Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2875—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the invention relates to the field of oncology, in particular to methods and pharmaceutical compositions for the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL).
• T-ALL T Cell Acute Lymphoblastic Leukemia
• Acute lymphoblastic leukemia is the most frequent childhood cancer. Particularly, T cell lineage ALL (T-ALL), which represents 10% of pediatric and 25% of adult ALL cases, are of poorer prognosis and with relapse being of dismal prognosis (Inaba et al., 2020). T cell acute lymphoblastic leukemia (T-ALL) results from the leukemic transformation of T cell precursors and abnormal blockade in differentiation (Reviewed in Belver & Ferrando, 2016; Girardi et al., 2017, Cordo et al., 2020).
• T- ALL Cytogenetic and global transcriptomic analyses allowed to classify T- ALL into molecular groups, characterized by abnormal expression of specific transcription factors (such as TAL; LM01/2; TLX1/3; HOXA and others) and a block of T cell precursors differentiation at specific stages.
• the TAL/LMO group is the largest one and often corresponds to a maturation arrest at the double positive (DP) to single positive (SP) transition (TCR“ ⁇ + , mature T-ALL).
• the maturation stage arrest of TLX 1 and TLX3 T-ALL cells occurs at the cortical stage, preceding TCR“ ⁇ expression (immature T-ALL) or often entails expression of TCR y5 .
• ETP early T-cell precursor
• T-ALL has been recognized as heterogeneous entities arrested at an early differentiation stage and harboring a signature of stem or early myeloid progenitors (reviewed in Girardi et al, 2017).
• tumor suppressive genes e.g., PTEN and CDKN2A
• oncogenic pathways e.g. NOTCH1, IL7R/JAK
• T-ALL new therapeutic strategies the inventors have previously shown that treatment of TCR“' l+ and TCR y5+ T-ALL patients-derived xenografts, which represents over 50% of T-ALL pediatric cases, with anti-CD3 monoclonal antibodies (human anti-CD3 mAbs), including the clinically relevant Teplizumab or Foralumab, results in leukemic cells apoptosis within hours, profound anti-leukemic effects over time, durable remission, and improved host survival (Trinquand et al., 2016; Tran Quang et al., 2020).
• anti-CD3 monoclonal antibodies human anti-CD3 mAbs
• the inventors also showed that these anti-CD3 monoclonal antibodies strongly cooperate with chemotherapy in T-ALL patients-derived xenografts (Tran Quang et al., 2020). Since teplizumab and foralumab, which are first-in-class human anti-CD3 mAb, both show only minor adverse effects in humans (for review Kuhn et al., 2016; Herold et al., 2019) and regarding the previous obtained encouraging results, these anti-CD3 monoclonal antibodies offer a novel immune-therapeutic option for the treatment of T-ALL.
• T- ALL PDX TCR + T-ALL patients-derived xenografts
• Teplizumab or Foralumab results in leukemia relapse and resistance to TCR-directed therapy.
• T-ALL in the context of TCR- directed therapies and in particular overcome leukemia relapse and resistance
• the inventors investigated specific combinations which would be effective in T-ALL.
• combination of anti-CD3 monoclonal antibodies with specific immunotherapeutic agents like other specific monoclonal antibodies, allows T-ALL cells death and anti-leukemic effect in a synergistic manner.
• an anti-CD3 agent and at least one immunotherapeutic agent for treating T-ALL particularly combination of an anti- CD3 monoclonal antibody and at least one immunotherapeutic agent for treating T-ALL.
• the invention relates to methods and pharmaceutical compositions for the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL).
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to an anti-CD3 agent for use in combination with at least one immunotherapeutic agent in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• said anti-CD3 agent for use according to the invention, it can optionally include one or more of the following characteristics alone or in combination : said anti-CD3 agent is a small organic molecule, a polypeptide, an aptamer, an oligonucleotide (such as an antisense oligonucleotide, a siRNA, a shRNA, a DNA aptamer, or an RNA aptamer), or an antibody; said antibody is a monoclonal antibody (mAb) selected from the group comprising: foralumab, teplizumab, otelixizumab, visilizumab, blinatumomab, and catumaxomab or any combination thereof; said immunotherapeutic agent is a monoclonal antibody or a Fc-fusion protein; said immunotherapeutic agent is a monoclonal antibody targeting at least one target selected from the group consisting of: PD1, 4- IBB, 0X40, PDL1,
• said immunotherapeutic agent is a combination of an anti-4- IBB agonistic mAb and an anti-OX40 agonistic mAb.
• said immunotherapeutic agent is a combination of at least one anti-4- IBB agonistic mAb, at least one anti-OX40 agonistic mAb and at least one anti-PDl or anti PDL1 antagonistic mAb.
• said immunotherapeutic agent protein is etanercept.
• the invention relates to a pharmaceutical composition for use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL), comprising a therapeutically effective amount of the anti-CD3 agent according to the invention and at least one immunotherapeutic agent according to the invention, and a pharmaceutically acceptable excipient.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the pharmaceutical composition for use in the treatment of T Cell Acute Lymphoblastic Leukemia can optionally include one or more of the following characteristics alone or in combination: said immunotherapeutic agent is selected among an anti-4- IBB agonistic mAh, an anti- 0X40 agonistic mAh, an anti-PDl antagonistic mAh, an anti-PDLl antagonistic mAh and an TNFa/LTa antagonistic mAh, or a combination thereof; said immunotherapeutic agent is a combination of at least two immunotherapeutic agents selected from an anti-4-lBB agonistic mAh, an anti-PDl antagonistic mAh, an anti-PDLl antagonistic mAh, an anti-OX40 agonistic mAh, and an anti-TNFa/LTa antagonistic mAh; a therapeutically effective amount of an anti-CD3 agent as specified above in combination with at least one anti-4- IBB agonistic mAh and at least one anti-PDl antagonistic mAh; a therapeutically effective amount of an anti-
• an anti-CD3 agent for use in combination with at least one immunotherapeutic agent in a method for inducing cell death of T Cell Acute Lymphoblastic Leukemia (T-ALL) cells.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the inventors performed microarray expression analysis and validation by Nanostring array and flow cytometry analysis of T-ALL PDX treated with a combination of the invention comprising anti- CD3 monoclonal antibodies with specific immunotherapeutic agents.
• TNFR2/TNFRSF1B 4-1BB/TNFRSF9, OX40/TNFRSF4, Fas/TNFRSF6, DR3/TNFRSF25, GITR/TNFRSF18
• ligands TNFa, LTa, TRAIL/TNFSF10, CD3OL/TNFSF8, LIGHT/TNFSF14
• effectors TRAF1, 3, 4, BIRC3 of the TNFR Super Family signaling pathways ( Figure 1A, B, C, D), while TNFR1/TNFRSF1A is constitutively expressed at the cell surface in T-ALL cells.
• mice carrying TCR + T-ALL patient-derived xenograft were co-treated with muromonab-CD3 mAb (also called anti-CD3s OKT3) and either anti-PDl antagonistic mAbs (pembrolizumab or nivolumab), or with an anti-4- IBB agonistic mAb (urelumab) or with respective control IgG2a/IgG4 monoclonal antibodies, or with the TNFa/LTa inhibitor etanercept (ENBREL).
