USRE38313E1 - Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies - Google Patents

Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies Download PDF

Info

Publication number
USRE38313E1
USRE38313E1 US09/931,103 US93110301A USRE38313E US RE38313 E1 USRE38313 E1 US RE38313E1 US 93110301 A US93110301 A US 93110301A US RE38313 E USRE38313 E US RE38313E
Authority
US
United States
Prior art keywords
lag
pro
soluble polypeptide
arg
ala
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 - Lifetime
Application number
US09/931,103
Other languages
English (en)
Inventor
Florence Faure
Thierry Hercend
Bertrand Huard
Frédéric Triebel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Serono SA
Institut Gustave Roussy (IGR)
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Institut Gustave Roussy (IGR)
Institut National de la Sante et de la Recherche Medicale INSERM
Applied Research Systems ARS Holding NV
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institut Gustave Roussy (IGR), Institut National de la Sante et de la Recherche Medicale INSERM, Applied Research Systems ARS Holding NV filed Critical Institut Gustave Roussy (IGR)
Priority to US09/931,103 priority Critical patent/USRE38313E1/en
Application granted granted Critical
Publication of USRE38313E1 publication Critical patent/USRE38313E1/en
Assigned to LABORATOIRES SERONO SA reassignment LABORATOIRES SERONO SA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.
Assigned to MERCK SERONO SA reassignment MERCK SERONO SA CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: LABORATOIRES SERONO S.A.
Assigned to MERCK SERONO SA reassignment MERCK SERONO SA CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: LABORATOIRES SERONO SA
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to soluble forms derived from the LAG-3 membrane protein which are useful as immunosuppressants, as well as antibodies capable of preventing the specific binding of the LAG-3 protein to MHC (major histocompatibility complex) Class II molecules as immunostimulants.
  • the LAG-3 protein is a protein selectively expressed by NK cells and activated T lymphocytes. Similarity of the amino acid sequence, the comparative exon/intron organization and the chromosomal localization show that LAG-3 is related to CD4. The initial characterization of the LAG-3 gene has been described by TRIEBEL et al. (1).
  • LAG-3 is a member of the immunoglobulin superfamily.
  • the mature protein comprises 476 amino acids (SEQ ID No. 1) with a theoretical molecular weight of 52 kD.
  • the extracellular region contains 8 cysteine residues and 4 potential N-glycosylation sites.
  • this ligand for MHC Class II was detected with higher levels on activated CD8 + lymphocytes (MHC Class I-restricted) than on activated CD4 + lymphocytes.
  • MHC Class II-restricted activated CD8 + lymphocytes
  • CD4 + lymphocytes activated CD4 + lymphocytes.
  • LAG-3 + cells were to be found in non-hyperplastic lymphoid tissue comprising the primary lymphoid organs, that is to say thymus and bone marrow.
  • LAG-3 + cells were to be found in hyperplastic lymphoid nodules and tonsils, as well as among peripheral blood mononuclear cells (PBMC) of patients receiving injections of high doses of IL-2.
  • PBMC peripheral blood mononuclear cells
  • LAG-3 is an activation antigen in contrast to CD4 expressed in a subpopulation of resting lymphocytes and other cell types, in particular macrophages.
  • the MHC comprises Class I and Class II molecules which are membrane glycoproteins which present fragments of protein antigens to the T lymphocyte receptors (TCR).
  • Class I molecules are responsible for the presentation to CD8 + cytotoxic cells of peptides derived in large part from endogenously synthesized proteins, while Class II molecules present to CD4 + helper lymphocytes peptides originating in the first place from foreign proteins which have entered the endocytic, that is to say exogenous, pathway.
  • T helper lymphocytes regulate and amplify the immune response, while cytotoxic lymphocytes are needed to destroy cells irrespective of the tissues expressing “non-self” antigens, for example viral antigens.
  • the mechanism of recognition involves intercellular signals leading to an effective activity of T lymphocytes.
  • T (CD4 + ) lymphocytes the foreign antigens must be captured and internalized in the form of peptides by specialized cells, the antigen presenting cells (APC).
  • the resulting antigenic peptides are reexpressed at the surface of the antigen presenting cells, where they are combined with MHC Class II molecules.
  • This MHC Class I II/peptide complex is specifically recognized by the T lymphocyte receptor, resulting in an activation of the T helper lymphocytes.
  • mice deficient in MHC Class II molecules (3) and possessing almost no peripheral CD4 + T lymphocytes and having only a few immature CD4 + lymphocytes in the thymus have proved to be completely incapable of responding to T-dependent antigens.
  • CD4 ⁇ / ⁇ mutant mice (4) have a substantially decreased T lymphocyte activity but show normal development and function of the CD8 + T lymphocytes, demonstrating that the expression of CD4 on the daughter cells and CD4 + CD8 + thymocytes is not obligatory for the development. Compared to normal mice, these CD4-deficient mice have a large amount of CD4 ⁇ CD8 ⁇ cells.
  • mice When they are infected with Leishmania, these mice show a population of functional T helper lymphocytes despite the absence of CD4. These cells are restrictive to MHC Class II and produce interferon- ⁇ when they are activated by the antigen. This indicates that the lineage of the T lymphocytes and their peripheral function need not necessarily depend on the function of CD4.
  • MHC Class II region proteins encoded by MHC Class II region are involved in many aspects of immune recognition, including the interaction between different lymphoid cells such as lymphocytes and antigen presenting cells. Different observations have also shown that other mechanisms which do not take place via CD4 participate in the effector function of T helper lymphocytes.
  • chimeric molecules composed of the extracytoplasmic domain of proteins capable of binding to ligands and a constant region of human immunoglobulin (Ig) chains for obtaining soluble forms of proteins and of cell receptors which are useful, in particular, as therapeutic agents.
  • Ig immunoglobulin
  • soluble forms of CD4 have proven their efficacy in inhibiting an HIV infection in vitro in a dose-dependent manner.
  • the extracytoplasmic region of LAG-3 represented by the sequence SEQ ID No. 1 comprises the domains D1, D2, D3 and D4 extending from amino acids 1 to 159, 160 to 239, 240 to 330 and 331 to 412, respectively.
  • the subject of the invention is a soluble polypeptide fraction consisting of all or part of at least one of the 4 immunoglobulin type extracellular domains of the LAG-3 protein (amino acid 1 to 159, 160 to 239, 240 to 330 and 331 to 412 of the sequence SEQ ID No. 1), or of a peptide sequence derived from these domains by replacement, addition and/or deletion of one or more amino acids, and which possesses a specificity at least equal to or greater than that of LAG-3 for its ligand.
  • the present invention encompasses, in particular, soluble polypeptide fractions having a sequence derived from the native LAG-3 sequence originating from the well-known phenomenon of polytypy.
  • the soluble polypeptide fraction is characterized in that it comprises the peptide region of LAG-3 responsible for the affinity of LAG-3 for MHC Class II molecules.
