useful in the detection of the gp130 antigen and human cells including melanoma which contain this antigen.
  • HLA Human immunodeficiency protein
  • HLA-A Leukocyte Antigen
  • HLA-B The products of the HLA genes are commonly called "antigens”.
  • the genes of the A, B, and C loci encode the classical transplantation antigens whereas the genes of the D and DR loci most probably encode antigens that control immune responsiveness.
  • HLA antigens are present in the membranes of human -cells. Some are present in most cells of the body whereas others are present only in specific kinds of cells. For instance,
  • HLA-DR antigens have been identified in B cells' but not in resting T cells.
  • HLA antigens are categorized into types that vary from individual to individual. HLA typing is used in paternity determinations, transplant and transfusion compatibility testing, blood component therapy, anthropological studies, and in disease association correlation to diagnose diseases or to predict susceptibility to disease.
  • Current HLA-DR typing techniques consist of two basic methods. One involves separating B cells from a total lymphocyte sample, e.g. peripheral blood lymphocytes (PBL) , treating the B cells with anti-DR sera and complement, and reading the resultant cytotoxicity as an index of reactivity. The B cells are separated from the total lymphocyte population because DR antigens are present only in B cells and B cells constitute only a small proportion, typically 10% to 25%, of PBL.
  • PBL peripheral blood lymphocytes
  • the second basic HLA-DR typing method is the two colour fluorescence technique.
  • a PBL preparation is incubated with a fluorochrome labelled anti-human lg, washed, and then dispensed in tissue typing trays.
  • the test results are read by determining the percent of viable B cells remaining by adding a fluorescent vital dye and measuring percent viability only of those cells having ring immunofluorescence.
  • this method avoids a B cell separation step, it requires that the cells be stained with anti-human lg. It also is practical only when read under high power microscopy and, therefore, has a more demanding reading step than the B cell separation method.
  • the serologically-defined HLA-DR4 specificity is complex and has recently been reported to have eight allelic variants or subtypes. These subtypes have been defined mainly by T cell recognition methods and have been confirmed by DNA typing techniques. From analysis of sequence data it is apparent that the serologically defined DR4 specificity can be attributed to amino acid differences in the first and second hypervariable regions of the first domain of the DR4 molecule, whereas the subtypic differences are all located in the third hypervarible region. Some subtypes vary by as little as one amino acid and at the most by three; yet these differences are enough to be recognized by T cells.
  • RA in different ethnic groups for example, DR1 in the
  • a principal object of the present invention is to provide a simple and effective HLA-DR typing technique that: (1) does not involve a B cell separation step or a lymphocyte staining step; and (2) is based on cytotoxicity function such that the sera and complement used in available lymphocytotoxicity tests may be used in the invention method.
  • Another object of this invention is to provide murine monoclonal antibodies recognizing polymorphic determinants of HLA-DP.
  • the DR4 specificity was originally defined by alloantisera derived from multiparous females but attempts to subtype with such reagents have been mostly unsatisfactory. Attempts to make murine monoclonal antibodies to HLA antigens generally proved to be more difficult than had been anticipated. This is thought to be due to the type of immunogen, usually whole cells, which express an enormous array of different molecules including at least six different HLA antigens. The murine immune system recognizes most of these molecules as foreign and even when purified HLA molecules are used, the bulk of the antigen-specific cells will be against the species-specific or monomorphic determinants present on the histocompatibility molecules.
  • mice transfectant cell lines expressing human histocompatibility molecules seemed to be a tremendous advance in this technology, particularly with respect to an anti-DP moab made using a transfectant as an immunogen.
  • the only foreign molecule expressed on the surface of the transfectant should be a HLA molecule. Therefore, the bulk of the antigen-specific B cells should be directed to HLA molecules and some of these should be directed to polymorphic determinants.
  • the present invention provides a set of monoclonal antibodies that react with epitopes on DR4 molecules. Specifically the present invention provides monoclonal antibodies which are specific for HLA-DR4 molecules.
  • the present invention also provides the use of the murine monoclonal antibodies to detect subtypes of DR4.
  • the present invention also provides for the production of 14 such monoclonal antibodies, and for the characterization of the properties thereof.
  • the present invention also provides for the producing and of analyzing the specificities of moabs to the subtypes of HLA-DR4 using transfectants.
  • the present invention also provides two other antibodies with DR4 subtypic specificity, that were produced from mice immunized with human molecules.