• muromonab-CD3 mAb also called anti-CD3s OKT3
• anti-PDl antagonistic mAbs pembrolizumab or nivolumab
• an anti-4- IBB agonistic mAb urelumab
• respective control IgG2a/IgG4 monoclonal antibodies or with the TNFa/LTa inhibitor etanercept (ENBREL).
• combination of an anti-CD3 agent with at least one immunotherapeutic agent allows a synergistic anti-leukemic effect between the two agents. Furthermore, such a combination enables induction of high caspase 8 activity in T-ALL cells and cancer cell death (Figure 2).
• the invention relates to an anti-CD3 agent for use in combination with at least one immunotherapeutic agent in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to an anti-CD3 agent for use in combination with at least one immunotherapeutic agent in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the terms “subject”, “individual” or “patient” are interchangeable and refer to a mammal, even more preferably to a human, including adult and child.
• a subject according to the invention refers to any subject, preferably human.
• the term “subject” refers to a subject afflicted or at risk to be afflicted with cancer.
• the term “subject” refers to a subject afflicted or at risk to be afflicted with Acute Lymphoblastic Leukemia (ALL).
• ALL Acute Lymphoblastic Leukemia
• T-ALL T Cell Acute Lymphoblastic Leukemia
• treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or disorder or suspected to have contracted the disease or disorder, as well as subjects who are ill or have been diagnosed as suffering from a disease or disorder, and includes suppression of clinical relapse.
• the treatment may be administered to a subject having a disease or disorder or who ultimately may acquire the disease or disorder, to: prevent, cure, delay the onset of, reduce the severity of, improve one or more symptoms of, delay or slow the progression of, prevent further progression of, in order to improve a subject’s conditions and/or prolong its survival beyond that expected in the absence of such treatment.
• therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
• a therapeutic regimen may include an induction regimen and a maintenance regimen.
• the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
• the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
• An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
• maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
• a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
• combination means a combination whose base is made of an anti-CD3 agent and at least one immunotherapeutic agent co-administered to the subject for the purpose of achieving a biological effect.
• An anti-CD3 agent and at least one immunotherapeutic agent in this combination can be administered together or separately, concomitantly or sequentially. When administered together, they can be administered in a single composition comprising an anti-CD3 agent and at least one immunotherapeutic agent. In other words, said an anti-CD3 agent and at least one immunotherapeutic agent are thus formulated together. Alternatively, they can be administered separately to the subject, by the same or a different route of administration.
• said at least one immunotherapeutic agent can be administered orally and said an anti-CD3 agent and at least one other compound can be injected into the subject, for example, intravenously or subcutaneously.
• said an anti-CD3 agent and said at least one immunotherapeutic agent are administered orally to the subject, but separately.
• the sequence of administration of the active ingredients of the combination is such that said active ingredients or the active metabolites exert their biological effects at the same time, such that the subject benefits from the maximum effect of said combination.
• an anti-CD3 agent and at least one immunotherapeutic agent are administered so as to reach their maximum concentration in the general blood circulation at the same time.
• an “agent” when referred to the treatment of T-ALL according to the invention refers indifferently to the anti-CD3 agent or the immunotherapeutic agent individually, or even to the combination of said agents.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• T-ALLs are a heterogeneous group of diseases regarding immunophenotype, cytogenetics, molecular genetic abnormalities and clinical features, including response to therapy (Reviewed in Belver & Ferrando, 2016; Girardi et al., 2017, Cordo et al., 2020). Cytogenetics, molecular approaches and mouse modeling have identified driver mutations in T-ALL (for reviews, see Belver and Ferrando 2016 ; Girardi et al., 2017 ; Cordo et al., 2020).
• T-ALL Gene expression profiling is used to molecularly classify T-ALL in so-called immature/ETP-like, TLX, TLX1/NKX2.1 and TAL/LMO subgroups.
• Early T cell progenitors (ETP) T-ALL includes the most immature cases, showing an expression profile shared with hematopoietic stem cells and early myeloid progenitors; the TLX group includes immature cases lacking a functional TCR“ ⁇ but often express cell surface TCR y5 receptor.
• TLX1/NKX2.1 T-ALL share genomic rearrangement in either TLX1 or NKX2.1 and a differentiation arrest at the cortical stage of T cell development.
• TAL/LMO T-ALL represent about half pediatric T-ALL cases, dysregulation of TAL/LMO expression as the result of various genetic alterations, a differentiation arrest close to mature thymocytes and TCR 0 ' 1 expression. This molecular classification largely overlaps with differentiation arrest at different stages of T cell development as analyzed by immunophenotypic markers (Belver and Ferrando 2016).
• T-ALL T-ALL into four subgroups: (TI) the immature subgroup or pro-T-ALL is defined by the expression of only CD7; (Til) pre-T-ALL express in addition CD2 and/or CD5 and/or CD8; (Till) cortical T-ALL shows CD1 a positivity; (TIV) mature T-ALL is characterized by the presence of surface CD3 and CD1 a negativity.
• TI the immature subgroup or pro-T-ALL is defined by the expression of only CD7
• Til pre-T-ALL express in addition CD2 and/or CD5 and/or CD8
• Till cortical T-ALL shows CD1 a positivity
• TIV mature T-ALL is characterized by the presence of surface CD3 and CD1 a negativity.
• CD3 Cluster of Differentiation 3
• the term “Cluster of Differentiation 3” or “CD3” has its general meaning in the art and refers to the protein complex associated with the T cell receptor, and which is essential to its intracellular signaling activities. It is composed of four distinct chains. In mammals, the complex contains a CD3-gamma (y) chain, a CD3-delta (6) chain and two CD3-epsilon (a) chains. These chains associate with the TCR and the zeta-chain (Q to generate an activation signal in T lymphocytes. The TCR, zeta-chain (Q and CD3 molecules together constitute the TCR complex.
• TCR T Cell Receptor
• MHC major histocompatibility complex
• anti-CD3 agent has its general meaning in the art and refers to any agent selected from the group consisting of, but not limited to, compounds targeting the protein complex CD3. Accordingly, the term “anti-CD3 agent” refers to compounds that bind to CD3, CD3-gamma (y) chain, CD3-delta (6) chain, CD3-epsilon (a) chains, or TCR complex and function as potent antagonists of CD3. The term “anti-CD3 agent” can also refer to a compound that selectively inactivates CD3, TCR complex, or CD3 intracellular signaling activities.
• an anti-CD3 agent is a small organic molecule, a protein, a polypeptide, a peptide, an oligopeptide, an aptamer, an oligonucleotide (antisense oligonucleotides, siRNA, shRNA, DNA and RNA aptamers), or an antibody.
• Anti-CD3 agents are well-known in the art as such as described in Kuhn et al., 2016, Alegre et al., 1992 and Menon et al., 2023.
• anti-CD3 agent refers to any compound selected from but not limited to anti- CD3 monoclonal antibodies including but not limited to: foralumab (28F11-AE, NI-0401), teplizumab (hOKT3Yl, MGA031), muromonab-CD3 (anti-CD3 OKT3, OKT3, Orthoclone OKT3TM), otelixizumab (TRX4, ChAglyCD3, GSK2136525), visilizumab (NUVIONTM, HuM291), blinatumomab (BLINCYTOTM), catumaxomab (REMOVABTM), gOKT3-5 described in Kuhn et al., 2016 and Alegre et al., 1992.