  • the soluble polypeptide fraction comprises, in particular, a peptide sequence derived from these domains by replacement, addition and/or deletion of one or more amino acids, and which possess a specificity equal to or greater than that of LAG-3 for its ligand, for example the whole of the first two immunoglobulin type domains of LAG-3, or the 4 immunoglobulin type domains of the extracytoplasmic domain of LAG-3.
  • the soluble polypeptide fraction is comprised of all or part of at least one of the four immunoglobulin type extracellular domains of the LAG-3 protein (amino acid 1 to 149, 150 159 , 160 to 239, 240 to 330 and 331 to 412 of sequence SEQ ID No. 1) comprising one or more of the arginine (Arg) rests at the positions 73, 75 and 76 of sequence SEQ ID No. 1 substituted with glutamic acid (Glu).
  • the soluble polypeptide fraction comprises a loop in which the average position of the atoms forming the basic linkage arrangement is given by the position of amino acids 46 to 77 (SEQ ID No. 1) appearing in Table 1 or Table 2 or differs therefrom by not more than 5%.
  • the soluble polypeptide fraction advantageously comprises, in addition, the second immunoglobulin type extracellular domain (D2) of LAG-3 (amino acids 150 160 to 241 239 ).
  • the soluble polypeptide fraction comprises, besides the peptide sequence of LAG-3 as defined above, a supplementary peptide sequence at its C-terminal and/or N-terminal end, so as to constitute a fusion protein.
  • fusion protein means a portion of any protein permitting modification of the physicochemical features of the subfragments of the extracytoplasmic domain of the LAG-3 protein. Examples of such fusion proteins contain fragments of the extracytoplasmic domain of LAG-3 as are defined above, bound to the heavy chain —CH2—CH3 junction region of a human immunoglobulin, preferably an isotype IgG4 immunoglobulin.
  • Such fusion proteins may be dimeric or monomeric. These fusion proteins may be obtained by recombination techniques well known to a person skilled in the art, for example a technique such as that described by Traunecker et al. (5).
  • the method of production of these fusion proteins comprising an immunoglobulin region fused with a peptide sequence of LAG-3 as defined above consists in inserting into a vector the fragments of cDNA coding for the polypeptide regions corresponding to LAG-3 or derived from LAG-3, where appropriate after amplification by PCR, and the cDNA coding for the relevant region of the immunoglobulin, this cDNA being fused with cDNA coding for the corresponding polypeptide regions or derivatives of LAG-3, and in expressing after transfection the fragments cDNA in an expression system, in particular mammalian cells, for example hamster ovary cells.
  • the fusion proteins according to the invention may also be obtained by cleavage of a LAG-3/ Ig conjugate constructed so as to contain a suitable cleavage site.
  • the subject of the invention is also a therapeutic composition having immunosuppressant activity comprising a soluble polypeptide fraction according to the invention.
  • This composition will be useful for treating pathologies requiring immunosuppression, for example autoimmune diseases.
  • the subject of the invention is also the use of antibodies directed against LAG-3 or soluble polypeptide fractions derived from LAG-3 as are defined above, or fragments of such antibodies, in particular the Fab, Fab′ and F(ab′) 2 fragments, for the preparation of a therapeutic composition having immunostimulatory activity.
  • Immunostimulatory means a molecular entity capable of stimulating the maturation, differentiation, proliferation and/or function of cells expressing LAG-3, that is to say T lymphocytes or active NK cells.
  • the anti-LAG-3 antibodies may be used as potentiators of vaccines or immunostimulants in immunosuppressed patients, such as patients infected with HIV or treated with immunosuppressant substances, or be used to stimulate the immune system by elimination of self cells displaying abnormal behaviour, for example cancer cells.
  • Such antibodies may be polyclonal or monoclonal; however, monoclonal antibodies are preferred.
  • the polyclonal antibodies may be prepared according to well-known methods, such as that described by BENEDICT A. A. et al. (6).
  • Monoclonal antibodies are preferred, on account of the fact that they are specific for a single epitope and yield results with better reproducibility.
  • Methods of production of monoclonal antibodies are well known from the prior art, especially the one described by KOHLER and MILSTEIN. This method, together with variants thereof, are described by YELTON et al. (7).
  • the subject of the invention is also anti-idiotype antibodies directed against the antibodies according to the invention, which contain the internal image of LAG-3 and are consequently capable of binding to MHC Class II.
  • Such antibodies may be used, in particular, as immunosuppressants, and, for example, in autoimmune pathologies.
  • compositions according to the present invention comprise soluble LAG-3 proteins or antibodies as are defined above, as well as a pharmaceutically acceptable vehicle. These compositions may be formulated according to the usual techniques.
  • the vehicle can vary in form in accordance with the chosen administration route: oral, parenteral, sublingual, rectal or nasal.
  • the vehicle will generally comprise sterile water as well as other possible ingredients promoting the solubility of the composition or its ability to be stored.
  • the parenteral administration routes can consist of intravenous, intramuscular or subcutaneous injections.
  • the therapeutic composition can be of the sustained-release type, in particular for long-term treatments, for example in autoimmune diseases.
  • the dose to be administered depends on the subject to be treated, in particular on the capacity of his/her immune system to achieve the desired degree of protection.
  • the precise amounts of active ingredient to be administered may be readily determined by the practitioner who will initiate the treatment.
  • compositions according to the invention can comprise, in addition to soluble LAG-3 or the antibodies according to the invention, another active ingredient, where apprto LAG-3 or to anmical appropriate, bound via a chemical bond to LAG-3 or to an antibody according to the invention.
  • soluble LAG-3 proteins according to the invention fused to a toxin, for example ricin or diphtheria toxoid, capable of binding to MHC Class II molecules and of killing the target cells, for example leukaemic or melanoma cells, or fused to a radioisotope.
  • FIG. 1 shows a comparison of the proliferation of T cells incubated with F(ab) fragments of 17B4 to the proliferation of T cells incubated with intact 17B4 monoclonal antibody.
  • FIG. 2 shows the proliferation of Clone 28 in response to tetanus toxoid when co-cultured with 17B4 or control antibody 10H3.
  • FIG. 3 shows the expression vector pCDM7 used for manufacturing the recombinant LAG-3 proteins and a recombinant CD8 immunoadhesin control.
  • FIG. 4 shows the pCLH3 AXS V2 DHFR h ⁇ IVS vector used to express amplified LAG-3 sequences.
  • FIG. 5A shows the inhibition of MHC Class II interaction with LAG-3 by recombinant LAG-3 D1-D4 and FIG. 5B shows the potential inhibition of MHC Class II interaction with CD4.
  • FIG. 6 shows the inhibition of Clone 28 proliferation by recombinant LAG-3 D1-D4.
  • FIG. 7 shows the inhibition of Clone 154 proliferation by LAG-3 Ig.
  • FIG. 8 shows the binding of LAG-3 to B cell lines expressing MHC class II haplotypes or human class II-transfected mouse cells.
  • FIG. 9 shows the binding of LAG-3 Ig to MHC Class II expressing Daudi cells.
  • FIG. 10 shows that preincubation of HLA class II expressing cells with 17B4 inhibits LAG-3 Ig binding.
