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WO1994023747A1 - Medicament contenant des anticorps, utilise dans le traitement de reactions immunes specifiques des lymphocytes t et de leucemies a lymphocytes t - Google Patents

Medicament contenant des anticorps, utilise dans le traitement de reactions immunes specifiques des lymphocytes t et de leucemies a lymphocytes t Download PDF

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Publication number
WO1994023747A1
WO1994023747A1 PCT/EP1994/001129 EP9401129W WO9423747A1 WO 1994023747 A1 WO1994023747 A1 WO 1994023747A1 EP 9401129 W EP9401129 W EP 9401129W WO 9423747 A1 WO9423747 A1 WO 9423747A1
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Prior art keywords
cells
medicament according
antibodies
treatment
cell
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PCT/EP1994/001129
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German (de)
English (en)
Inventor
Rolf Schuh
Stefan Thierfelder
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Fresenius Ag
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Priority to AU66778/94A priority Critical patent/AU6677894A/en
Publication of WO1994023747A1 publication Critical patent/WO1994023747A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A61K51/1039—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants against T-cell receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a medicament for the treatment of T-cell-specific immune reactions and T-cell leukemias, which contains, as active ingredient, a combination of at least four monoclonal antibodies of different specificity, two of the antibodies against different, non-over - Lapping epitopes of the human lymphocyte marker CD5 and the other two are directed against different, non-overlapping epitopes of the human lymphocyte marker CD7.
  • the medicament has proven to be extremely effective for the treatment of leukemia, autoimmune diseases and for the suppression of immune reactions in organ or bone marrow transplants without producing the side effects observed in connection with antibody preparations known to date in the prior art.
  • the "lymphocyte marker” CD5 is an antigenically active molecule which is expressed on practically all normal peripheral T cells and on a subpopulation of normal peripheral B cells, the so-called polyreactive IgM antibodies to produce.
  • the "lymphocyte marker” CD7 is an antigenic active molecule which is expressed within the lymphocyte population on most normal peripheral T and NK cells (N.atural Killer Cells).
  • the medicament according to the invention thus covers a broad but precisely defined spectrum of target cells, as a result of which an extremely effective suppression of immune reactions in organ or bone marrow transplants is achieved.
  • T-lymphocytes develop a cellular immune response after contact with foreign antigens.
  • the host's T cells recognize the donor organ cells as foreign; the patient develops host versus donor disease.
  • the T cells transferred with the donor bone marrow or ripening therefrom recognize the host as foreign; the patient develops a graft-versus-host-disease (GvHD).
  • GvHD graft-versus-host-disease
  • the patient's T cells recognize the body's own tissue as foreign due to an incorrect regulation.
  • the antigens recognized as foreign cause a proliferation of the T lymphocytes and the induction of helper and cytotoxic T cells represents the first stage of the T cell-specific immune reactions.
  • T cells can be chemotactic effective, ie among other things attracting macrophages, as well as mitogenic substances and ⁇ -interferon are released.
  • cytotoxic substances are released, which kill the cells containing the foreign antigen.
  • this cell destruction has a macroscopic effect on rejection of the transplant.
  • NK-Zel.len can also be considered as another effector cell population for mediating rejection symptoms - at least with GvHD.
  • CD5 + B cells could be involved in inducing a humoral immune response.
  • One way to influence rejection symptoms prophylactically or therapeutically is to destroy or inactivate the effector cells. This is possible by using specific polyclonal or monoclonal antibodies. By binding antibodies to their target cells, cell-eliminating mechanisms, such as the complement system, can be started in vivo and in vitro.
  • the complement system In addition to the lytic function, the complement system also contributes to the initiation of cellular effects and mechanisms.
  • complement receptors on macrophages are known, by means of which these effector cells recognize the target cells on which complement activation has taken place (Tenner and Cooper, J. Immunol. 1981, 126, 1174). Stimulation of macrophage-mediated, antibody-dependent cytotoxicity has also been described (Leu et al., J. Immunol. 1989, 143, 3250).
  • Clq The first component of the classic component cascade is Cl, a pentameric molecule whose largest subunit, Clq, mediates the bond between Cl and the Fc parts of immunoglobulins (Cooper, Advances in Immunol. (Editor: Dixon) Academic Press, New York, 1985 , 151).
  • Clq consists of six movable collagen arms, each with a globular binding head.