• foralumab 28F11-AE, NI-0401
• teplizumab hOKT3Yl, MGA031
• muromonab-CD3 anti-CD3 OKT3, OKT3, Orthoclone
• Tests and assays for determining whether a compound is an anti-CD3 agent are well known by the skilled person in the art such as described in Kuhn et al., 2016, Alegre et al., 1992 and Menon et al., 2023. Determining whether a compound is an anti-CD3 agent may also be performed by using recombinant CD3 proteins, competitive binding assays and measuring the binding affinities, measuring the association of CD3 protein with the T cell receptor, measuring CD3 intracellular signaling activities, measuring the activation signal in T lymphocytes or assays such as described in the examples.
• the anti-CD3 agent for use according to the invention is an antibody, i.e., an anti-CD3 antibody.
• antibody includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods of the art to reduce its immunogenicity in man.
• Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of the target. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
• Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.
• Other suitable adjuvants are well-known in the field.
• the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
• the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in the target.
• lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma.
• cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
• cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
• Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
• an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment
• an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated a Fab fragment
• Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
• the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
• CDRs complementarity determining regions
• FRs framework regions
• CDR1 through CDRS complementarity determining regions
• non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or hetero specific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
• compositions and methods that include humanized forms of antibodies.
• humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules.
• Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.
• the above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria, which may be used in designing the humanized antibodies.
• the first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies.
• the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
• the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
• the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three-dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
• the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
• One of ordinary skill in the art will be familiar with other methods for antibody humanization.
• humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
• Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
• a "humanized" antibody retains a similar antigenic specificity as the original antibody.
• the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.
• Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
• monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
• HAMA human anti-mouse antibody
• the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
• the present invention also includes so-called single chain antibodies.
• the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
• IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
• the agent of the invention is a Human IgG4.
• the antibody according to the invention is a single domain antibody.
• the term “single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in camelid mammals, which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
• VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
• CDRs complementarity determining region
• CDR complementarity determining region
• VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation.
• VHH variants and modified form thereof may be produced under any known technique in the art such as in vitro maturation.
• VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
• Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
• immobilized antigen e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
• VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
• VHHs from immune libraries are attributed to the natural selection of variant VHHs during clonal expansion of B -cells in the lymphoid organs of immunized animals.
• the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
• VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies.
• VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
• the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
• the “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
• affinity variants of anti-CD3 agents can be obtained starting from these antibodies.
• Affinity variants useful for the invention can exhibit an improved antagonistic activity toward CD3.
• these affinity variants can be antigen -binding fragments of the above listed anti-CD3 antibodies as described above (an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, and a single domain antibody fragment (DAb)), or results from recombinant means to combine features of the above described anti-CD3 antibodies.
• VL antibody light chain variable domain
• VH antibody heavy chain variable domain
• scFv single chain antibody
• F(ab')2 fragment a Fab fragment
• Fd fragment an Fv fragment
• DAb single domain antibody fragment
• the anti-CD3 agent for use in a combination according to the invention is an affinity variant which competes for binding to CD3 with an antibody selected from the group consisting of foralumab, teplizumab, muromonab-CD3, otelixizumab, visilizumab, blinatumomab, catumaxomab (REMOVABTM), and/or gOKT3-5 as described above.
• a candidate competing antibody is considered an antibody that binds substantially to the same epitope or that competes for binding to the same epitope as the anti-CD3 antibody described herein, if the candidate competing antibody can block binding of the anti-CD3 antibody by at least 20%, preferably by at least 20-50%, even more preferably, by at least 50% as compared to a control, as using techniques well known in the art..
• binding in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10-10 M or less, or about 10-11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte.
• SPR surface plasmon resonance
• BIACORE® GE Healthcare, Piscaataway, NJ
• BIACORE® is one of a variety of surface plasmon resonance assay format that are routinely used to epitope bin panels of monoclonal antibodies.
• an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen.
• a non-specific antigen e.g., BSA, casein
• An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.
• SPR plasmon resonance
• Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard antigen binding assays.
• the ability of a test antibody to inhibit the binding of antibodies of the present invention to the target demonstrates that the test antibody can compete with that antibody for binding to the target; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the target as the antibody with which it competes.
• another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein.
• an antibody “competes” for binding when the competing antibody inhibits the target binding of an antibody or antigen binding fragment of the invention by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% in the presence of an equimolar concentration of competing antibody.
• the antibodies or antigen binding fragments of the invention bind to one or more epitopes of the target.
• the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes. In other embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are non-linear, conformational epitopes.
• Antibodies directed against an antigen of interest can be raised according to known methods by administering the appropriate antigen or epitope e.g., a full-length CD3 protein or antigenic peptide fragment of CD3 in the case of CD3) to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, camelids (camel, dromedary, llama, vicuna) and mice, among others.
• a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, camelids (camel, dromedary, llama, vicuna) and mice, among others.
• Various adjuvants known in the art can be used to enhance antibody production.
• the anti-CD3 agent for use according to the invention is a monoclonal antibody (mAb).
• the term “monoclonal antibody” refers to an immunoglobulin (https://www.genscript.com/antibody-basics.html), generated from a single B-cell clone and which recognizes unique epitopes on a single antigen (such as CD3).
• Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV- hybridoma technique using the method of Kohler (Kohler and Milstein 1975).
• anti-CD3 monoclonal antibodies are known in the art, including, but not limited to: foralumab (28F11-AE, NI-0401), teplizumab (hOKT3Yl, MGA031), muromonab-CD3 (anti- CD3 0KT3, 0KT3, Orthoclone 0KT3TM), otelixizumab (TRX4, ChAglyCD3, GSK2136525), visilizumab (NUVIONTM, HuM291), blinatumomab (BLINCYTOTM), catumaxomab (REMOVABTM), and gOKT3-5 described in Kuhn et al., 2016 and Alegre et al., 1992.
• foralumab 28F11-AE, NI-0401
• teplizumab hOKT3Yl, MGA031
• muromonab-CD3 anti- CD3 0KT3, 0KT3, Orthoclone 0KT
• the anti-CD3 agent for use according to the invention is a monoclonal antibody selected from the group comprising: foralumab, teplizumab, otelixizumab, visilizumab, blinatumomab, and catumaxomab, or any combination thereof.
• antibodies and particularly monoclonal antibodies, can be mono or bispecific.
• the anti-CD3 agent for use according to the invention can be a monospecific monoclonal antibody, selected from the group comprising: foralumab, teplizumab, otelixizumab and visilizumab, or any combination thereof.
• the anti-CD3 agent for use according to the invention can be a bispecific monoclonal antibody, selected from the group comprising: blinatumomab and catumaxomab, or any combination thereof.
• the inventors show that combination between an anti-CD3 agent with at least one immunotherapeutic agent directly targets T-ALL cells and improves the therapeutic efficacy of said anti-CD3 agent in T-ALL in an unexpectedly synergistic manner.
• the invention relates to an anti-CD3 agent for use in combination with at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen immunotherapeutic agents. As shown in the experiments combining, within the combinatory treatment according to the invention, several immunotherapeutic agents is of particular interest.