  • FIGS. 11A-11C show the inhibition of clone T154 proliferation by crosslinked LAG-3 Ig.
  • FIG. 12 compares inhibition of T cell proliferation by anti-Class II antibodies to inhibition of T cell proliferation by LAG-3 Ig.
  • FIGS. 13A-13D show that T cell proliferation in response to OKT3 (FIG. 13 A), lectins (FIG. 13B) and low-concentration IL 2 (FIG. 13C) is inhibited by LAG-3 Ig but proliferation in response to high-concentration IL 2 is not inhibited by LAG-3 Ig (FIG. 13 D).
  • FIG. 14 shows clone S1B5 cytotoxicity towards Epstein-Barr virus transformed human B cells.
  • FIG. 15 shows peripheral blood lymphocyte cytotoxicity towards HLA Class I ⁇ Daudi cells.
  • the anti-LAG-3 monoclonal antibodies used were 17B4, described in BAIXERAS et al. (2) and deposited at the CNCM under No. I-1240 on Jul. 10, 1992, and 11E3, described in HUARD et al. (8).
  • These antibodies belong to the isotype IgG1. These antibodies were tested for their biological effects on activated T lymphocytes, stimulated by specific antigenic peptides or processed antigens presented by MHC Class II molecules expressed by autologous antigen presenting cells, expressing LAG-3.
  • An anti-CD48 monoclonal antibody designated 10 H3 was used as irrelevant IgG1 antibody (negative control).
  • the saturating concentrations of anti-LAG-3 and anti-CD48 antibodies were determined by immunofluorescence on PHA (phytohaemagglutinin)-blasts and cell lines transformed by Epstein-Barr virus (EBV). In the proliferation tests, the monoclonal antibodies were added in the proportion of 5 times the saturating concentration.
  • the T lymphocyte lines used were, on the one hand the clone 154 derived from peripheral blood lymphocytes, raised against a peptide mimicking an influenza haemagglutinin (HA) fragment having an amino acid sequence extending from amino acid 306 to 329 (p20 peptide), and on the other hand the clone 28, a T lymphocyte clone derived from peripheral lymphocytes of a single human donor, raised against diphtheria toxoid (DT).
  • the antigen presenting cells (APC) corresponding to clone 154 were EBV-transformed B lymphocytes of the same donor (DR3/DR11) as T 154.
  • the antigen presenting cells corresponding to clone 28 were EBV-transformed B lymphocytes of the same donor. This clone was restricted to HLA DR7.
  • the APC (5 ⁇ 10 6 ) were incubated at 37° C. for one and a half hours with variable doses of the p20 peptide, then washed and irradiated (10,000 rad).
  • the cells were plated out on 96-well microtitration plates at the same time as the clone 154 cells (0.5 ⁇ 10 5 to 10 ⁇ 10 5 cells/ml) in a 3:1 ratio.
  • the responding cells/stimulating cells ratio was 1.
  • the HLA DR7/EBV APC cells were either treated with mitomycin or irradiated, then added to the T lymphocytes in the presence of DT (which remained in the culture).
  • the final concentration of clone 28 cells was 100,000 cells/ml.
  • [ 3 H]Thymidine (1 ⁇ Ci/well) was added at varying time intervals from day 2 to day 10 of culture.
  • the results were expressed as the mean cpm and after subtraction of the cpm found in the negative control (T lymphocytes cocultured with APC unladen with immunogens).
  • the proliferation tests were carried out on 96-well plates. The absorption of tritiated thymidine in the individual 200 ⁇ l wells was measured after adding 1 ⁇ Ci of thymidine for the last 18 hours of culture. The results were expressed in the form of the mean of 3 tests. The standard deviation was usually less than 12% (a little more in the case of very low cpm measurements).
  • mixed culture (clone 154/APC) supernatants were combined, filtered through 0.22 ⁇ m membranes, divided into samples and frozen at ⁇ 20° C. until the time of titration using commercial immunoassay kits: Immunotech IL-2 and INF- ⁇ titration kit, Genzyme IFN- ⁇ kit and Cayman Chemicals IL-4 kit.
  • a dose determination study was carried out to establish the proliferation profiles of clone 154 brought into contact with the p20 specific antigen at varying concentrations and in the presence or absence of anti-LAG-3 monoclonal antibodies or irrelevant monoclonal antibodies (negative control).
  • Clone 28 was also stimulated with the antigen (tetanus toxoid 10 ⁇ g/ml) in the presence of 17B4 monoclonal antibodies after coculture with the corresponding APC in the presence of DT. The results are shown in FIG. 2 .
  • Tests were carried out designed to measure the miscellaneous cellular events occurring after the antigenic stimulation of clone 154 cells incubated in the presence of anti-LAG-3 monoclonal antibodies.
  • the cells were harvested during conventional antigenic stimulation of clone 154 in the presence of anti-LAG-3 or anti-CD48 monoclonal antibodies or in the absence of antibodies, and tested for the expression of LAG-3 and CD25 transmembrane receptors, and samples of culture supernatants were collected at different time intervals after stimulation and tested for the presence of IFN- ⁇ , TNF- ⁇ , IL-4 and IL-2.
  • LAG-3 plays a regulatory role for CD4 + cells.
  • anti-LAG-3 monoclonal antibodies increase proliferation, and hence act as immunopotentiators, suggest that LAG-3 is involved in the “deactivation” of CD4 + T lymphocytes with a negative role of LAG-3 on the antigen-dependent stimulation.
  • Soluble proteins derived from LAG-3 were obtained by a recombinant DNA technique using suitable vectors comprising DNA coding for LAG-3 and DNA coding for an immunoglobulin fragment.
  • the transient expression system consisted of transfected Cos cells. This system makes it possible to produce several mg of recombinant fusion proteins.
  • Recombinant DNA techniques were carried out as described by MANIATIS et al. (22). The modifications were made as recommended by the manufacturer.
  • Fragments coding for the D1D2 or D1-D4 regions were amplified (30 cycles) from a fragment of cDNA (FDC sequence) encompassing LAG-3 cDNA (TRIEBEL et al. (1)), using Taq polymerase free from 5′-endonuclease activity and relatively resistant to an exposure to very high temperature; the amplification was followed by a denaturation at 98° C. (with a Perkin Elmer Cetus “DNA thermal cycle”). Specific primers were used as recorded in the table below.
  • the resulting amplified fragments (739 bp and 1312 bp for LAG-3 D1-D2 and LAG-3 D1-D4, respectively) were inserted into a pBS plasmid (Stratagene).
  • Inserts were prepared after digestion with XhoI and BglII and introduced into the XhoI/BamHI sites of the vector pCDM7-CD8-IgG1 (pCDM7 being derived from pCDM8 marketed by Stratagene), as illustrated in FIG. 3, so as to exchange the DNA sequences coding for CD8 for those coding for the subfragments of LAG-3.
  • the resulting expression vectors contained the sequences coding for D1D2 or D1-D4 fused to the DNA sequences coding for the —CH 2 —CH 3 junction region of a human IgG1 chain.
  • CDM7 is a eukaryotic expression vector derived from the vectors developed by SEED et al. (10) for the cloning of DNA and its expression in E. coli and eukaryotic cells.
  • CDM7 possesses the following features: (i) the human cytomegalovirus promoter for transient expression in mammalian cells; (ii) a viral origin of SV40 for an autosomal replication of mammalian cells expressing T antigen; (iii) ⁇ VX (type Col E1) as plasmid origin for a high copy number; (iv) a Sup F selection for resistance to ampicillin and tetracycline in Tet amb and Amp amb E. coli strains; (v) an origin of replication of M13 for the release of a single strand; (vi) a T7 RNA promoter; and (vii) a polylinker for an efficient cloning of heterologous DNA.