  • the isotope conveys the affinity of Clq to the Fc part of the antibodies.
  • mouse IgG2b and mouse IgG2a mouse IgG2b and human IgGl and human IgG3 have a high Clq affinity (Füst et al., Immunol. Lett. 1980 , i, 249; Schumaker et al., Biochemistry 1976, 5, 5175).
  • the expression density of the antigens determines the possible Clq binding sites. Therefore, the antigen density on the target cell must theoretically exceed a certain threshold so that the average distance between two adjacent immunoglobulins is within the range of a Clq molecule (Hughes-Jones et al., Eur. J. Immunol. 1985, 15, 976 ).
  • a Clq molecule can also bind to two different antibodies which are directed against two non-overlapping epitopes on the same antigen (so-called ⁇ ynergistic pair of antibodies).
  • ⁇ ynergistic pair of antibodies According to Hughes-Jones et al. (Eur. J. Immunol. 1984, 14, 974), regardless of the expression density, each antigen represents a potential Clq binding site if suitable antibody pairs are present which bind two epitopes of this antigen within the range of Clq.
  • a certain correlation between the in vitro measurable binding of Clq to a given antibody preparation and its cell-depleting potency in vivo was established by Kummer et al. (J. Immunol. 1987, 138, 4069). So far, however, no reliable evidence for a direct causal relationship has been provided.
  • either the bone marrow to be transplanted is treated in vitro before an autologous bone marrow transplantation, or in vivo treatments can be used to achieve remission.
  • the number of immune-competent cells can be reduced by in vivo treatment of the transplant recipient in order to prevent or suppress rejections.
  • Autoimmune diseases can be treated with comparable therapy.
  • the antibody preparations used in the clinic can either lead to an elimination of the target cells by activating the body's own effector mechanisms or change the readiness of the target cells to function, for example by blocking or activating functionally relevant receptors, for example IL2 receptors.
  • functionally relevant receptors for example IL2 receptors.
  • toxin or radionuclide conjugated antibodies is also practiced. While antibodies themselves activate natural effector mechanisms (elimination of the target cells or change in their functional state), in the case of the antibody-toxin conjugates, a toxic component can be transported directly to the target cells (in vitro and in vivo).
  • Antibodies that are genetically or chemically modified, for example by exchanging constant regions, by coupling antibodies of different or the same specificities, by adding or removing functional components, such as by adding enzymes or removing the Fc part, are also suitable the immunoglobulins and others
  • Suitable immunosuppressive antibody preparations are, for example, monoclonal antibodies against the human CD3 T cell antigen (OKT3) and polyclonal antiserum against human T cells (ATG).
  • ATG Acute thrombocytopenias and granulocytopenias also occur, which in some cases make it necessary to stop immunosuppressive antibody therapy with ATG.
  • the massive cell elimination releases a large number of cell constituents and immune mediators, which in addition to incompatibility reactions, in particular at the beginning of the treatment, leads to a drop in blood pressure, chest pain, fever reactions, diarrhea and itching (ATG-Fresenius, information brochure).
  • monoclonal antibodies have a high specific titer, so that undesired reactions, which may be initiated by the special antigen / antibody interaction, can take place massively and unchecked.
  • the antibody preparation 0KT3 shows clear side effects in vivo, which are based on the initial stimulation of the CD3 + T cells and the associated massive release of immune mediators. These extremely diverse side effects, such as fever (73%), chills (57%), shortness of breath (21%), pectanginous symptoms (14%), spasticity (11%), nausea (11%), vomiting (13%) and diarrhea (20%) can be life-threatening (Todd and Brogden, Drugs 1989, 2 871).
  • OKT3 induces the modulation of the CD3 antigen and thus causes a non-functional, so-called anergic state.
  • CD5-specific antibodies are known in the prior art which are cost-stimulating in vitro (Ledbetter et al., Mol. Immunol. 1989, 2nd r 137; Vandenberghe and Ceuppens, Eur. J. Immunol. 1991, 2nd r 251) or a stimulating (Spertini et al., J. Immunol. 1991, 146, 47) effect on T cells.
  • a stimulating Spertini et al., J. Immunol. 1991, 146, 47
  • the stimulation potency also with CD5-specific antibodies depends on the respective Fc receptor type on the accessory cells. There does not seem to be a correlation between the epitope specificity of the antibodies used.