• the term “immunotherapeutic agent” is also used for “immunomodulating agent” and refers to an agent able to treat Acute Lymphoblastic Leukemia, preferably T Cell Acute Lymphoblastic Leukemia (T-ALL) either by directly killing leukemic cell and/or modulating the immune system of the subject afflicted by the T-ALL, by stimulating or suppressing the patient's immune system to attack the malignant tumor cells.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the immunotherapeutic agent according to the invention can be a small organic molecule, a protein, a fusion protein, a polypeptide, a peptide, an oligopeptide, an aptamer (Soldevilla et al., 2016), an oligonucleotide (such as antisense oligonucleotides, siRNA, shRNA, DNA and RNA aptamers), or an antibody.
• the term “immunotherapeutic agent” also refers to a cancer therapeutic treatment using the immune system to reject cancer. The therapeutic treatment stimulates the patient's immune system to attack the malignant tumor cells.
• immunotherapeutic agent also refers to immune checkpoint therapy such as checkpoint inhibitors including, but are not limited to programmed death-1 (PD-1) inhibitors, programmed death ligand- 1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte-activation gene 3 (LAG3) inhibitors, T-cell immunoglobulin and mucin-domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyte attenuator (BTLA) inhibitors, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptors (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors and carcinoembryonic antigen-related cell
• PD-1 programme
• checkpoint inhibitors include antibodies anti-PDl, anti-PD-Ll, anti-CTLA4, anti-TIM-3, anti-LAG3.
• Immune checkpoint therapy also includes co- stimulatory antibodies delivering positive signals through immune- regulatory receptors including but not limited to ICOS, CD137, CD27, 0X40 and GITR.
• Cell surface glycoprotein 4-1BB (also known as CD137 and TNFRSF9) is a member of the tumor necrosis factor receptor super family (TNFRSF) that is expressed on activated T cells, among others.
• TNFRSF tumor necrosis factor receptor super family
• Activation of 4-1BB by its 4-1BB ligand delivers potent costimulatory signals to CD8+ cytotoxic T cells, promoting cell proliferation, facilitating differentiation into memory cells, and delivering important survival signal.
• 4- IBB signaling can deliver an apoptotic signal to activated T-cells (Ma BY et al., 2005).
• 4-1BB agonistic monoclonal antibodies such as urelumab (BMS-663513) or utomilumab (PF-05082566).
• anti-4-lBB agonistic mAb such as urelumab (BMS-663513) or utomilumab (PF-05082566).
• BMS-663513 urelumab
• PF-05082566 utomilumab
• Eskiocak et al. showed that anti-4- IBB agonistic mAb are pre-clinically efficient at blocking growth of solid tumors through their ability to co-activate anti-tumor T cells cytotoxic activity and NK cells ADCC (Eskiocak et al., 2020).
• Novel therapeutic strategies aimed to minimize cytotoxicity of available agonistic mAbs are under development (Chester C. et al., 2018).
• 0X40 also known as TNFRSF4 or CD 134 is another member of the TNFR super family (TNFRSF) and is type I transmembrane glycoproteins characterized by the presence of cysteine- rich domains (CRDs).
• the only ligand known for 0X40 is OX40L.
• OX40L/OX40 interaction delivers co-stimulatory signals to antigens-stimulated T cells but can also deliver an apoptotic signal (Ma BY et al., 2005)
• anti-OX40 agonistic monoclonal antibodies such as MEDI6469 (9B12), MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916 (RG7888), GSK3174998 or MEDI6383.
• anti-OX40 agonistic monoclonal antibodies such as MEDI6469 (9B12), MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916 (RG7888), GSK3174998 or MEDI6383.
• PD-1 Programmed cell death 1 PD-1 (also called CD279) is an immune checkpoint receptor playing a critical role in immunity. Briefly, it regulates the threshold of antigen responses of T cells and B cells. Its ligands are PDL1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273) and both PD-1 and PDL1 are type I transmembrane proteins that belong to the immunoglobulin (Ig) superfamily.
• Ig immunoglobulin
• anti-PD1 antagonistic mAb such as cemiplimab, dostarlimab, nivolumab, pembrolizumab or pidilizumab (CT-011).
• Antagonistic anti-PDl or anti-PDLl (atezolizumab, durvalumab, avelumab) therapeutic antibodies are widely used clinically to relieve the Immune T cells exhaustion program in patients and restore an active anti-tumor response (Pardoll et al., 2012).
• TNFa tumor necrosis alpha
• LTa lymphotoxin alpha
• cytoplasmic complexes are formed (complex 2a and b) including FAS- associated death domain (FADD), RIPK1, RIPK3 and pro-caspase 8, that leads to cell death.
• FADD FAS- associated death domain
• RIPK1 RIPK3
• pro-caspase 8 that leads to cell death.
• Binding of TNFa/ETa to TNFR2 recruits TRAF2/ c-IAPl/c-IAP2 complexes, activation of both canonical and non-canonical NF-kB, MAP Kinase and AKT, associated with cell survival/proliferation.
• TNFR2 can also induce cell death either directly or indirectly by crosstalk with TNFR1.
• TNFa/ETa Several inhibitors of TNFa/ETa have been and continue to be developed including monoclonal antibodies (infliximab, adalimumab, certolizumab, golimumab), the TNFR2-Fc fusion protein etanercept (ENBRELTM) or low-molecular weight small chemicals (reviewed in Jang et al., 2021; Javaid N. et al., 2022).
• Example of anti-4- IBB antibodies include, but are not limited to, urelumab or utomilumab.
• Example of anti-OX40 antibodies include, but are not limited to, MED 16469 (9B12), MED 10562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916 (RG7888), GSK3174998 or MED 16383.
• Example of anti-PDl antibodies include, but are not limited to, cemiplimab (REGN2810 or REGN-2810, LIBTAYOTM), tislelizumab (BGB-A317), tislelizumab, spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR- 042, AGEN2034, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL7335 or OPDIVOTM), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or KEYTRUDATM), dostarlimab (JEMPERLITM), pidilizumab (CT-011) and antibodies described in International patent applications W02004004771, W02004056875, W02006121168, WO2008156712, W02009014708, W0
• Example of anti-PD- L1 antibodies include, but are not limited to, LY3300054, Atezolizumab, Durvalumab and Avelumab.
• Example of TNFa/LTa antibodies include, but are not limited to, infliximab, adalimumab, certolizumab, golimumab.
• Example of anti-CTLA4 antibodies include, but are not limited to, Ipilimumab (see, e.g., US patents US6,984,720 and US8,017,114), tremelimumab (see, e.g., US patents US7, 109,003 and US8, 143,379), single chain anti-CTLA4 antibodies (see, e.g., International patent applications WO1997020574 and WO2007123737) and antibodies described in US patent US8,491,895.
• Example of anti-VISTA antibodies are described in US patent application US20130177557.
• Example of inhibitors of the LAG3 receptor are described in US patent US5,773,578.
• Example of KIR inhibitor is IPH4102 targeting KIR3DL2.
• the at least one immunotherapeutic agent to be combined with an anti- CD3 for use according to the invention is a monoclonal antibody targeting at least one target selected from the group consisting of: 4-1BB, 0X40, PD-1, PDL1, TRAIL, TNFR1, TNFR2, TNFa/LTa, CD30, CD38, and/or CTLA4.