  • Cos cells (5 ⁇ 10 6 ) were transfected with 30 ⁇ g of DNA of suitable expression vectors (coding for either LAG-3 D1D2 Ig, or LAG-3 D1-D4 Ig, or CD8 Ig) by electroporation (200 V, 1500 ⁇ F, 30-40 msec) using a Cellject apparatus (Eurogentech, Liège, BE). The cells were plated out again and cultured on a medium containing 5% of foetal calf serum. The supernatants were withdrawn 6 days after transfection.
  • suitable expression vectors coding for either LAG-3 D1D2 Ig, or LAG-3 D1-D4 Ig, or CD8 Ig
  • electroporation 200 V, 1500 ⁇ F, 30-40 msec
  • the cells were plated out again and cultured on a medium containing 5% of foetal calf serum. The supernatants were withdrawn 6 days after transfection.
  • the synthesis of the resulting fusion proteins was analysed from the supernatants as well as from cell extracts of transfected cells, by Western blot analysis with the 17B4 monoclonal antibodies. Immunoreactive materials were observed in the supernatant of cells transfected with DNA coding for LAG-3 D1D2 Ig or LAG-3 D1-D4 Ig.
  • CD8 immunoadhesin (CD8 Ig) was obtained as negative control using the same expression system and the expression vector pCDM7-CD8 (FIG. 3 ).
  • LAG-3 D1D2 Ig. LAG-3 D1-D4 Ig and CD8 Ig were purified by means of the standard method on protein A-Sepharose. The resulting material was analysed by SDS-PAGE, followed by Coomassie staining or a Western blot analysis using anti-human Ig antibody.
  • DHFR dihydrofolate reductase
  • Transfection of these cells with expression vectors containing the DHFR gene permits the secretion of recombinant DHFR-resistant clones, and the culturing of these cells on selective media containing increasing amounts of MTX results in amplification of the DHFR gene and the DNA associated therewith.
  • Fragments of DNA coding for the D1, D1D2 or D1-D4 regions were amplified using a PCR method identical to the one described previously, using the primers specified in the table below.
  • the resulting amplified fragments were digested with SalI and inserted into the SalI site of pUC 18 (Stratagene).
  • This vector is a eukaryotic expression vector which is multifunctional for the expression C cDNA and its amplification in eukaryotic cells. It possesses the following features: (i) the murine promoter of the metallothionein-1 gene and a polyadenylation sequence SV 40 (comprising a donor-acceptor splicing site) to bring about transcription of the gene of interest, (ii) a human intervening sequence A containing the donor-acceptor splicing site of the gene for the subunit of ⁇ glycoprotein for obtaining high levels of transcription of cDNA, (iii) the pML sequence containing the origin of replication of pBR322 and a gene for resistance to aampicillin ampicillin for bacterial amplification, and (iv) a DHFR transcription unit of SV 40 to bring about transcription of the sequences used for selection and amplification of the transfectants.
  • LAG-3 D1, LAG-3 D1D2 and LAG-3 D1-D4 were used to transfect CHO DUKX cells, and these cells were cultured on a selective medium. Cells capable of multiplying under these conditions were combined and cultured on a medium containing increasing amounts of MTX. Levels of expression were measured by Western blot analysis using the 17B4 monoclonal antibody. Clones producing high levels of recombinant soluble molecules derived from LAG-3 were propagated in bioreactors, and the material derived from LAG-3 was purified by ion exchange chromatography and immunoaffinity.
  • Target cells (4 ⁇ 10 5 ) were incubated for 30 minutes at 4° C. in the presence of LAG-3 D1-D4 Ig, CD8 Ig, a murine monoclonal antibody, (949) anti-human MHC Class II (DR, DP, DQ) conjugated to FITC (isothiocyanate fluoride) from a Coulter clone, or murine Ig-FITC; an irrelevant immunoglobulin G conjugated to FITC.
  • FITC isothiocyanate fluoride conjugated to FITC
  • the cells were washed and incubated at 4° C. for 30 minutes with either a goat anti-human Ig polyclonal F(ab′) 2 conjugated to fluorescein or a goat anti-mouse Ig polyclonal antibody conjugated to fluorescein (Coulter clone).
  • LAG-3 D1-D4 Ig was incubated with MHC Class II-positive or -negative cells.
  • B lymphocyte lines expressing MHC Class II(L31, Phil EBV, Raji, Sanchez and Personnaz) were treated with anti-Class II monoclonal antibody 949, or the supernatants for Cos cells transfected with DNA coding either for LAG-3 D1-D4 Ig or for CD8 Ig.
  • the five cell lines expressing the different haplotypes of MHC Class II molecules were recognized by LAG-3 Ig in the same way as by the anti-Class II monoclonal antibodies (positive control), while the supernatant containing CD8 Ig (negative control) did not bind to these cell lines, as could be expected.
  • MHC Class II-negative cell lines CEM, RJ, HSB2, K562 were treated with the same reagents as above. None reacted, either with the anti-MHC Class II (negative control) or with LAG-3 D1-D4 Ig, showing that the binding of LAG-3 D1-D4 is specific to MHC Class II molecules.
  • mice fibroblasts transfected or otherwise with genes coding for human DR7 or human DP4, (ii) mouse cells expressing or otherwise MHC Class II molecules, (iii) activated human CD4 + or CD8 + cells, and (iv) T lymphocyte lines expressing the different haplotypes of MHC Class II molecules (FIG. 8 ).
  • LAG-3 D1-D4 Ig binds to all cells expressing MHC Class II as efficiently as the anti-MHC Class II monoclonal antibody 949.
  • LAG-3 D1-D4 Ig binds to all DR and DP haplotypes tested, to human MHC Class II molecules expressed by transfected mouse cells, to murine MHC Class II molecules and also to MHC Class II molecules expressed by CD4 + or CD8 + T lymphocytes.
  • LAG-3 D1D2 bound to cells expressing MHC Class II in as specific a manner and with the same efficiency as LAG-3 D1-D4.
  • the capacity of this immunoadhesin to bind to cell ligands is measured using a fluorescein-labeled goat serum directed against human immunoglobulins.
  • the target cells are first incubated with a human monoclonal antibody or an immunoadhesin for 30 min at 4° C. in RPMI 1640 containing 10% of FCS (foetal calf serum). The cells are then incubated with an FITC-labelled goat anti-mouse immunoglobulin serum (Coulter) for the murine monoclonal antibodies or with an FITC-labelled goat anti-human immunoglobulin serum (Tago) for the immunoadhesins. The fluorescence is measured after two washes, analyzing 3,000 cells with an Elite cytometer (Coultronics, Hialeah, Fla.).
  • FIG. 9 shows the degrees of binding of LAG-3Ig, CD8Ig, antibody 949 or antibody OKT3 (anti-CD3, ATCC), represented by the number of cells counted as a function of the logarithm of the measured fluorescence intensity.
  • LAG-3Ig binds to mouse fibroblasts transfected for the gene for the HLA DR 4 molecule, and does not bind to untransfected cells.
  • CD8Ig is incapable of binding to HLA DR 4 + fibroblasts under the same conditions.
  • the cellular distribution of the ligands for LAG-3Ig was evaluated on a cell population sample by immunofluorescence.
  • LAG-3Ig is visualized on all positive Class II cells tested, including B cell lines transformed by Epstein-Barr virus (derived from genetically unrelated donors, including 10 homozygous lines of DR 1 to DR 10 typing), as well as on activated T and NK cells.
  • FIG. 9 shows, by way of example, the binding of LAG-3Ig to Daudi cells which are positive for Class II antigens.
  • the mean fluorescence intensity with LAG-3Ig is similar to that observed with antibody 949 which is specific for Class II antigens.
  • the binding of LAG-3Ig to DR 4 (FIG. 9 ).
  • DR 2 , DR 7 or DPw4 (not shown) expressed at the surface of mouse fibroblasts is, in contrast, weaker than that observed for antibody 949.
  • T origin peripheral blood T cells, CEM, HSB2, REX lines
  • B origin RJ 2.2.5 line