  • the medicament according to claim 1 which has at least four monoclonal antibodies of different specificity as active substance. contains, two of which are directed against non-overlapping epitopes of the CD5 lymphocyte marker and two against non-overlapping CD7 lymphocyte marker epitopes.
  • the medicinal product according to the invention surprisingly shows not only in number and extent drastically reduced side effects, but it has an unexpected, in contrast to monoclonal antibody preparations known from the prior art, significantly increased effectiveness in the treatment of T-cell-specific immune reactions. Due to the effective elimination or inactivation of the T cells, the medicament according to the invention is suitable for the effective treatment or delaying of clinically manifest rejection reactions in organ and / or bone marrow transplants Treatment of autoimmune diseases and CD5 and / or CD7 positive leukemias.
  • the medicament according to the invention not only covers practically all T cells, but advantageously also the NK cells which carry the CD7 antigen and which are jointly responsible for rejection reactions.
  • T cells around 10% of the B cells (polyreactive, IgM-producing B cells) also have the CD5 antigen and thus also represent a target cell population for the medicament according to the invention Reaction to foreign antigens suppressed extremely effectively.
  • the use of the medicament according to the invention initially leads to a clear elimination of the CD5 and / or CD7 positive target cells from the peripheral blood.
  • some target cells lose their target antigens through modulation.
  • these modulated and therefore CD5 and / or CD7 negative target cells are surprisingly further reduced significantly after about 5 to 10 days when the medicament according to the invention is used further.
  • a prerequisite for this is a dosage which ensures that the serum level for the individual components of the medicament according to the invention does not drop to zero.
  • Antibodies to CD3 or CD5 neither individually nor in any combination for a significant stimulation of T- Cells in vitro. An Fc receptor heterogeneity caused by the blood cell donor could be excluded.
  • the medicament according to the invention thus allows an effective treatment of T-cell-specific immune reactions, with which clinically manifest rejection reactions can be effectively delayed or suppressed without causing the side effects which occur in the case of previously used antibody preparations and which are sometimes life-threatening.
  • the medicament according to the invention preferably contains antibodies of the rat IgG2b subclass.
  • monoclonal antibodies of the mouse IgG2a, mouse IgG2b, human IgGl and human IgG3 sub-class have proven to be particularly advantageous for use in the medicament according to the invention.
  • IgG subclasses of other animals that have a high affinity for Clq are also suitable for the medicament according to the invention.
  • the medicament can additionally contain two monoclonal antibodies against non-overlapping epitopes of another (third) lymphocyte marker.
  • a toxic component in the medicament according to the invention can be ricin, saporin, cis-platinum, or radionuclides, which are bound to the corresponding antibody in the form of a chemical bond.
  • one or more of the antibodies contained in the drug can be genetically modified. Examples of possible modifications are: 1. Exchange of constant regions for homologous sequences of antibodies from another subclass of the same or a different species, preferably of humans (chimerization, humanization); 2. Coupling of antibodies with one another for the purpose of efficient induction of effector mechanisms, preferably the binding of Clq (dimerization); 3. Elimination of parts of the antibody, preferably the Fc part or a Fab part (monovalent antibodies, antibody fragments).
  • a further modification relates to the direct and indirect coupling of at least one antibody to components or agents which allow physical separation or removal of the target cells or which generally enable enrichment or depletion.
  • Agents that can be coupled to the antibodies are those that allow coupling to membranes or magnetic particles.
  • the medicament according to the present invention is able to reduce the number of leukemia cells, and it is used in particular for the in vitro treatment of autologous bone marrow to be transplanted, the bone marrow preferably with the medicament and a lytic complement source is incubated. Furthermore, the use of the drug to achieve remission in vivo is advantageous if the drug is administered to the leukemia patient iv in the form of daily doses.
  • the preparation of monoclonal antibodies against CD5 and CD7 according to the invention enables the number of immunocompetent cells to be reduced and / or treated for graft-versus-host disease (GvHD) even after allogeneic bone marrow transplantation. It is advantageous if the drug is used for the in vitro treatment of the donor bone marrow before the transplant, but it leads to at least the same effect if the drug is used for the prophylactic and therapeutic in vivo treatment of the bone marrow recipient becomes.
  • the medicament according to the invention also enables the number of immunocompetent cells to be reduced to prevent and / or treat rejections after organ transplants, the medicament being administered to the recipient of the organ transplant.