• the immunotherapeutic agent is an anti-TRAIL agent such as described in Chodorge et al., 2012, an anti-TNFRl agent, an anti-TNFR2 agent, an anti-TNFa/LTa agent such as infliximab, adalimumab, certolizumab, or golimumab, an anti-CD30 agent such as brentuximab, an anti-CD38 agent such as daratumumab, an anti-FAS agent or an anti-GITR agent.
• an anti-TRAIL agent such as described in Chodorge et al., 2012, an anti-TNFRl agent, an anti-TNFR2 agent, an anti-TNFa/LTa agent such as infliximab, adalimumab, certolizumab, or golimumab
• an anti-CD30 agent such as brentuximab
• an anti-CD38 agent such as daratumumab
• an anti-FAS agent an anti-GITR agent
• the immunotherapeutic agent is a monoclonal antibody targeting at least one target selected from the group consisting of: 4-1BB, 0X40, PD-1 and/or TNFa/LTa. In a particular embodiment, the immunotherapeutic agent is a monoclonal antibody targeting at least one target selected from the group consisting of: 4-1BB, 0X40 and/or PD-1.
• the immunotherapeutic agent is selected among anti-4- IBB mAb, anti- 0X40 mAb, anti-PDl mAb, anti-PDLl and anti-TNFa/LTa mAb, or any combination thereof.
• the immunotherapeutic agent is selected among anti-4- IBB agonistic mAb, anti-OX40 agonistic mAb, anti-PDl antagonistic mAb, anti-PDLl antagonistic mAb and/or anti-TNFa/LTa antagonist.
• the immunotherapeutic agent is selected from the group consisting of: urelumab, utomilumab, MEDI6469 (9B 12), MEDI0562, PF-04518600, INCAGN01949, B MS- 986178, MOXR0916 (RG7888), GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab and golimumab, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of: urelumab, nivolumab, and pembrolizumab, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of: urelumab, nivolumab, pembrolizumab, urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the at least one immunotherapeutic agent to be combined with an anti-CD3 for use according to the invention is a fusion protein.
• a fusion protein consists of two proteins that have been joined into single unit. This type of protein typically retains some characteristics of both original proteins. Fusion proteins can occur naturally but are often engineered to help purify a protein, or to aid in the study of its expression or localization.
• Fc-fusion proteins are specific type of fusion proteins and are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. Fc-fusion proteins can potentially be useful in some therapeutic approaches, such as treatment of HIV, inflammatory diseases, diabetes, or cancer.
• the linked protein may be an interested protein such as receptor, cytokine, enzyme, or peptide (peptibody).
• the immunotherapeutic agent is a Fc-fusion protein.
• the immunotherapeutic agent is a Fc-fusion protein selected among: etanercept (TNFR2-Fc fusion protein or ENBRELTM), alefacept, abatacept, rilonacept, romiplostim, belatacept, aflibercept, Eloctate, Alprolix, dulaglutide, trebananib, blisibimod, apocept, CNTO 528, CNTO 530, NKG2D-Fc or ALT-803.
• Fc-fusion protein selected among: etanercept (TNFR2-Fc fusion protein or ENBRELTM), alefacept, abatacept, rilonacept, romiplostim, belatacept, aflibercept, Eloctate, Alprolix, dulaglutide, trebananib, blisibimod, apocept, CNTO 528, CNTO 530, NKG2D-Fc or ALT-803.
• ENBRELTM e
• the at least one immunotherapeutic agent to be combined with an anti-CD3 for use according to the invention etanercept.
• anti-CD3 agent as previously described, with at least one immunotherapeutic agent, as previously described, can be used for the treatment of T-ALL.
• the anti-CD3 agent is used according to the invention in combination with at least one immunotherapeutic agent selected among anti-4- IBB agonistic mAb, anti-OX40 agonistic mAb, anti-PDl antagonistic mAb, anti-PDLl antagonistic mAb, anti-TRAIL agonistic mAb, anti-TNFRl antagonistic mAb, anti-TNFR2 antagonistic mAb, anti-TNFa/LTa antagonistic mAb, or any combination thereof.
• immunotherapeutic agent selected among anti-4- IBB agonistic mAb, anti-OX40 agonistic mAb, anti-PDl antagonistic mAb, anti-PDLl antagonistic mAb, anti-TRAIL agonistic mAb, anti-TNFRl antagonistic mAb, anti-TNFR2 antagonistic mAb, anti-TNFa/LTa antagonistic mAb, or any combination thereof.
• the at least one immunotherapeutic agent is selected among anti-4- IBB agonistic mAb, anti-OX40 agonistic mAb, anti-PDl antagonistic mAb, anti-PDLl antagonistic mAb and anti-TNFa/LTa antagonistic mAb or any combination thereof.
• the anti-CD3 agent is used according to the invention in combination with an anti-4- IBB agonistic mAb and an anti-OX40 agonistic mAb.
• the anti-CD3 agent is used according to the invention in combination with an anti-4- IBB agonistic mAb and an anti-PDl antagonistic mAb.
• the anti-CD3 agent is used according to the invention in combination with an anti-4- IBB agonistic mAb and an anti-PDLl antagonistic mAb.
• the anti-CD3 agent is used according to the invention in combination with an anti-4- IBB agonistic mAb and an anti-TNFa/LTa antagonistic mAb.
• the anti-CD3 agent is used according to the invention in combination with an anti-4- IBB agonistic mAb, an anti-OX40 agonistic mAb and an anti-PDl antagonistic mAb.
• the anti-CD3 agent is used according to the invention in combination with an anti-4-lBB agonistic mAb, an anti-OX40 agonistic mAb and an anti-PDLl antagonistic mAb.
• the anti-CD3 agent is used according to the invention in combination with an anti-4-lBB agonistic mAb, an anti-OX40 agonistic mAb and aTNFa/LTa antagonist.
• the anti-CD3 agent for use according to the invention is selected from the group consisting of foralumab, teplizumab, otelixizumab, visilizumab, blinatumomab, and catumaxomab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is
• the anti-CD3 agent for use according to the invention is selected from the group consisting of foralumab, teplizumab, otelixizumab and visilizumab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomil
• the anti-CD3 agent for use according to the invention is selected from the group consisting of foralumab and teplizumab, or any combination thereof
• the immunotherapeutic agent is selected from the group consisting of foralumab, teplizumab, otelixizumab and/or visilizumab
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimum
• the anti-CD3 agent for use according to the invention is foralumab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949,
• the anti-CD3 agent for use according to the invention is teplizumab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN019
• the anti-CD3 agent for use according to the invention is muromonab-CD3 and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN0
• the anti-CD3 agent for use according to the invention is otelixizumab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN0
• the anti-CD3 agent for use according to the invention is visilizumab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949,
• the anti-CD3 agent for use according to the invention is blinatumomab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN0
• the anti-CD3 agent for use according to the invention is catumaxomab and the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab (CT-011), atezolizumab, durvalumab, avelumab, infliximab, adalimumab, certolizumab, golimumab and etanercept, or any combination thereof.
• the immunotherapeutic agent is selected from the group consisting of urelumab, utomilumab, MEDI6469, MEDI0562, PF-04518600, INCAGN019
• the anti-CD3 agent for use according to the invention is teplizumab and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the anti-CD3 agent for use according to the invention is foralumab and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the anti-CD3 agent for use according to the invention is selected from the group consisting of foralumab and teplizumab, or any combination thereof, and the immunotherapeutic agent is selected from the group consisting of urelumab, nivolumab, pembrolizumab, and etanercept or any combination thereof.