  • Embodiments of such antibodies include the following: NFLD.D1 which binds to all DR4 molecules; NFLD.D12, which binds only to the Dw4 subtype of DR4;
  • NFLD.D14 which binds to Dw4 and Dw14 subtypes
  • NFLD.D7 which binds to all DR4 and DR2 molecules but also, less strongly with several non-DR4 molecules
  • NFLD.D2, NFLD.D3, NFLD.D4, NFLD.D8 and NFLD.D9 which bind strongly to Dw4 and Dw14, but not at all to the subtype of DR4 called Dw10, which give moderate to low reactions with some other DR4 subtypes, and also which react with DR1, DR2, and DR14 (Dw16);
  • NFLD.D10 which reacts with the Dw9 subtype of DR14 as well as binding weakly to some of the DR3-, DR7-, and DR9- typed B cell lines.
  • Figure 1 is a histogram which shows the reactions in CELISA of the antibody NFLD.D10. Each bar represents the reaction against a particular transfectant line, whose specificities are shown at the bottom of the figure. The heights of the bars represent the adjusted optical densities, which have (a) had the background subtracted and then (b) been converted into a percentage figure with reference to a positive control antibody, in this case L243, an antibody reactive with all DR molecules;
  • Figure 2 is a histogram which shows the reaction in CELISA of the antibody NFLD.D7;
  • FIG. 3 is a diagram which shows the differing specificities of this series of antibodies.
  • Each bar represents the reactions of one antibody, which identity is given on the vertical axis.
  • the relevant DR subtypes are each assigned one interval; below these are given the DR grouping in which the subtypes are contained; thus DR4 contains Dw4, Dw14, Dw10, Dw13, and Kt. Filled parts of the bars indicate strong reactions of an antibody with the DR or Dw type shown; latched indicates smaller but still significant reactions. White indicates negative reactions. 5) Description of Preferred Embodiments
  • the DR4 transfectants used for the immunizations and most of the analysis are now included in the transfectants distributed by the organizers of the llth IHW (see Table 1 below).
  • DRw53 (DR4Dw15) L17.8** R. Karr/J. Silver
  • the transfectants as described in the Table 1 above include L89.2. (Dw13); L164.11 (Dw10); L165.6 (Dw14); Dw4 transfectant (DAP3DR4); two other transfectants, L243.6 (DR4Dw4) and L259.1 (DR4Dw13). All transfectants were grown in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS), 5 ⁇ 10 -3 mM 2-mercaptoethanol, penicillin and streptomycin (Flow Laboratories).
  • DMEM Dulbecco's modified Eagles medium
  • the cells were grown on either 10 cm dishes (FALCON TM ) or 75 cm flasks (LINBRO TM ) and were harvested in log phase using trypsin (Flow Laboratories) and left in standard type bacteriological petri dishes for one to three days. Expression was assayed by CELISA or FACS analysis using the moabs Tu39 or GSP4.1 prior to immunization.
  • the first approach consisted of indiscriminate standard-type immunizations where young adult C3H mice were immunized twice intraperitoneally (IP) followed by a final boost intravenously (IV) or IP three days prior to fusion.
  • IP intraperitoneally
  • IV intravenously
  • IP three days prior to fusion.
  • CFA complete Freund's adjuvant
  • I intraspenically
  • CELISA Cellular enzyme-linked immunospecific assay
  • the supernatants were tested against the immunizing cells and all positive were differentially screened on the following day against the immunizing cell and non-transfected L cells. Those that were positive only with the immunizing cells were selected for further testing against a small panel of transfected cells, including those expressing DP, DQ and informative DR. Hybridomas were then selected for cloning and further analyzed on both transfectants and B cell lines.
  • mice had been primed with affinity purified HLA molecules extracted from a lysate of the B cell line SAVC (10th Workshop #9034), using beads (DYNAL TM ) that had been coated with two antibodies: first, anti-mouse IgG had been coated by the manufacturer; secondly the beads were coated with a mouse IgG1 monoclonal antibody made in this laboratory (NFLD.M67) that detects a monomorphic determinant on HLA-DP molecules.