  • non-lymphoid origin human lines, K562 of erythromyoloid origin and line originating from melanoma cells (not shown)
  • LAG-3Ig binds to xenogeneic Class II molecules of the MHC, such as the antigens expressed by mouse lymphoid A 20 and the monkey Classes II expressed by phytohaemagglutinin-stimulated blasts (data not shown).
  • LAG-3Ig The specificity of binding of LAG-3Ig was also verified using the monoclonal antibodies 17B4, whose capacity to block LAG-3/MHC Class II interactions in cell adhesion tests was demonstrated beforehand (FIG. 10 ).
  • the LAG-3Ig molecules are preincubated for 30 minutes at 4° C. either with medium alone, or with 17B4 (1 mg/ml), or with OKT3 (1 mg/ml), before being brought into contact with Daudi cells.
  • FIG. 10 shows that a preincubation of LAG-3Ig with 17B4 inhibits the binding to Class II + cells, whereas no inhibition is detected with the OKT3 control.
  • LAG-3/MHC Class II interaction by the soluble fragments of LAG-3 may be observed directly in relation to the binding of LAG-3Ig by Class II MHC molecules, by competitive experiments with the soluble fragments.
  • Daudi cells are incubated with soluble LAG3-D 1 D 2 fragments so as to permit the binding of these molecules to the MHC Class II antigens expressed at the surface of the Daudi cells.
  • the cells are incubated in the presence of LAG-3D 1 D 4 Ig in dimeric form or LAG-3D 1 D 2 Ig in monomeric form.
  • the binding of these immunoadhesins derived from LAG-3 is measured using a goat anti-human Ig F(ab′) 2 conjugated to fluorescein (GAH-FITC).
  • control groups are represented by Daudi cells incubated with dimeric LAG-3D 1 D 4 Ig or monomeric LAG-3D 1 D 2 Ig without preincubation with the soluble LAG-3D1D2 fragments.
  • the method described in this publication was modified by replacing the visualization and counting of Cos cells binding to B lymphocytes by counting the radioactivity remaining after incubation of 51 Cr-labelled B lymphocytes with Cos cells expressing LAG-3 (binding assay).
  • Cos cells transfected with a suitable expression vector (coding for wild-type LAG-3 or for CD4). Two days later, the Cos cells were treated with trypsin and plated out again on the basis of 0.05 ⁇ 106 cells/well on a flat-bottomed 12-well tissue culture plates, 24 hours later. 51 Cr-labelled Daudi cells (5.5 ⁇ 10 6 ) were incubated on this monolayer of Cos cells (final vol.: 1 ml) for 1 hour. The target B cells were then aspirated off and the wells washed 5 to 7 times, gently adding 1 ml of medium dropwise. The edges of the wells were washed by suction using a Pasteur pipette.
  • the remaining cells were lysed with 1 ml of PBS, 1% Triton for 15 minutes at 37° C.
  • the lysates were centrifuged at 3000 rpm for 10 minutes, and 100 ⁇ l of the resulting supernatant were counted.
  • LAG-3 D1-D4 Ig was used to inhibit LAG-3/MHC Class II and CD4/MHC Class II interaction in the 51 Cr binding assay.
  • Human CD8 Ig and IgG1 were tested in parallel and used as negative controls.
  • LAG-3/Class II interaction A significant inhibition of LAG-3/Class II interaction by LAG-3 D1-D4 Ig was detected (FIG. 5 A).
  • the LAG-3/MHC Class II interaction can be partially and non-specifically inhibited by human CD8 Ig and IgG1.
  • LAG-3 Ig proved to be a potential inhibitor of CD4/Class II interaction (FIG. 5B) under experimental conditions in which CD4/MHC Class II interaction was not modified by human CD8 Ig or IgG1. This suggests that LAG-3/Class II interaction is weaker than CD4/Class II interaction.
  • LAG-3 D1-D4 Ig showed a strong inhibition of the proliferation of clone 28, while human CD8 Ig and IgG had no effect (FIG. 6 ).
  • Similar experiments were carried out with clone 154 (FIG. 7 ), and showed a partial inhibition in the presence of LAG-3 Ig.
  • a control carried out with anti-LAG-3 monoclonal antibodies had the reverse effects, as observed previously.
  • LAG-3 D1-D4 Ig is a potential immunosuppressant of the proliferation of T lymphocytes stimulated by an antigen, and indicate that LAG-3 might act as an “extinguisher” of the secondary immune response induced by activated CD4 + T helper lymphocytes.
  • the T cells are incubated beforehand with a saturating amount of LAG-3Ig (100 nM). The cells are then washed twice with cold RPMI and incubated with 10 ⁇ g/ml of goat antibodies directed against human immunoglobulins (Tago) at 4° C. for 30 minutes.
  • the cells are resuspended in RPMI containing 10% of foetal calf serum and incubated for 2 hours at 37° C. before adding the signal.
  • cross-link a goat anti-mouse antibody at a concentration of 10 ⁇ g/ml (Tago) is used.
  • FIG. 11 depicts an experiment in which clone T154 has been preincubated with LAG-3Ig bound (“cross-linked”) to a second reactant (polyclonal serum specific for the constant region of human immunoglobulins). The degree of binding of LAG-3Ig to the cells is measured by immunofluorescence (FIG. 11 A).
  • FIG. 11B shows that a more than 50% inhibition of the proliferation of clone T154 is produced by LAG-3Ig. Under the same experimental conditions, no effect is observed with the control CD8Ig or with LAG-3Ig without “cross-linking” (not shown in the figure).
  • FIG. 11C also shows that no effect is observed when LAG-3Ig is used to bind (“cross-link”) the MHC Class II molecules expressed by antigen-presenting B cells.
  • LAG-3Ig The role of LAG-3Ig in relation to cell cytotoxicity is studied on two types of effector cells:
  • PBL peripheral blood lymphocytes
  • S1B5 line cells (clone of human NK cells).
  • the cytotoxic activity of these cells is measured by counting the 51 Cr released into the medium by previously labelled target cells, in the presence or absence of LAG-3Ig in the medium.
  • FIG. 14 shows the degree of cytotoxicity of S1B5 for a line of human B cells transformed by Epstein-Barr virus and carrying major histocompatibility complex Class I and II antigens (LAZ 388 line), as a function of different reactants added to the cultures.
  • the negative controls consist of medium alone (MED), the immunoadhesin CD8Ig and the monoclonal antibody 17.B4 (anti-LAG-3).
  • the positive controls consist of three different monoclonal antibodies:
  • Anti-HLA Class I (W632) or Class II (L243) antibodies increase the lysis of the target cells (and not the 17B4 control).
  • the immunoadhesin LAG-3Ig increases the lysis.
  • the CD8Ig control has no effect.
  • FIG. 15 shows the results of an experiment similar to the above, in which the cytotoxicity of PBL with respect to Daudi cells (HLA Class I ⁇ ) is measured, for effector/target ratios of 50:1 (clear columns) and 15:1 (shaded columns).
  • the reactants added to the medium are the same as the ones used in the first experiment, except for antibody 9.49 and antibody 17.B4.
  • Antibody 10H3 is an isotype IgG1 immunoglobulin specific for the CD45 surface antigen. It is used as negative control.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Transplantation (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
US09/931,103 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies Expired - Lifetime USRE38313E1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/931,103 USRE38313E1 (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
FR9405643 1994-05-06
FR9405643 1994-05-06
US09/931,103 USRE38313E1 (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
PCT/FR1995/000593 WO1995030750A2 (fr) 1994-05-06 1995-05-05 Fractions polypeptidiques solubles de la proteine lag-3; procede de production; composition therapeutique; anticorps anti-idiotype
US08/737,271 US5955300A (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US08/737,271 Reissue US5955300A (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies

Publications (1)

Publication Number Publication Date
USRE38313E1 true USRE38313E1 (en) 2003-11-11

Family

ID=9463004

Family Applications (3)

Application Number Title Priority Date Filing Date
US09/931,103 Expired - Lifetime USRE38313E1 (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
US08/737,271 Ceased US5955300A (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
US09/058,555 Expired - Lifetime US6143273A (en) 1994-05-06 1998-04-13 Therapeutic composition containing antibodies to soluble polypeptide fractions of LAG-3 protein

Family Applications After (2)

Application Number Title Priority Date Filing Date
US08/737,271 Ceased US5955300A (en) 1994-05-06 1995-05-05 Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
US09/058,555 Expired - Lifetime US6143273A (en) 1994-05-06 1998-04-13 Therapeutic composition containing antibodies to soluble polypeptide fractions of LAG-3 protein

Country Status (18)

Country Link
US (3) USRE38313E1 (es)
EP (1) EP0758383B1 (es)
JP (1) JP3700859B2 (es)
KR (1) KR100257466B1 (es)
CN (1) CN1110557C (es)
AT (1) ATE352617T1 (es)
BR (1) BR9507618A (es)
CA (1) CA2189657C (es)
DE (1) DE69535375T2 (es)
DK (1) DK0758383T3 (es)
ES (1) ES2281899T3 (es)
IL (1) IL113617A (es)
MX (1) MX9605365A (es)
NO (1) NO325828B1 (es)
PT (1) PT758383E (es)
RU (1) RU2178306C2 (es)
WO (1) WO1995030750A2 (es)
ZA (1) ZA953629B (es)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080003235A1 (en) * 2004-04-13 2008-01-03 Frederic Triebel Vaccine Composition Comprising a Class II Cmh Lignd Coupled With an Antigen, Method for the Preparation and the Use Thereof
US20100196394A1 (en) * 2003-02-28 2010-08-05 The Johns Hopkins University Anti-cancer vaccine composition
US20110008331A1 (en) * 2007-10-05 2011-01-13 Immutep Use of recombinant lag-3 or the derivatives thereof for eliciting monocyte immune response
US9505839B2 (en) 2012-07-02 2016-11-29 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
WO2017009842A2 (en) 2015-07-16 2017-01-19 Biokine Therapeutics Ltd. Compositions and methods for treating cancer
US9908936B2 (en) 2014-03-14 2018-03-06 Novartis Ag Antibody molecules to LAG-3 and uses thereof
US10081681B2 (en) 2013-09-20 2018-09-25 Bristol-Myers Squibb Company Combination of anti-LAG-3 antibodies and anti-PD-1 antibodies to treat tumors
US10188730B2 (en) 2014-08-19 2019-01-29 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US10344089B2 (en) 2008-08-11 2019-07-09 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US10736940B2 (en) 2013-12-19 2020-08-11 Immutep S.A.S. Combined preparations for the treatment of cancer
US10874713B2 (en) 2015-01-09 2020-12-29 Immutep S.A.S. Combined preparations for the treatment of cancer or infection
US11045547B2 (en) 2015-12-16 2021-06-29 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US11723975B2 (en) 2017-05-30 2023-08-15 Bristol-Myers Squibb Company Compositions comprising an anti-LAG-3 antibody or an anti-LAG-3 antibody and an anti-PD-1 or anti-PD-L1 antibody
US11807686B2 (en) 2017-05-30 2023-11-07 Bristol-Myers Squibb Company Treatment of LAG-3 positive tumors
US11827704B2 (en) 2014-01-24 2023-11-28 Novartis Ag Antibody molecules to PD-1 and uses thereof

Families Citing this family (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0843557B1 (en) 1995-07-21 2002-11-13 Applied Research Systems ARS Holding N.V. Methods for detecting, identifying, isolating, and selectively labelling and targeting th1 lymphocytes by means of the lag-3 protein
AU728911B2 (en) * 1996-11-28 2001-01-18 Institut Gustave Roussy Mutants of the LAG-3 proteins, products for the expression of these mutants and use
DE69632967T2 (de) 1996-11-29 2005-08-11 Applied Research Systems Ars Holding N.V. Verfahren zur Verhinderung der Transplantabstoßung bei einer Transplantation und zur Herstellung einer universalen Gentherapie-Wirtszelle unter Anwendung von Lymphocyten-Aktivierungsgen (LAG-3)
EP0900841A1 (en) * 1997-06-18 1999-03-10 Institut National De La Sante Et De La Recherche Medicale (Inserm) LAG-3 splice variants
EP0893507A1 (en) * 1997-07-25 1999-01-27 Institut Gustave Roussy Use of MHC class II ligands (CD4 and LAG-3) as adjuvant for vaccination and of LAG-3 in cancer treatment
US6660843B1 (en) * 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
US6434012B2 (en) * 2000-02-11 2002-08-13 Tyco Electronics Logistics Ag Circuit board interconnect
WO2001083525A2 (en) * 2000-05-03 2001-11-08 Amgen Inc. Modified peptides, comprising an fc domain, as therapeutic agents
EP2388265A1 (en) * 2002-02-22 2011-11-23 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
RS61678B1 (sr) 2014-05-28 2021-05-31 Agenus Inc Anti-gitr antitela i postupci za njihovu primenu
TWI693232B (zh) 2014-06-26 2020-05-11 美商宏觀基因股份有限公司 與pd-1和lag-3具有免疫反應性的共價結合的雙抗體和其使用方法
MA41463A (fr) 2015-02-03 2017-12-12 Anaptysbio Inc Anticorps dirigés contre le gène d'activation 3 des lymphocytes (lag-3)
TWI773646B (zh) 2015-06-08 2022-08-11 美商宏觀基因股份有限公司 結合lag-3的分子和其使用方法
PE20231958A1 (es) 2015-07-30 2023-12-06 Macrogenics Inc Moleculas de union a pd-1 y metodos de uso de las mismas
US20180230431A1 (en) 2015-08-07 2018-08-16 Glaxosmithkline Intellectual Property Development Limited Combination Therapy
TWI756187B (zh) 2015-10-09 2022-03-01 美商再生元醫藥公司 抗ｌａｇ３抗體及其用途
MX2018005389A (es) * 2015-11-20 2018-09-05 Regeneron Pharma Animales no humanos que tienen un gen 3 de activacion linfocitaria humanizado.
JP7089470B2 (ja) 2015-12-02 2022-06-22 アジェナス インコーポレイテッド 抗体およびその使用方法
TW202208440A (zh) 2015-12-14 2022-03-01 美商宏觀基因股份有限公司 對於pd-1和ctla-4具有免疫反應性的雙特異性分子及其使用方法
KR20180104149A (ko) * 2016-02-04 2018-09-19 트리아니, 인코포레이티드 면역글로불린의 증대된 생성
CN109475603B (zh) 2016-06-20 2023-06-13 科马布有限公司 抗pd-l1抗体
AU2017282892B2 (en) 2016-06-23 2023-10-26 Jiangsu Hengrui Medicine Co., Ltd. LAG-3 antibody, antigen-binding fragment thereof, and pharmaceutical application thereof
CA3037380A1 (en) 2016-10-11 2018-04-19 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
KR20230136711A (ko) 2016-10-13 2023-09-26 치아타이 티안큉 파마수티컬 그룹 주식회사 항-lag-3 항체 및 조성물
TW201829462A (zh) 2016-11-02 2018-08-16 英商葛蘭素史克智慧財產（第二）有限公司 結合蛋白
EA201991673A1 (ru) 2017-02-10 2020-01-17 Регенерон Фармасьютикалз, Инк. Меченные радиоактивным изотопом антитела к lag3 для иммуно-пэт-визуализации
JP2020513009A (ja) 2017-04-05 2020-04-30 シムフォゲン・アクティーゼルスカブＳｙｍｐｈｏｇｅｎ Ａ／Ｓ Ｐｄ−１、ｔｉｍ−３、およびｌａｇ−３を標的とする併用治療
WO2018208868A1 (en) * 2017-05-10 2018-11-15 Smet Pharmaceutical Inc Human monoclonal antibodies against lag3 and uses thereof
KR20200042937A (ko) 2017-08-30 2020-04-24 페인스 테라퓨틱스 인코포레이티드 항-lag-3 항체 및 이의 용도
RU2771384C2 (ru) * 2017-12-22 2022-05-04 Цзянсу Хэнжуй Медсин Ко., Лтд. Фармацевтическая композиция, содержащая антитело к lag-3, и ее применение
CA3136568A1 (en) 2019-05-13 2020-11-19 Regeneron Pharmaceuticals, Inc. Combination of pd-1 inhibitors and lag-3 inhibitors for enhanced efficacy in treating cancer
CN112010972B (zh) * 2019-05-31 2023-01-10 瑞阳(苏州)生物科技有限公司 与人lag-3蛋白结合的抗体及其编码基因和应用
CN110950966B (zh) * 2019-12-13 2020-12-11 启辰生生物科技(珠海)有限公司 融合蛋白、编码核酸和细胞及用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0325224A2 (en) 1988-01-22 1989-07-26 ZymoGenetics, Inc. Methods of producing secreted receptor analogs and biologically active peptide dimers.
WO1991008298A2 (en) 1989-11-22 1991-06-13 Genentech, Inc. Fusion proteins consisting of a ligand binding protein and a stable plasma protein
WO1991010682A1 (fr) 1990-01-08 1991-07-25 Institut National De La Sante Et De La Recherche Medicale Proteines produites par les lymphocytes humains, sequence d'adn codant pour ces proteines et applications pharmaceutiques et biologiques
WO1992000092A1 (en) 1990-07-02 1992-01-09 Bristol-Myers Squibb Company Ligand for cd28 receptor on b cells and methods
US5976877A (en) 1990-01-08 1999-11-02 Institut National De La Sante Et De La Recherche Medicale (Inserm) Proteins produced by human lymphocytes DNA sequence encoding these proteins and their pharmaceutical and biological uses