• the anti-CD3 agent for use according to the invention is foralumab and the immunotherapeutic agent is selected from the group consisting of urelumab, nivolumab, pembrolizumab, and etanercept or any combination thereof.
• the anti-CD3 agent for use according to the invention is teplizumab and the immunotherapeutic agent is selected from the group consisting of urelumab, nivolumab, pembrolizumab, and etanercept or any combination thereof.
• the anti-CD3 agent for use according to the invention is muromonab-CD3 and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the anti-CD3 agent for use according to the invention is otelixizumab and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the anti-CD3 agent for use according to the invention is visilizumab and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the anti-CD3 agent for use according to the invention is blinatumomab and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the anti-CD3 agent for use according to the invention is catumaxomab and the immunotherapeutic agent is selected from the group of combinations consisting of: urelumab and nivolumab, nivolumab and pembrolizumab, and urelumab and pembrolizumab.
• the invention relates to an anti-CD3 agent for use in combination with at least one immunotherapeutic agent in a method for inducing cell death of T Cell Acute Lymphoblastic Leukemia.
• the anti-CD3 agent and immunotherapeutic agent are defined as previously described.
• the invention relates to a method for treating T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-CD3 agent in combination with at least one immunotherapeutic agent as described above.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to a method for treating T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-CD3 monoclonal antibody in combination with at least one monoclonal antibody and/or at least one Fc-fusion protein as immunotherapeutic agent.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to a combination of an anti-CD3 agent as described above with a SMAC-mimetic for use in the treatment of T-ALL.
• IAP apoptosis protein
• SMAC second mitochondrial-derived activator of caspases
• SMAC is a mitochondrial protein that binds inhibitor of apoptosis proteins, inhibiting IAPS ability to bind caspases, which are pro-apoptotic proteins. IAPs antagonistically bind caspases, thus, SMAC induces a pro-apoptotic effect. Endogenous SMAC binds the Bir-domain of c-IAPl, c- IAP2 and XIAP via an lAP-binding motif (IBM). Endogenous SMAC homodimerizes and is bivalent.
• IBM lAP-binding motif
• the SMAC mimetic is an IAP antagonist, in particular an c-IAPl/2 inhibitor.
• the invention relates to a synergistic combination of an anti-CD3 agent as described above with a SMAC-mimetic for use in the treatment of T-ALL.
• the invention relates to a synergistic combination of an anti- CD3 agent as described above with an IAP antagonist, in particular an c-IAPl/2 inhibitor, for use in the treatment of T-ALL. It has indeed been observed that cIAP inhibition synergizes with anti- CD3 in T-ALL.
• the SMAC mimetic can be selected in the group consisting of: birinapant (CAS registry No 1260251-31-7), APG-1387 (CAS registry No 1570231-89-8), Debio 1143 (also called xevinapant, CAS registry No 1071992-99-8), ASTX660 (CAS registry No 1799328-86-1), GDC-0152 (CAS registry No 873652-48-3), HGS- 1029/AEG40826 (CAS registry No 1107664-44-7), SM-164 (CAS registry No 957135-43-2), BV6 (CAS registry No 1001600-56-1), embelin 15 (CAS registry No 550-24-3), MV1 (CAS registry No 1001600-54-9), SM-1295 (CAS registry No 1562375-46- 5), SBP-0636457 (CAS registry No 1422180-49-1), GDC-0917 (CAS registry No 1446182-94-0), LCL161 (CAS registry No 1005342-46-0), XIAP/c
• the anti-CD3 agent for use according to the invention selected from foralumab, teplizumab, otelixizumab, visilizumab, blinatumomab, and catumaxomab or any combination thereof, is combined with an IAP antagonist, in particular an c-IAPl/2 inhibitor.
• the anti-CD3 agent for use according to the invention is foralumab and a SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• the anti-CD3 agent for use according to the invention is teplizumab and a SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• the anti-CD3 agent for use according to the invention is muromonab-CD3 and a SMAC mimetic is selected in the group consisting of: birinapant, APG- 1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• the anti-CD3 agent for use according to the invention is otelixizumab and SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065
• the anti-CD3 agent for use according to the invention is visilizumab and SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• the anti-CD3 agent for use according to the invention is blinatumomab and SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065
• the anti-CD3 agent for use according to the invention is catumaxomab and SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and AZD5582, or any combination thereof.
• SMAC mimetic is selected in the group consisting of: birinapant, APG-1387, debio 1143, ASTX660, GDC-0152, HGS- 1029/AEG40826, SM-164, BV6, embelin 15, MV1, SM-1295, SBP-0636457, GDC-0917, LCL161, XIAP/cIAPl antagonist-1, BI 891065 and
• the invention relates to a combination of an anti-CD3 agent as described above with birinapant for use in the treatment of T-ALL.
• the compound, combination and/or pharmaceutical composition of the invention may be used in combination with targeted therapy.
• targeted therapy refers to targeted therapy agents, drugs designed to interfere with specific molecules necessary for tumor growth and progression.
• targeted therapy agents such as therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors.
• Small molecules can penetrate the cell membrane to interact with targets inside a cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein such as for example proteasome inhibitor, tyrosine kinase or cyclin-dependent kinase inhibitor, histone deacetylase inhibitor.
• Targeted therapy may also use cytokines.
• Examples of such targeted therapy include with no limitations: Ado- trastuzumab emtansine (HER2), afatinib (EGFR (HER1/ERBB1), HER2), aldesleukin (Proleukin), alectinib (ALK), alemtuzumab (CD52), axitinib (kit, PDGFRbeta, VEGFR1/2/3), belimumab (BAFF), belinostat (HDAC), bevacizumab (VEGF ligand), blinatumomab (CD19/CD3), bortezomib (proteasome), brentuximab vedotin (CD30), bosutinib (ABE), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib
• the compound, combination and/or pharmaceutical composition of the invention may be used in combination with chemotherapy.
• chemotherapy or “chemotherapy” has its general meaning in the art and refers to a cancer therapeutic treatment using chemical or biochemical substances, in particular using one or several antineoplastic agents or chemotherapeutic agents.
• Chemotherapeutic agents include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; du
• calicheamicin especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin),
• the compound, combination and/or pharmaceutical composition of the invention is administered to the subject in combination with radiotherapy.
• radiation therapies include, but are not limited to external beam radiotherapy (such as superficial X-rays therapy, orthovoltage X-rays therapy, megavoltage X-rays therapy, radiosurgery, stereotactic radiation therapy, Fractionated stereotactic radiation therapy, cobalt therapy, electron therapy, fast neutron therapy, neutron-capture therapy, proton therapy, intensity modulated radiation therapy (IMRT), 3-dimensional conformal radiation therapy (3D-CRT) and the like); brachytherapy; unsealed source radiotherapy; tomotherapy; and the like.
• Gamma rays are another form of photons used in radiotherapy.
• Radiotherapy may be proton radiotherapy or proton minibeam radiation therapy.
• Proton radiotherapy is an ultra-precise form of radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy.
• Radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.