  • the monoclonal antibodies used according to the invention are in purified form, preferably dissolved in phosphate-buffered saline (PBS), optionally mixed with a surface-active agent such as, for example, Tween 80, in an amount of, for example, 0.02%, based on the total solution.
  • PBS phosphate-buffered saline
  • the solution can contain the antibody mixture in amounts of 0.5 to 5, preferably 1 to 3 and particularly preferably 1 mg / ml, the different types of antibodies preferably being present in approximately equal proportions.
  • Such a solution can be provided for intravenous administration, the daily dose being 10 to 30 mg of antibody preparation.
  • the production of the monoclonal antibody pairs against the human lymphocyte markers CD5 and CD7 takes place in principle by targeted immunization of rats or mice by ip, iv or sc application of the antigen, preferably in the form of whole human T cells (lymphocytes, T cell line) or of corresponding cell lysates or (cleaned) surface molecules.
  • B-lymphocytes After the immunization has taken place, a large number of B-lymphocytes are stimulated for proliferation and antibody production, depending on the complexity of the antigen, its antigenic action, the type and number of immunization steps and the physiological readiness of the animal.
  • the so-called B-blasts are obtained from the animal, preferably by removing the spleen and immortalized in vitro.
  • Immortalization is carried out by polyethylene glycol-induced cell hybridization or by electrofusion with an in vitro viable and dividable myeloma cell line.
  • Suitable myeloma lines preferably have an anzaguranin resistance which enables selection of the immortalized B cell / myeloma hybrids (hybridomas).
  • the cells are diluted with HAT selection medium and transferred to appropriate culture vessels (e.g. 96-well plates) so that individual clones can be created and manipulated separately.
  • appropriate culture vessels e.g. 96-well plates
  • Each individual clone typically secretes identical antibodies that are not always directed against the desired antigen.
  • the antibodies are examined for desired properties (eg isotype and / or specificity) using suitable test systems.
  • a sub-class-specific ELISA can be used to determine the isotype, and an immunoassay (eg EIA) to determine the specificity against human T cells, which detects the binding behavior of the antibodies to T and non-T cells (eg B- Cells).
  • EIA immunoassay
  • Clones which produce suitable antibodies are expanded in liquid medium and repeatedly reclined at the individual cell level until a stable cell line with almost constant properties is generated.
  • the exact antigen specificity of the antibodies can be examined in FACS double-labeling experiments on human blood lymphocytes with reference antibodies of the desired specificity (anti-CD5, anti-CD?), The distribution and location of the double and single positive cells being used for the assessment. The antigen specificity is confirmed after immunoprecipitates from the solubilizate of human T cells in comparison with the molecular weights precipitated by reference antibodies. The relative epitope specificity of the generated antibodies can be determined by cross-inhibition in the EIA.
  • the antibodies are secreted by the hybrid cells into the culture medium; the technical implementation of this process is called fermentation.
  • the cells are removed from the fermentation supernatant by centrifugation or filtration and the antibodies are obtained by purification.
  • Specific immunological and / or physico-chemical parameters of the antibodies are used in order to selectively enrich them, preferably using chromatographic methods.
  • the purified antibodies can be lyophilized or stored in solution in suitable buffers, either deep-frozen or preferably at 4 ° C.
  • RhCD5p RhCD5 ⁇ , RhCD7p and RhCD7q:
  • the fusion partner was the azaguanine-resistant, non-producing mouse myeloma line P3X63Ag8.653 (ATCC, CRL 1580), which had previously been expanded in a standard medium (RPMI 1640, 10% fetal calf serum, glutamine, non-essential amino acids, Na pyruvate).
  • RPMI 1640 10% fetal calf serum, glutamine, non-essential amino acids, Na pyruvate
  • the fusion was carried out according to standard procedures (Galfre et al., Nature 1977, 266, 550) with polyethylene glycol 1500. 100 ⁇ 10 6 rat spleen cells and 50 ⁇ 10 6 mouse myeloma cells were used per batch.
  • the cells were transferred to 96-well culture plates. For this purpose, they were diluted so that an average of one clone per hole was to be expected.