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0325224A2 (en) 1988-01-22 1989-07-26 ZymoGenetics, Inc. Methods of producing secreted receptor analogs and biologically active peptide dimers.
WO1991008298A2 (en) 1989-11-22 1991-06-13 Genentech, Inc. Fusion proteins consisting of a ligand binding protein and a stable plasma protein
WO1991010682A1 (fr) 1990-01-08 1991-07-25 Institut National De La Sante Et De La Recherche Medicale Proteines produites par les lymphocytes humains, sequence d'adn codant pour ces proteines et applications pharmaceutiques et biologiques
US5773578A (en) 1990-01-08 1998-06-30 Institut National De La Sante Et De La Recherche Medicale Proteins produced by human lymphocytes, DNA sequence encoding these proteins and their pharmaceutical and biological use
US5874250A (en) 1990-01-08 1999-02-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) DNA encoding for a protein containing the extracellular domain of lymphocyte activation gene 3
US5976877A (en) 1990-01-08 1999-11-02 Institut National De La Sante Et De La Recherche Medicale (Inserm) Proteins produced by human lymphocytes DNA sequence encoding these proteins and their pharmaceutical and biological uses
WO1992000092A1 (en) 1990-07-02 1992-01-09 Bristol-Myers Squibb Company Ligand for cd28 receptor on b cells and methods

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
Bertrand et al., "The lymphocyte activation gene product LAG-3 is a ligand for class II antigens" 8 International Congress of Immunology. Abstracts, p. 281, Budapest (Aug. 1992).
Biaxeras et al., "Characterizaton of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens", The Journal of Experimental Medicine, 176(2)327-337, New York (Aug. 1992).
De Santes et al., Cancer Research, 52:1916-1923 (Apr. 1, 1992).
Eilat et al., Proc. Natl. Acad. Sci. (USA), 89(15):6871-6875 (Aug. 1, 1992).
Huard et al., "Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand", Immunogenetics, 39(3)213-217, New York (Mar. 1994).
Loetscher et al., "Recombinant 55-kDa tumor necrosis factor (TNF) receptor stoichiometry of binding to TNFalpha and TNFbeta and inhibition of TNF activity", The Journal of Biological Chemistry, 266:18324-18329 (1991).
Loetscher et al., "Recombinant 55-kDa tumor necrosis factor (TNF) receptor stoichiometry of binding to TNFα and TNFβ and inhibition of TNF activity", The Journal of Biological Chemistry, 266:18324-18329 (1991).
Peppel et al., "A tumor necrosis factor (TNF) receptor-IgG Heavy chain chimeric protein as a bivalent antagonist of TNF activity", J. Exp. Med., 174:1483-1488 (1991).
Taetle et al., J. Natl. Cancer Inst., 80(13):1053-1059 (Sep. 7, 1988).
Triebel et al., "LAG-3, a novel lymphocyte activation gene closely related to CD4", The Journal of Experimental Medicine, 171(5)1393-1405 (May 1990).