• FLASH radiotherapy involves the ultra-fast delivery of radiation treatment at dose rates several orders of magnitude greater than those currently in routine clinical practice (ultra-high dose rate) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31 : 121-123. DOI: 10.105 l/medsci/20153102002); Patriarca A., Fouillade C. M., Martin F., Pouzoulet F., Nauraye C., el al., Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016/j.ijrobp.2018.06.403. Epub 2018 Jul 11).
• the compounds or combinations of the invention may be used or prepared in a pharmaceutical composition.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the compounds or combinations of the invention and a pharmaceutical acceptable carrier for use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of an anti-CD3 agent and at least one immunotherapeutic agent, and a pharmaceutical acceptable carrier for use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL).
• T-ALL T Cell Acute Lymphoblastic Leukemia
• a “therapeutically effective amount” of the agent of the present invention as above described is meant a sufficient amount of the agent at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the agents and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific agent employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific agent employed; the duration of the treatment; drugs used in combination or coincidental with the specific agent employed; and like factors well known in the medical arts.
• the daily dosage of the products may be varied over a wide range from 0.005 to 2,000 mg per adult.
• the compositions contain from 0.01 to 1000 mg, in particular from 0.1 to 500 mg of the agent of the present invention for the symptomatic adjustment of the dosage to the patient to be treated.
• a medicament typically contains from 0.01 mg to 1000 mg of the agent of the present invention, in particular from 0.1 mg to 500 mg of the agent of the present invention, in particular from 1 mg to 500 mg of the agent of the present invention, specifically 25 mg, 50 mg or 400 mg.
• An effective amount of the drug is ordinarily supplied at a dosage level from 0.005 mg/kg to 20 mg/kg of body weight, especially from 0.01 mg/kg to 10 mg/kg of body weight, especially from 0.1 mg/kg to 1 mg/kg of body weight.
• Dosage, mode and frequency of delivery of clinically approved therapeutic mAbs is a subject of intense ongoing research and clinical trials (Hirch I et al., 2022).
• the agent of the present invention is administered to the subject in the form of a pharmaceutical composition.
• the agent of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• pharmaceutically acceptable excipients such as a carboxylate, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, sorbitol, aditol, adiluent, encapsulating material or formulation auxiliary of any type.
• an active principle alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
• Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and intranasal administration forms and rectal administration forms.
• the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form In all cases, the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Solutions comprising agents of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the agent of the present invention can be formulated into a composition in a neutral or salt form.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
• the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized agents of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the agent of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
• aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• therapeutic effect it is meant an effect induced by an agent or a pharmaceutical composition comprising such agent, capable to prevent or to delay the appearance or development of a disease or disorder, or to cure or to attenuate the effects of a disease or disorder.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutic effective amount of an anti-CD3 agent in combination with an anti-4- IBB agonistic mAb and an anti-OX40 agonistic mAb and a pharmaceutical acceptable carrier for use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutic effective amount of an anti-CD3 agent in combination with an anti-4- IBB agonistic mAb, an anti-OX40 agonistic mAb and an anti-PDl antagonistic mAb and a pharmaceutical acceptable carrier for use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutic effective amount of an anti-CD3 agent in combination with a Fc-fusion protein, in particular etanercept and a pharmaceutical acceptable carrier for use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• the pharmaceutical composition for use according to the invention comprises a therapeutic effective amount of an anti-CD3 agent in combination with a SMAC mimetic, preferably birinapant.
• Every such formulation can also contain other pharmaceutically compatible and nontoxic auxiliary agents, such as, e.g., stabilizers, antioxidants, binders, dyes, emulsifiers, or flavoring substances.
• auxiliary agents such as, e.g., stabilizers, antioxidants, binders, dyes, emulsifiers, or flavoring substances.
• the pharmaceutical composition according to the invention is administered intravenously, orally, by mucosal route, or by nasal route.
• the pharmaceutical composition contains from 0.005 to 2,000 mg of an agent, in particular from 0.01 to 1,000 mg, especially from 0.1 to 500 mg. In a specific embodiment, the pharmaceutical composition contains from 1 mg to 400 mg of an agent, especially 25, 50 or 400 mg.
• the pharmaceutical composition contains an effective amount of the agent at a dosage level from 0.005 mg/kg to 20 mg/kg of body weight, especially from 0.01 mg/kg to 10 mg/kg of body weight, especially from 0.1 mg/kg to 5 mg/kg of body weight, advantageously from 0.1 mg/kg to 1 mg/kg of body weight, advantageously from 0.3 mg/kg to 0.8 mg/kg of body weight.
• the compound, agent, combination and/or pharmaceutical composition of the invention is administered to the patient every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks. In particular, every two to six weeks, especially every two or three weeks.
• the compound, agent, combination and/or pharmaceutical composition of the invention is administered to the patient at a rate of one, two, three, four, five, six, seven or eight times per week. In a particular embodiment, the compound, combination and/or pharmaceutical composition of the invention is administered to the patient at a rate of one or two times per week.
• the compound, combination and/or pharmaceutical composition of the invention is administered to the patient for a period of 1 to 12 months, in particular of 1 to 8 months, specially of 2 to 6 months.
• the compound, combination agent and/or pharmaceutical composition of the invention may further be administered in addition to other anti-cancer therapy, for example: chemotherapy, radiotherapy, CAR-T cell or CAR-NK cell treatments, immune checkpoint inhibitor treatment and/or therapeutic vaccines.
• anti-cancer therapy for example: chemotherapy, radiotherapy, CAR-T cell or CAR-NK cell treatments, immune checkpoint inhibitor treatment and/or therapeutic vaccines.
• said additional active compounds or agent may be contained in the same composition or administrated separately.
• the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of T Cell Acute Lymphoblastic Leukemia (T-ALL) in a subject in need thereof.
• T-ALL T Cell Acute Lymphoblastic Leukemia
• kits comprising the compounds of the invention.
• Kits containing the compounds of the invention find use in therapeutic methods.
• FIG. 1 Transcriptome profiling of T-ALL PDX in response to anti-CD3: Leukemic mice were injected with 40pg control IgG, anti-hCD3 OKT3 or anti-hCD3 teplizumab for 6 hours. Leukemic cells were then collected, flow-cytometry sorted, RNA isolated and analyzed using human Affymetrix arrays. A Hierarchical clustering on genes deregulated significantly more than 2-fold is shown for IgG2a, (IgG): teplizumab; (Tepli): OKT3 (OKT3)). B List of genes of interest found significantly upregulated in response to OKT3 and teplizumab. FC: Fold Change.
• RNA expression analysis (digitalized numeration of transcripts - mRNA) of genes of interest in M106 xenograft overtime after OKT3 treatment using Nanostring arrays: TNFa, LTa, TNFRSF9 (4- 1BB), TNFRSF1B (TNFR2), TNFRSF4 (0X40) and PDCD1 (PD1).
• Figure 3 A Combination of 0KT3 (muromonab-CD3) and urelumab - upper panel : Leukemia burden at time of death of the control arm in NSG mice injected with leukemic cells from a diagnostic T-ALL case (M106) and treated with control IgG, lOmg/kg of urelumab , 4pg of 0KT3 per mice or combination of 0KT3 and urelumab ; ns non-significant, *p ⁇ 0.05; ***p ⁇ 0.001, ANOVA, Tukey’s Multiple Comparison test.