  • beads DYNAL TM
  • NFLD.M67 mouse IgG1 monoclonal antibody made in this laboratory
  • Protocols varied from experiment to experiment, but a typical protocol is as follows: 10 7 transfectant cells were injected subcutaneously, dividing the dose between four sites on the back, together with Freunds complete adjuvant, 0.1 ml per site. After a wait of 4-8 weeks, the mice were boosted by 10 7 cells, either given introperitonealy or intrasplenically, and the spleen removed three days later. In some experiments, a primary immunization with human B cell line cells or with DP transfectants was made by intravenous injection; three days later the spleen was removed and a fusion performed. Fusions
  • Fusions were performed three days after the last injection of antigen and were carried out with the fusion partner SP2/0-Ag14 (Shulman M, Wilde CD, Kohler G. Nature 1978: 276: 289.). Fusions in the presence of polyethylene glycol were done according to a standard method, (Drover S, Marshall WH, Youghusband HB. Tissue Antigens 1985: 26: 340.). Usually 2 ⁇ 10 5 cells per well were plated in a 96 well plate with flat bottomed wells. Screening and Specificity Testing
  • the immunizing cell was used as a target.
  • differential testing was done on the transfectant that had been used as immunogen and on L cells.
  • human B cell lines were the immunogen
  • the second screen was done on several B cell lines plus a human T cell line that fails to express class II HLA molecules (MoIt/4).
  • hybrids were lost through what appeared to be overcrowding and lysis due to cytotoxic T cells. This problem was partially alleviated by plating the fused cells at a maximum density of 2 ⁇ 10 5 cells per wall and eliminating spleen cells or thymocytes as feeder cells.
  • mice An attempt was made at neonatally tolerizing C3H mice by injecting them with non-DR4 transfectants at various times from age 24 hours to 6 weeks. Prior to immunization, serum samples were obtained from these mice as well as from non-tolerized litter mates. The sera were titered in CELISA on the tolerizing cells and on non-transfected L cells. The CELISA data showed evidence of antibody activity to the tolerizing cells, indicating that tolerance to DR had not been achieved. It was decided to use some of the mice for fusions and, at age 8 to 16 weeks, they and some of their non-tolerized litter mates were immunized with DR4-expressing transfectants.
  • mice immunized with transfectants were all isotyped as IgGl. This is a non-complement fixing subclass so all specificity analysis has been done using CELISA.
  • Homozygous B cell lines from the 10th IHW were used for specificity analysis on eight monoclonal antibodies produced from mice immunized with DR4-expressing transfectants. These tests were done using optimally- diluted supernatants from cloned hybridomas.
  • two monoclonal antibodies from uncloned hybridomas resulting from mice immunized with human B cell lines were also studied (see below). The antibodies were also tested on a panel of L-cell transfectants expressing various DR4 and non-DR4 molecules.
  • the HLA class II types and splits were obtained from references 23 to 26.
  • the numbers in brackets refers to the ratio of % reactivity for each cell divided by the % reactivity for the immunizing substype, DW13 for NFLD.D1 and DW4 for all the other noabs.
  • This moab was derived from a mouse (Rll) immunized with DR4-Dw13 expressing transfectants as shown in Table 3. It appears completely monospecific for the DR4 specificity since it reacts with all the subtypes, although Dwl5 has not so far been tested (Table 6). This specificity was confirmed by testing on a small panel of transfectants as is shown in Table 7. In addition, testing supernatant from the uncloned hybrid against additional transfectants provided by the llth IHW (data not shown) revealed no extra reactivity.
  • NFLD.D3 All except NFLD.D3 were obtained from different microculture plates of the same fusion (R19, see Table 4) and are believed to be derived from different clones although their specificities are similar.
  • This antibody derived from a different fusion, R23 (Table 4), has a similar activity to those derived from R19 but reacts more strongly with DW13, DR1, DR2 and DR14 (w16). Unlike the preceding antibodies, it also reacts with the DR14 subtype Dw9 and weakly with some DR17, DR7 and DR9 cells. From data obtained by testing undiluted supernatant on the llth IHW transfectants ( Figure 1), this pattern was essentially confirmed. However, the weak reactivity observed with DR3 was not apparent when the appropriate DR3 transfectant was tested. No DR7 transfectant with good expression was available for testing. In addition the antibody reacted weakly with the DR10 transfectant.
  • Dw4 is again a target molecule but with this antibody there are significant reactions also with one of the four lines expressing the Dw14 subtype of DR4. Minor reactions are also noted with the remaining two members of the Dw14 subtypes as well as with the Dw10 subtype. Reactions with the remaining cells in the panel were mostly either negative or trivial.
  • mice have been immunized with and without adjuvant, by various routes (subcutaneous, intraperitoneal, intravenous, intrasplenic) and with varying antigen doses.