  • the HAT supplement which enables the selection of fused cells, was added to the standard medium. Approximately 24 hours before the fusion, these culture plates were coated with a cell lawn from mouse peretoneal macrophages, which favor the growth of hybridoma cells in the sensitive phase after the fusion. Table 1: Immunization scheme
  • Antibody immunogen 1 2. 3. Fusion
  • RhCD5p stim PBL + Day 0: Day 44: Day 83: Day 86
  • RhCD5q stim PBL Day 0: Day 84: Day 112: Day 115
  • RhCD7q stim PBL Day 0: Day 49: Day 112: Day 115
  • the target line and antigen specificity was determined in a double-marking experiment with reference antibodies against suitable CD antigens (see Example 2, Test III).
  • the hybridoma cell lines that secreted the CD5- or CD7-specific antibodies were thawed again and cloned in 96-well culture plates until a monoclonality or a stable production rate was reached at the individual cell level.
  • a subclass-specific ELISA see Example 2, Test I
  • a cell binding test see Example 2, Test II
  • RhCD5p and RhCD5q are directed against the human CD5 antigen, which is expressed on practically all peripheral T. cells and approximately 10% of the B cells in peripheral blood; RhCD7p and RhCD7q are directed against the human CD7 antigen, which is expressed on most human T and NK cells in peripheral blood and on myeloid progenitor cells in the bone marrow.
  • the relative epitope specificity of the antibodies was investigated by binding inhibition experiments (see Example 2, Test IV). As a result, neither RhCD5p and RhCD5q nor RhCD7p and RhCD7q were significantly inhibited. They therefore bind to two different epitopes on the CD5 and CD7 antigen.
  • a cell line was cryopreserved as a master cell bank (MCB).
  • MCB master cell bank
  • WB working cell bank
  • Rat IgG 2i antibodies (ATCC 174).
  • a detection antibody a polyclonal, peroxidase-conjugated mouse anti-rat IgG antibody (Dianova) was used. "The test was developed by adding o-phenylenediamine dihydrochloride (OPD) and evaluated in an ELISA reader.
  • OPD o-phenylenediamine dihydrochloride
  • This test was performed as a cell immunoassay in a 96-hole round bottom. plates carried out.
  • a T cell line e.g. Molt-3; ATCC, CRL 1552
  • a non-T cell line e.g. HL-60; ATCC, CCL 240
  • the cells were incubated with the culture supernatant to be tested and then washed.
  • the binding of the antibody to the target cell was demonstrated by incubation with a polyclonal, peroxidase-conjugated mouse anti-rat IgG antibody (Dianova).
  • the test was developed by adding OPD and evaluated in an ELISA reader.
  • the cell parameters were measured in the cytofluorometer and the data saved. In the analysis, only the cells were taken into account by setting a corresponding window in the forward versus sidescatter representation, which fell into the lymphocyte population due to their size and granulation.
  • test antibody is not T cell specific.
  • the antibody to be tested formed a flat distribution with one or more reference antibodies: in addition, a significant number of only single-positive cells can also occur: the test antibody showed a similar distribution pattern to the reference antibody and recognizes T cells.
  • the single-positive cells can be single-labeled B- or
  • NK cells act, which gives a further indication of the antigen specificity of the test antibody.
  • reference and test antibodies bind to two different, but coupled, expressed epitopes, which are probably on the antigen defined by the reference antibody.
  • Suitable target cells eg lymphocytes from human tonsils stimulated with phytohemagglutinin for 3 days
  • the rat antibodies RhCD5p, RhCD5q, RhCD7p and RhCD7q were used as detection antibodies, which had previously been purified via Protein-G-Sephadex (Pharmacia) and labeled with a 100-fold molar excess of NHS-LC-Biotin (Pierce).
  • the biotin-labeled rat antibodies were detected by peroxidase-conjugated avidin (Dianova).
  • the test was developed with OPD and evaluated with an ELISA reader.
  • the maximum binding of the detection antibody was determined in control batches without a competent test antibody. The extent of the inhibition was achieved using the following formula:
  • a human T cell-containing cell population e.g. lymphocytes from tonsils stimulated with phytohemagglutinin for 3 days
  • NHS-LC-Biotin Pierce
  • NP-40 lysed with NP-40
  • the antibodies to be tested were purified or via a Capture antibodies attached to the solid phase in a 96-well ELISA plate and incubated with the biotin-labeled cell solubilisate. Excess material was removed by intensive washing.
  • the specifically bound antigen was eluted with a pH 2.7 citric acid buffer in the presence of 1% SDS and subjected to gel electrophoresis.