• M106 diagnostic T-ALL case
• Figure 4 A Flow cytometry analysis of cell death (PI staining) in control (black symbols) and PD1 knock-down cells (two independents shRNA were used, open and filled circles) in absence (-) or response (+) to anti-CD3 stimulation.
• B Combination of 0KT3 (muromonab-CD3) and nivolumab - upper panel Leukemia burden at time of death of mice from the control arm in NSG mice injected with leukemic cells from a diagnostic T-ALL case (M106) and treated with control IgG , 4pg of 0KT3 per mice (oc-CD3), lOmg/kg of nivolumab (OPDIVO; oc-PDl) or combination of 0KT3 and Nivolumab (oc-CD3 + oc-PDl) ; ns non- significant, **p ⁇ 0.01; ***p ⁇ 0.001, ANOVA, Tukey’s Multiple Comparison test.
• mice from the same experiment and treated with control IgG (control IgG, dotted black line), 0KT3 ( 0C-CD3, dark grey line), lOmg/kg of nivolumab (OPDIVO®; oc-PDl (nivolumab, dotted black line) or combination of 0KT3 and nivolumab (OPDIVO®; 0C-CD3+0C-PD1, light grey line) .
• Figure 5 Survival of NSG mice injected with leukemic cells from a diagnostic T-ALL case and treated with control IgG, 4pg of OKT3per mice, 20mg/kg of birinapant (Biri), enbrel (Enbrel, 10mg/kg,) or combination of either 0KT3 and birinapant (OKT3+Biri) or 0KT3 and enbrel (OKT3+Enbrel).
• Figure 6 Interest of combining several immunotherapeutic agents with an anti-CD3 agent : Leukemia burden at time of death of mice from the control arm in NSG mice injected with leukemic cells from a diagnostic T-ALL case (M106) and treated with control IgG (Ctr), teplizumab (Tepli, anti-CD3), pembrolizumab (Pembro, PD1 antagonist), urelumab (Ure, 4- IBB agonist) alone or their teplizumab-based combinatory treatment combination according to the invention.
• T-ALL case M106
• NSG female mice i.e., devoid of T cells, B cells, NK cells; age: 2 months; Charles River Laboratories
• mice were maintained under specific pathogen-free conditions.
• Experiments were carried out in accordance with the European Union and French National Committee recommendations, under agreement APAFIS #7393-2016102810475144-vl for the care and use of laboratory animals.
• Leukemic mice are euthanized when terminally ill, as evidenced by weakness caused by leukemic dissemination in vital organs (bone marrow, lung, and liver).
• mice 1 x 10 6 fresh leukemic cells obtained from primary NSG mice engrafted with patient- derived cells from T-ALL cases (M106 or UPNT420) are intravenously injected in 2-month-old NSG mice. Tumor load is monitored in blood of engrafted mice using flow cytometry analysis (FSC hl , hCD7 + , hCD45 + cells). When tumor load reached 1% of nucleated cells (meaning around 10-20% of leukemic load in BM), mice are injected intravenously with either anti-CD3s OKT3 mAb (muromonab-CD3, 4 pg/mouse) or the isotype matched (IgG2a) mAb.
• OKT3 mAb muromonab-CD3, 4 pg/mouse
• IgG2a isotype matched
• teplizumab a humanized, mutated version of OKT3 which is FDA-approved in prevention of type I diabetes (Herold et al., 2019) and with foralumab, a fully human anti-CD3 mAb (Tran Quang et al., 2020).
• Etanercept Enbrel
• the c-IAPl/2 inhibitor birinapant (20mg/kg) was administrated every 3-4 days for 3 weeks.
• mice are simultaneously injected once with 4pg anti-CD3s OKT3 mAb and 200pg of either pembrolizumab or nivolumab (anti- PD1 mAbs) or respective control antibodies.
• Mice are monitored weekly by flow cytometry for leukemic load (FSC hl , hCD7 + , hCD45 + cells) in peripheral blood.
• FSC hl , hCD7 + , hCD45 + cells leukemic load
• Statistical analyses and survival curves are calculated using Prism 5 (GraphPad). One-way ANOVA was performed using Tukey’s test and used to analyze leukemia burden. Kaplan-Meier survival curves were compared using the log-rank test. Differences were considered statistically significant at p ⁇ 0.05 (*), p ⁇ 0.01 (**) or p ⁇ 0.001 (***).
• Jurkat cell line is cultured in RPMI-1640 medium supplemented with 50 pg/ml streptomycin, 50 IU penicillin, and 15% fetal bovine serum (Gibco, Life Technology). Lentivirus are produced as described in Passaro (Passaro et al., 2015) and used to transduce Jurkat cell lines. GFP + - transduced cells are sorted using ARIA-FACS sorter. Validation of PD1 knock-down is analyzed by flow cytometry.
• Apoptosis in response to anti-CD3/CD28 beads is analyzed using annexin V-APC and propidium iodide (PI) (BD Biosciences).
• cRNA synthesis and hybridization of GeneChip Human Gene 2.0ST Array (Affymetrix) were according to the manufacturer’s instructions, as described (http://www-microarrays.u-strasbg.fr).
• a paired Student’s t-test was performed to compare gene intensities in the different biological replicates. Genes were considered significantly regulated when fold-change was > 2 and p value ⁇ 0.05.
• Hierarchical clustering analysis and pathway analyses on regulated genes were performed by Genosplice company.
• mice carrying TCR + T-ALL patient derived xenograft (PDX) with anti- CD3 mAbs Monotherapy of mice carrying TCR + T-ALL patient derived xenograft (PDX) with anti- CD3 mAbs, while inducing durable remission, ultimately results in leukemia relapse and resistance to TCR-directed therapy (Ghysdeal et al., 2016; Ghysdeal et al., 2020).
• the inventors thus sought to identify molecular pathways induced by anti-CD3 mAbs in T-ALL cells with the aim to therapeutically target these pathways and create improved vulnerability to anti-CD3 mAbs therapy.
• the inventors compared the in vivo global transcriptome induced by Muromonab-CD3 and the clinically relevant anti-CD3 mAb teplizumab in T-ALL PDX cells.
• the muromonab-CD3/teplizumab samples cluster together and identify a TCR-induced signature in T-ALL of ca 600 deregulated genes (2X; p-value ⁇ 0.05) common to both monoclonal antibodies (Fig. 1A).
• the top signature identified is massive deregulation in ligands (e.g., TRAIL, TNFa, LTa), receptors (e.g., TNFR2, 4- IBB, 0X40), adaptors (TRAFs) and effectors (e.g., cIAP2, TNFAIP3/A20) of the TNF signaling pathway as well as the ITIM-containing, inhibitory receptor PD-1 (Fig. IB).
• ligands e.g., TRAIL, TNFa, LTa
• receptors e.g., TNFR2, 4- IBB, 0X40
• TRAFs adaptors
• effectors e.g., cIAP2, TNFAIP3/A20
• Fig. 6 shows said improvement is further increased when using combined immunotherapeutic agents, e. g. 2 immunotherapeutic agents, one anti-PDl antagonistic mAb and one 4- IBB agonistic mAb.
• immunotherapeutic agents e. g. 2 immunotherapeutic agents, one anti-PDl antagonistic mAb and one 4- IBB agonistic mAb.
• TNF-* 1 Tumor Necrosis Factor Alpha

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