  • the antigenic material has mostly been in the form of L-cell transfectants expressing HLA-DP molecules, but some immunizations have been done with EBV-transformed human B cell lines; a few immunizations have been done with affinity-purified HLA-DP molecules and with synthetic peptides designed to reproduce small polymorphic parts of the HLA-DP molecules.
  • immunization with peptides has been a fruitless procedure.
  • the antibodies described here and summarized in Tables 8a and 8b, represent those selected after exhaustive screening and testing of many thousands of hybridomas, typically on the order of 1000 per fusion (one fusion means one mouse spleen).
  • IgG1 subclass NFLD.M67
  • IgG2 NFLD.M68
  • IgM NFLD.m65
  • NFLD.M58 One antibody, NFLD.M58, was produced in an early experiment. This antibody showed a striking resemblance to two other published antibodies. In our laboratory the same specificity has been found again, either with a
  • NFLD.M58 is considered to be recognizing an epitope requiring the amino acid sequence DE at positions 55 and 56 on the HLA-DP beta chain. Tests to prove this conclusion are planned, using mutated beta chain molecules.
  • a fourth antibody, different from all the others is NFLD.M77.
  • This antibody binds to cells that express DP molecules containing the amino acid sequence "QL" at positions 10 and 11 on the beta chain. The single exception to this is that cells expressing the DPB1*1301 gene are not recognized by this antibody. Since there are no available examples of homozygous cell lines expressing DPB1.1101, the antibody has not been evaluated for its reaction to the product of DPB1*1101; according to the present interpretation it should bind, unless the DP molecular structure is influenced by polymorphic sequences in the adjacent chain, which may be the case for the non-binding DPB1*1301 product. In preliminary studies the NFLD.M77 antibody binds also, as predicted, to transfectant cells expressing the DPB1*0901 gene.
  • the epitope must also be influenced by amino acids outside the third hypervariable region; otherwise one would expect it to react with either DR1 or Dwl6 in the same way as does the CCCL20 moab (Dejelo CL, Braun WE, Zachary AA, Teresi, GA, Smerglia AR & Clark LV. Hum Immunol 1986: 17: 135-136.) which reacts mainly with Dw14, Dw4, DR1 and DR14 (Dw16).
  • the reactivity pattern for D7 is considerably more complex (Table 6, Figures 2 and 3). In addition to reacting with all DR4 cells tested, it also reacts moderately with DR2 cells (all subtypes). At the dilution used for specificity analysis on the cell lines (Table 6) it reacted weakly or not at all with numerous cells expressing DR52. However, testing on transfectants using undiluted supernatant from an uncloned culture was positive for the two DR52 transfectants ( Figure 2). Since the DRB8 gene, which encodes the DR52 specificity, is constitutively expressed at lower levels than the DRB1 gene, this simply may be a dilution problem. It is also possible that the culture from which D7 was derived was not clonal. More testing on the transfectants using supernatant from a cloned culture, as well as testing the cell lines with antibody in excess, should clarify this.
  • the fifth pattern shown in Figure 3 is for five moabs, but the specificities are not quite as simple as portrayed in the figure, due to graduations of reactivity. This is particularly apparent in the data in Table 6.
  • Both NFLD.D2 and NFLD.D3 are considerably less reactive with the Dw13 expressing transfectant (L259.1) than are NFLD.D4, NFLD.D8, and NFLD.D9 moabs, all of which show the same degree of reactivity.
  • NFLD.D2 differs from the others in that it reacts poorly with the Dwl6 transfectant (L182.1) and with the DR2a transfectant lines.
  • the final pattern shown in Figure 3, produced by NFLD.D10 is similar to the pattern produced by NFLD.D4, NFLD.D8, and NFLD.D9 but it binds Dw13 more strongly. In addition it also binds to DR14 (Dw9) molecules and gives weak reactions with some DR3, DR7, and DR9 molecules. When used undiluted on the transfectants, it is also weakly bound to DRw10 ( Figure 1).
  • the HLA-DP system discovered in a remarkable series of experiments by Shaw et al. (see Shaw S, Johnson AH, Shearer GM. J Exp Med 1980: 152: 565.) was revealed by a primed lymphocyte test (PLT) procedure. In a PLT, the polymorphism is recognized by T-lymphocytes and not by antibodies. By 1984, PLT had revealed six probable alleles. An uncertainty with the DP system was that it might not be accessible to classical serology. However, various observations have contributed to showing that it is accessible. A monoclonal antibody was made by Heyes et al. (see Heyes J, Austin P, Bodmer J, et al.