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100196394A1 (en) * 2003-02-28 2010-08-05 The Johns Hopkins University Anti-cancer vaccine composition
US10934354B2 (en) 2003-02-28 2021-03-02 The Johns Hopkins University Methods of increasing T cell immune response in the treatment of cancer
US8551481B2 (en) 2003-02-28 2013-10-08 The Johns Hopkins University Anti-cancer vaccine composition comprising an anti-CD223 antibody and kit comprising an anti-cancer vaccine and an anti-CD223 antibody
US9005629B2 (en) 2003-02-28 2015-04-14 St. Jude Children's Research Hospital Inc. Method for treating cancer with an antibody to CD223 protein
US10787513B2 (en) 2003-02-28 2020-09-29 The Johns Hopkins University Methods for treatment comprising an antibody that binds CD223 protein
US20080003235A1 (en) * 2004-04-13 2008-01-03 Frederic Triebel Vaccine Composition Comprising a Class II Cmh Lignd Coupled With an Antigen, Method for the Preparation and the Use Thereof
US9579382B2 (en) 2007-10-05 2017-02-28 Immutep Use of recombinant LAG-3 or the derivatives thereof for eliciting monocyte immune response
US10232038B2 (en) 2007-10-05 2019-03-19 Immutep Use of recombinant LAG-3 or the derivatives thereof for eliciting monocyte immune response
US20110008331A1 (en) * 2007-10-05 2011-01-13 Immutep Use of recombinant lag-3 or the derivatives thereof for eliciting monocyte immune response
US11583582B2 (en) 2007-10-05 2023-02-21 Immutep Use of recombinant LAG-3 or the derivatives thereof for eliciting monocyte immune response
US11530267B2 (en) 2008-08-11 2022-12-20 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11236163B2 (en) 2008-08-11 2022-02-01 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11236164B2 (en) 2008-08-11 2022-02-01 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11001630B2 (en) 2008-08-11 2021-05-11 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation Gene-3 (LAG-3), and uses thereof
US10988535B2 (en) 2008-08-11 2021-04-27 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US10344089B2 (en) 2008-08-11 2019-07-09 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US10988536B2 (en) 2008-08-11 2021-04-27 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11512130B2 (en) 2008-08-11 2022-11-29 E.R. Squibb & Sons, L.L.C. Human antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11236165B2 (en) 2008-08-11 2022-02-01 E.R. Squibb & Sons, L.L.C. Human antibodies that bind Lymphocyte Activation Gene-3 (LAG-3), and uses thereof
US9505839B2 (en) 2012-07-02 2016-11-29 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11345752B2 (en) 2012-07-02 2022-05-31 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US10377824B2 (en) 2012-07-02 2019-08-13 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US10266591B2 (en) 2012-07-02 2019-04-23 Bristol-Myers Squibb Company Optimization of antibodies that bind lymphocyte activation gene-3 (LAG-3), and uses thereof
US11274152B2 (en) 2013-09-20 2022-03-15 Bristol-Myers Squibb Company Combination of anti-LAG-3 antibodies and anti-PD-1 antibodies to treat tumors
US10081681B2 (en) 2013-09-20 2018-09-25 Bristol-Myers Squibb Company Combination of anti-LAG-3 antibodies and anti-PD-1 antibodies to treat tumors
US10736940B2 (en) 2013-12-19 2020-08-11 Immutep S.A.S. Combined preparations for the treatment of cancer
US11827704B2 (en) 2014-01-24 2023-11-28 Novartis Ag Antibody molecules to PD-1 and uses thereof
US10711060B2 (en) 2014-03-14 2020-07-14 Novartis Ag Antibody molecules to LAG-3 and uses thereof
US9908936B2 (en) 2014-03-14 2018-03-06 Novartis Ag Antibody molecules to LAG-3 and uses thereof
US10188730B2 (en) 2014-08-19 2019-01-29 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US11207406B2 (en) 2014-08-19 2021-12-28 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US11278620B2 (en) 2014-08-19 2022-03-22 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US10898571B2 (en) 2014-08-19 2021-01-26 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US10940181B2 (en) 2015-01-09 2021-03-09 Immutep S.A.S. Combined preparations for the treatment of cancer or infection
US10874713B2 (en) 2015-01-09 2020-12-29 Immutep S.A.S. Combined preparations for the treatment of cancer or infection
US11684654B2 (en) 2015-01-09 2023-06-27 Immutep S.A.S. Combined preparations for the treatment of cancer or infection
EP3943098A2 (en) 2015-07-16 2022-01-26 Biokine Therapeutics Ltd. Compositions and methods for treating cancer
EP3744340A2 (en) 2015-07-16 2020-12-02 Biokine Therapeutics Ltd. Compositions and methods for treating cancer
WO2017009842A2 (en) 2015-07-16 2017-01-19 Biokine Therapeutics Ltd. Compositions and methods for treating cancer
US11045547B2 (en) 2015-12-16 2021-06-29 Merck Sharp & Dohme Corp. Anti-LAG3 antibodies and antigen-binding fragments
US11723975B2 (en) 2017-05-30 2023-08-15 Bristol-Myers Squibb Company Compositions comprising an anti-LAG-3 antibody or an anti-LAG-3 antibody and an anti-PD-1 or anti-PD-L1 antibody
US11807686B2 (en) 2017-05-30 2023-11-07 Bristol-Myers Squibb Company Treatment of LAG-3 positive tumors

Also Published As

Publication number Publication date
NO964650L (no) 1997-01-06
CA2189657A1 (fr) 1995-11-16
DE69535375T2 (de) 2007-11-08
KR100257466B1 (ko) 2000-07-01
EP0758383B1 (fr) 2007-01-24
IL113617A0 (en) 1995-08-31
BR9507618A (pt) 1997-08-19
CN1110557C (zh) 2003-06-04
US6143273A (en) 2000-11-07
ES2281899T3 (es) 2007-10-01
AU708825B2 (en) 1999-08-12
RU2178306C2 (ru) 2002-01-20
PT758383E (pt) 2007-05-31
WO1995030750A2 (fr) 1995-11-16
IL113617A (en) 2007-09-20
NO964650D0 (no) 1996-11-04
WO1995030750A3 (fr) 1995-12-21
US5955300A (en) 1999-09-21
DK0758383T3 (da) 2007-05-29
MX9605365A (es) 1997-12-31
JPH09508023A (ja) 1997-08-19
ATE352617T1 (de) 2007-02-15
NO325828B1 (no) 2008-07-28
ZA953629B (en) 1996-11-05
AU2570195A (en) 1995-11-29
WO1995030750A8 (fr) 1999-07-29
CA2189657C (fr) 2002-03-12
KR970702917A (ko) 1997-06-10
CN1155904A (zh) 1997-07-30
JP3700859B2 (ja) 2005-09-28
DE69535375D1 (de) 2007-03-15
EP0758383A1 (fr) 1997-02-19

Similar Documents

Publication Publication Date Title
USRE38313E1 (en) Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
US7034121B2 (en) Antibodies against CTLA4
AU694760B2 (en) B7-1-specific ligands, and their use for the induction of T cell anergy
US5885579A (en) CTLA4 receptor and uses thereof
ES2247585T3 (es) Procedidmiento de regulacion de la respuesta inmunitaria usando moleculas de union a il4 y a ctla4.
KR100238712B1 (ko) 씨티엘에이4 수용체,그를 함유한 연합단백질 및 그의 용도
US5844095A (en) CTLA4 Ig fusion proteins
JP3520272B2 (ja) リンパ球機能関連抗原３のｃｄ２結合ドメイン
US5869050A (en) Methods of blocking T-cell activation using anti-B7 monoclonal antibodies
US6338851B1 (en) Method of suppressing an immune response to a transplanted organ or tissue by administering an OX-2 protein
US5620889A (en) Human anti-Fas IgG1 monoclonal antibodies
US20070065875A1 (en) NTB-A, a surface molecule involved in natural killer cells activity
EP0787151A1 (en) Monoclonal antibody fragments having immunosuppressant activity
Hercend et al. Soluble polypeptide fractions of the LAG-3 protein, production method, therapeutic composition, anti-idiotype antibodies
AU5938899A (en) LAG-3 protein soluble polypeptide fractions, method of production, therapeutic composition and anti-idiotype antibody
KR20000005060A (ko) 단백질의 가용성 2가 및 다가 헤테로이량체 유사체

Legal Events

Date Code Title Description
FPAY Fee payment

Year of fee payment: 8

SULP Surcharge for late payment

Year of fee payment: 7

AS Assignment

Owner name: LABORATOIRES SERONO SA,SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.;REEL/FRAME:019966/0026

Effective date: 20070827

Owner name: LABORATOIRES SERONO SA, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.;REEL/FRAME:019966/0026

Effective date: 20070827

XAS Not any more in us assignment database

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.;REEL/FRAME:019808/0379

AS Assignment

Owner name: MERCK SERONO SA, SWITZERLAND

Free format text: CHANGE OF NAME;ASSIGNOR:LABORATOIRES SERONO S.A.;REEL/FRAME:023000/0862

Effective date: 20070625

AS Assignment

Owner name: MERCK SERONO SA,SWITZERLAND

Free format text: CHANGE OF NAME;ASSIGNOR:LABORATOIRES SERONO SA;REEL/FRAME:023569/0120

Effective date: 20081212

Owner name: MERCK SERONO SA, SWITZERLAND

Free format text: CHANGE OF NAME;ASSIGNOR:LABORATOIRES SERONO SA;REEL/FRAME:023569/0120

Effective date: 20081212

FPAY Fee payment

Year of fee payment: 12

SULP Surcharge for late payment

Year of fee payment: 11