  • a colored protein mixture (LMW Biorad) served as the molecular weight standard.
  • the protein from the gel was then transferred to nitrocellulose paper and visualized with real dye via streptovidin-coupled phosphatase (Dianova).
  • Leu 1 (CD5) and Leu 9 (CD7; both Becton Dickinson) served as reference antibodies.
  • the antigen specificity could be demonstrated by comparing the band patterns between reference and test antibodies.
  • the cell lines characterized according to Examples 1 and 2 and established as MCB or WCB were thawed individually in serum-containing medium (RPMI 1640, 10% fetal calf serum, glutamine, non-essential amino acids, Na pyruvate) and in plastic culture bottles at 37 ° C and 5% C0 2 grown. The cells were then switched to serum-free LP medium (Gibco), expanded and transferred to a corresponding number of 10-well stack (NUNC).
  • serum-containing medium RPMI 1640, 10% fetal calf serum, glutamine, non-essential amino acids, Na pyruvate
  • the fermentation took place over a period of approx. 2 months in a volume of approx. 2 l per stack of trays.
  • the tub stacks were harvested semi-continuously three times a week, with twice 1.5 1 and once 1 1 cell-containing culture supernatant was harvested and replaced by fresh medium.
  • the daily harvests of the parallel tub stack of a cell line were filtered together to remove the cells from the culture supernatant using a glass fiber pre-filter and a sterile filter (0.22 ⁇ m).
  • the filtered culture supernatants of the total fermentation of a cell line were concentrated after the completion of the fermentation via a hollow fiber module with an exclusion limit of 30 kD to an antibody content of approximately 0.5 g / l.
  • the antibody concentration was measured in a subclass-specific ELISA (see Example 2, Test I), with the samples being titrated out in a serial dilution.
  • the antibody content was determined by calculation by comparing the dilution series of the sample to be examined with the dilution series of a standard antibody of the same subclass and known concentration.
  • the cleaning was carried out using an FPLC system and was divided into two sub-steps:
  • Sub-step 2 ( Figure 2) related to a further treatment of the entire mixture. This consisted of a chromatographic Method and a further virus inactivation step and ended in the production of the sterile end product.
  • the sterile end product (mATG) was stored at 4 ° C in plastic bottles. If necessary, the corresponding volumes were transferred to siliconized, sterile 30 ml glass bottles with rubber stoppers and stored in an upright position at 4 ° C.
  • a 32-year-old female patient (A) was transplanted from her HLA-DR different mother in the beginning recurrence of a secondary AML.
  • the AML first appeared 6 months earlier and had to be regarded as a secondary AML in connection with chemotherapy and radiotherapy performed 6 years earlier due to a M. Hodgkin. Due to the rapid relapse of the AML, the search for an optimally matched donor was dispensed with and, despite the increased GvHD risk, the HLA-DR haploidentic mother was chosen as the bone marrow donor.
  • the patient developed GvHD grade II of the skin and the liver with accompanying cytomegalovirus-associated interstitial pneumonia.
  • the GvHD was initially treated with high-dose steroids and a monoclonal antibody against TNF ⁇ .
  • the skin symptoms turned out to be primarily refractory and required a further 7-day treatment with 0KT3 on day 20, and after only a short response, a 10-day treatment with polyclonal ATG on day 29.
  • the concomitant therapy with corticosteroids and ciclosporin was continued in maintenance doses, in addition, methotrexate was administered in a low dose once a week.
  • the patient had slight dyspnea 1 hour after the end of the infusion, which stopped after a further 30 minutes without additional measures.
  • the following applications of the antibody preparation were tolerated without acute reactions.
  • the skin rash faded, the improvement became apparent from the 4th day of treatment.
  • Serum alkaline phosphatase (AP) initially expressed as liver GvHD, decreased from 469 U / ml to 339 U / ml at the end of the cycle.
  • the leukocytes remained stable under the administration of antibodies, the pre-existing thrombopenia and the plasma coagulation were not adversely affected.
  • a 31-year-old male patient (B) was transplanted with bone marrow from an HLA-identical donor in the specified phase of chronic myeloid leukemia.
  • myelofibrosis with secondary hapato and spenomegaly and signs of liver damage were complicating.
  • this patient developed a GvHD grade II-III of skin and liver, which was initially treated with anti-TNF antibody and high-dose steroid administration while continuing the cyclosporin prophylaxis. If there was a progressive increase in bilirubin and transaminases under this treatment, a 7-day cycle with ATG was added on day 20.