  • the monoclonal antibodies may not be good at recognizing alleles, since there is so much sharing of polymorphic portions of the molecule between alleles, but they should be excellent at detecting epitopes, which after all is what are important in provoking immune responses, either of graft rejection or of graft versus host disease.
  • NFLD.D1 binds to all DR4 molecules, whilst others bind only to subtypes of DR4.
  • the shortest, NFLD.D12 binds only to the Dw4 subtype of DR4;
  • NFLD.D14 binds to Dw4 and Dw14 subtypes;
  • NFLD.D7 binds to all DR4 and DR2 molecules but also, less strongly with several non-DR4 molecules.
  • NFLD.D2, D3, D4, D8 & D9 have approximately the same pattern as each other, they all bind strongly to Dw4 and Dw14, but not at all to the subtype of DR4 called DwlO; they give moderate to low reactions with some other DR4 subtypes; they also react with DR1, DR2, and DR14 (Dw16).
  • the final pattern, that of NFLD.D10 resembles the one just described; in addition, it reacts with the Dw9 subtype of DR14 as well as binding weakly to some of the DR3-, DR7-, and DR9-typed B cell lines.
  • cell line refers to various embodiments including, but not limited to individual cells, harvested cells and cultures containing cells so long as these are derived from cells of the cell line referred to.
  • derived is meant progeny or issue. It is, further, known in the art that spontaneous or induced changes can take place in karyotype during storage or transfer. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and any cell line referred to includes such variants.
  • CELISA Cellular Enzyme- linked Immunospecific Assay

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Abstract

On décrit de nouveaux anticorps monoclonaux comprenant une série d'anticorps monoclonaux qui réagissent aux épitopes se présentant sur les molécules DR4.
PCT/CA1992/000105 1991-03-11 1992-03-11 Anticorps monoclonaux murins reconnaissant des determinants polymorphes de l'antigene leucocytaire humain hla WO1992015698A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999037684A1 (fr) * 1998-01-26 1999-07-29 Genentech, Inc. Anticorps du recepteur 4 de la mort cellulaire (dr4) et leurs utilisations
US7252994B2 (en) 2001-07-03 2007-08-07 Genentech, Inc. Human DR4 antibodies and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4471056A (en) * 1982-04-02 1984-09-11 The Board Of Trustees Of The Leland Stanford Junior University Method for HLA-DR typing of total human lymphocyte sample
EP0204522A2 (fr) * 1985-05-30 1986-12-10 Genetic Systems Corporation Série d'anticorps monoclonaux pour la caractérisation de la compatibilité histologique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4471056A (en) * 1982-04-02 1984-09-11 The Board Of Trustees Of The Leland Stanford Junior University Method for HLA-DR typing of total human lymphocyte sample
EP0204522A2 (fr) * 1985-05-30 1986-12-10 Genetic Systems Corporation Série d'anticorps monoclonaux pour la caractérisation de la compatibilité histologique

Non-Patent Citations (4)

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Title
HUMAN IMMUNOLOGY, Vol. 32, No. 2, October 1991, New York, USA, pages 110-118; L. BARBER et al., "Contribution of T-cell receptor-contacting and peptide-binding residues of the class II molecule HLA-DR4 Dw10 to serologic and antigen-specific T-cell recognition". *
JOURNAL OF IMMUNOLOGY, Vol. 143, No. 7, 1 October 1989, Baltimore MD, USA, pages 2248-2255; C. ALBER et al., "Multiple regions of HLA-DRbetal chains determine polymorphic epitopes recognized by monoclonal antibodies", see page 2251. *
TISSUE ANTIGENS, Vol. 26, No. 1, July 1985, Copenhagen, Denmark, pages 25-34; T. CREPALDI et al., "The monoclonal antibody AC1.59 defines a new polymorphic determinant on HLA-DR molecules". *
TISSUE ANTIGENS, Vol. 26, No. 5, November 1985, Copenhagen, Denmark, pages 340-343; S. DROVER et al., "A Mouse monoclonal antibody with HLA-DR4 associated specificity". *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999037684A1 (fr) * 1998-01-26 1999-07-29 Genentech, Inc. Anticorps du recepteur 4 de la mort cellulaire (dr4) et leurs utilisations
US7252994B2 (en) 2001-07-03 2007-08-07 Genentech, Inc. Human DR4 antibodies and uses thereof
US7744881B2 (en) 2001-07-03 2010-06-29 Genentech, Inc. Human DR4 antibodies and uses thereof

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