  • thalidomide was additionally used on the 83rd day after KMT, this treatment had to however, due to transient neurological symptoms in the sense of paraesthesia in the trigeminal region, they are stopped again after a few days.
  • the indication for the administration of the preparation according to the invention (cocktail) was therefore seen after the conventional therapeutic measures had been exhausted and discussed in detail with the patient. Therefore, from the 91st to the 98th day after KMT, 2 x 7.5 mg of the cocktail were applied, here too, short-term dyspnea due to spasticity and urticaria, which responded quickly to Fenistil, occurred under the first antibody administration. With good tolerance, the skin rash gradually regressed.
  • the 20-year-old patient D was referred for allogeneic KMT in the second remission of a T-ALL.
  • he When admitted, he already showed a recurrence of the underlying disease with multiple to five-mark-sized skin infiltrates in the trunk area and an increase in the number of blasts in the peripheral blood to 4%. Since the patient had suffered severe complications in the sense of pneumonia with ARDS during previous chemotherapy, chemotherapy again seemed too risky before the KMT was carried out. On the other hand, an unfavorable starting situation for the KMT was to be feared at the beginning of the conditioning without previous tumor reduction.
  • the cocktail was administered in one dose for three days of 2 x 7.5 mg / day.
  • prednisolone was administered in a dose of 100 mg from the 1st application: Immediately after the end of the 1st cocktail infusion, the patient complained of itching, which followed within a few minutes from the formation of patchy urticaria, partly in the area of the leukemia focus, partly in the healthy skin area.
  • the patient indicated that breathing was difficult, which necessitated the administration of oxygen.
  • antihistamines and 2 x 100 mg prednisolone urticaria improved within 10 minutes and dyspnea after about an hour. No further reactions such as an increase in fever or cardiac arrhythmia were observed, and the further administration of antibodies was well tolerated without any reaction.
  • the larger skin infiltrates showed a significant decrease in both the extent and the thickness, which continued in the further course.
  • the day of the first whole body exposure to the KMT they were practically no longer detectable. bar.
  • the peripheral blood count on the second day of administration of the monoclonal antibody preparation according to the invention showed a disappearance of the circulating blasts, and the bone marrow on the fourth day of treatment showed complete remission.
  • the application of the cocktail led to the intended tumor reduction in the absence of severe reactions in the sense of a tumor lysis syndrome.
  • the further course of the transplantation was low in complications. A short time later, the patient could be discharged to the outpatient clinic with normal haematological reconstitution and a full remission picture.
  • the monoclonal antibody preparation according to the invention achieved the desired effect of full remission with sufficient acute tolerability and that no further toxicities with regard to haematopoiesis or important organ functions occurred beyond the changes described.

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Abstract

L'invention concerne un médicament utilisé dans le traitement d'immunoréactions spécifiques des lymphocytes T et aux leucémies à lymphocytes T. Ce médicament contient au moins quatre anticorps monoclonaux de spécificité différente contre les marqueurs de lymphocytes humains CD5 et CD7. Deux des anticorps monoclonaux sont dirigés contre deux épitopes du marqueur de lymphocytes CD5, qui ne se chevauchent pas, et les deux autres sont dirigés contre deux épitopes du marqueur de lymphocytes CD7. Ce médicament permet de supprimer efficacement des réactions immunes survenant lors de transplantations d'organes ou de moëlle osseuse sans entraîner d'effets secondaires, comme c'était le cas avec les préparations à base d'anticorps utilisées jusqu'à maintenant.
PCT/EP1994/001129 1993-04-14 1994-04-11 Medicament contenant des anticorps, utilise dans le traitement de reactions immunes specifiques des lymphocytes t et de leucemies a lymphocytes t WO1994023747A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU66778/94A AU6677894A (en) 1993-04-14 1994-04-11 Drug containing antibodies for treatment of t cell specific immune reactions and t cells leukemias

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19934312916 DE4312916C2 (de) 1993-04-14 1993-04-14 Arzneimittel zur Behandlung von Immunreaktionen
DEP4312916.1 1993-04-14

Publications (1)

Publication Number Publication Date
WO1994023747A1 true WO1994023747A1 (fr) 1994-10-27

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PCT/EP1994/001129 WO1994023747A1 (fr) 1993-04-14 1994-04-11 Medicament contenant des anticorps, utilise dans le traitement de reactions immunes specifiques des lymphocytes t et de leucemies a lymphocytes t

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AU (1) AU6677894A (fr)
DE (1) DE4312916C2 (fr)
WO (1) WO1994023747A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010022737A1 (fr) * 2008-08-29 2010-03-04 Symphogen A/S Anticorps anti-cd5
WO2020216947A1 (fr) 2019-04-24 2020-10-29 Heidelberg Pharma Research Gmbh Conjugués anticorps-médicaments d'amatoxine et leurs utilisations
US12251448B2 (en) 2017-11-29 2025-03-18 Heidelberg Pharma Research Gmbh Compositions and methods for the depletion of CD5+ cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD267737A1 (de) * 1987-12-09 1989-05-10 Univ Leipzig Verfahren zur herstellung monoklonaler antikoerper gegen epitope eines 67 kda zelloberflaechenantigens humaner t-lymphozyten und b-cll
WO1992022324A1 (fr) * 1991-06-14 1992-12-23 Xoma Corporation Fragments d'anticorps produits par des microbes et leurs conjugues

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD267737A1 (de) * 1987-12-09 1989-05-10 Univ Leipzig Verfahren zur herstellung monoklonaler antikoerper gegen epitope eines 67 kda zelloberflaechenantigens humaner t-lymphozyten und b-cll
WO1992022324A1 (fr) * 1991-06-14 1992-12-23 Xoma Corporation Fragments d'anticorps produits par des microbes et leurs conjugues

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Title
"Leucocyte Typing IV. White Cell Differentiation Antigens (eds. W. Knapp et al.)", 1989, OXFORD UNIVERSITY PRESS, OXFORD, GROSSBRITANNIEN *
B. DOWELL ET AL.: "A monoclonal antibody reactive with a second epitope of the 67,000-dalton human T cell antigen.", HUMAN IMMUNOLOGY, vol. 7, 1983, NEW YORK, NY, USA, pages 95 - 104 *
C. BINDON ET AL.: "Therapeutic potential of monoclonal antibodies to the leucocyte common antigen.", ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY, vol. 186, 1985, NEW YORK, NY, USA, pages 805 - 812 *
F. UCKUN ET AL.: "Autologous bone marrow transplantation in high-risk remission T-lineage acute lymphoblastic leukemia using immunotoxins plus 4-hydroperoxycyclophosphamide for marrow purging.", BLOOD, vol. 76, no. 9, 1 November 1990 (1990-11-01), NEW YORK, NY, USA, pages 1723 - 1733 *
G. KÖGLER ET AL.: "High efficiency of a new immunological magnetic cell sorting method for T cell depletion of human bone marrow.", BONE MARROW TRANSPLANT, vol. 6, no. 3, 1990, pages 163 - 168 *
M. JONKER ET AL.: "Immunosuppression by monoclonal T cell antibodies.", TRANSPLANTATION PROCEEDINGS, vol. 18, no. 5, October 1986 (1986-10-01), NEW YORK, NY, USA, pages 1287 - 1290 *
R. DI STEFANO ET AL.: "Anti-interleukin 2 receptor monoclonal antibodies spare phenotypically distinct T suppressor cells in vivo and exert synergistic biological effects.", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 167, no. 6, 1 June 1988 (1988-06-01), NEW YORK, NY, USA, pages 1981 - 1986 *
S. QIN ET AL.: "CD4 monoclonal antibody pairs for immunosuppression and tolerance induction.", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 17, no. 8, August 1987 (1987-08-01), WEINHEIM, GERMANY, pages 1159 - 1165 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010022737A1 (fr) * 2008-08-29 2010-03-04 Symphogen A/S Anticorps anti-cd5
US12251448B2 (en) 2017-11-29 2025-03-18 Heidelberg Pharma Research Gmbh Compositions and methods for the depletion of CD5+ cells
WO2020216947A1 (fr) 2019-04-24 2020-10-29 Heidelberg Pharma Research Gmbh Conjugués anticorps-médicaments d'amatoxine et leurs utilisations

Also Published As

Publication number Publication date
DE4312916A1 (de) 1994-10-20
DE4312916C2 (de) 1995-03-23
AU6677894A (en) 1994-11-08

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