Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57426—Specifically defined cancers leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the invention is in the field of oncology and more particularly treatment of T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• T-cell acute lymphoblastic leukemias are malignant proliferations of thymocytes by multi-step oncogenic processes leading to T-cell differentiation arrest.
• Targeted therapy including abrogation of a block to cell maturation, is a promising therapeutic approach in cancer.
• the mechanism underlying the therapeutic effect of OKT3 monoclonal antibody in said T-ALL patient is based on the stimulation of the immune system as suggested by the induction of IL-2 responsiveness of the malignant cells leading to propose a sequential application of OKT3 and IL-2 to render certain T cell tumours more susceptible to chemotherapy.
• the invention relates to a non-mitogenic anti-CD3 antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• the invention in a second aspect, relates to a non-mitogenic anti-CD3 antibody for use in a method for inducing cell death of T-ALL cells.
• the invention in a third aspect, relates to a pharmaceutical composition comprising a non-mitogenic anti-CD3 antibody for use in a method for treating T-ALL.
• the invention in a fourth aspect, relates to an in vitro method for predicting the responsiveness of a patient affected with a T-ALL to a treatment with a TCR-activating agent comprising a step of determining the expression of TCR/CD3 complex at the surface of T-ALL cells obtained from a patient, wherein the absence of the TCR/CD3 complex is indicative of the non-responsiveness to said treatment.
• the invention relates to a TCR-activating agent for use in a method for treating T-ALL in a patient classified as responder according to the method of the invention.
• the invention relates to an anti-TCR antibody for use in a method for treating T-ALL.
• OKT3 monoclonal antibody acts directly on T-ALL blasts. They demonstrated indeed that targeting CD3 both in vitro and in vivo, induces human primary T-ALL cell death and a molecular program that mimics thymic negative selection. Importantly, the inventors identified a new method for stratifying T-ALL patients and predicting their responsiveness to a treatment with an anti-CD3 antibody since they observed massive T-ALL cell death and TCR signaling activation in TCR-expressing T-ALL (also referred herein as CD3 -positive primary T-ALL cells) regardless of their molecular oncogenic subtype, but not in TCR-negative cases.
• the present data provide strong and unexpected rationale for targeted therapy based on non-mitogenic anti-CD3 treatment of T-ALLs in patients identified as responder.
• the invention provides methods and compositions (such as pharmaceutical compositions) for use in a method for treating T-ALL.
• the invention also provides methods and compositions for use in a method for inhibiting proliferation and/or inducing cell death of T-ALL cells.
• the invention also provides methods and compositions for use in a method for inducing irreversible differentiation of T-ALL cells.
• the invention relates to an anti-CD3 antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• CD3 Cluster of Differentiation 3 refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3-epsilon, CD3 -delta, CD3-zeta, and CD3 -gamma.
• antibody that binds CD3 or "anti-CD3 antibody” include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers).
• the antibodies and antigen-binding fragments of the invention may bind soluble CD3 and/or cell surface expressed CD3.
• anti-CD3 antibody includes both monovalent antibodies with a single specificity, as well as bispecific antibodies comprising a first arm that binds CD3 and a second arm that binds a second (target) antigen.
• the invention relates to an anti-TCR antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• TCR T Cell Receptor
• MHC major histocompatibility complex
• antibody that binds TCR or “anti-TCR antibody” include antibodies and antigen-binding fragments thereof that specifically recognize the constant region of the a chain of the TCR or otherwise specifically binds the a chain regardless of clonal origin of the T-cell as well as antibodies, or antigen binding fragment thereof, that specifically binds the constant region of the ⁇ chain of the TCR or otherwise specifically binds the ⁇ chain regardless of clonal origin of the T-cell.
• antibody or “immunoglobulin” have the same meaning, and will be used equally in the present invention.
• the term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
• the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.
• two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k).
• the heavy chain includes two domains, a variable domain (VL) and a constant domain (CL).
• the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
• VL variable domain
• VH variable domain
• CH constant domain
• the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
• the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
• the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
• Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site.
• Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
• the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
• An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
• Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
• Fab denotes an antibody fragment having a molecular weight of about
• F(ab')2 refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
• Fab refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
• a single chain Fv (“scFv”) polypeptide is a covalently linked VH: : VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
• dsFv is a VH:: VL heterodimer stabilised by a disulfide bond.
• Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
• a peptide linker such as divalent sc(Fv)2.
• the term "diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy- chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL).
• 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) to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, Camelidae (camel, dromedary, llama) and mice, among others.
• a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, Camelidae (camel, dromedary, llama) and mice, among others.
• a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, Camelidae (camel, dromedary, llama) and mice, among others.
• Various adjuvants known in the art can be used to enhance antibody production.
• antibodies useful in practicing the invention can be
• 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.
• techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies against the CD3.
• Useful antibodies according to the invention also include antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
• Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to the CD3.
• the anti-CD3 or the anti-TCR antibodies can be polyclonal or monoclonal.
• the antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human.
• Human antibodies avoid certain of the problems associated with antibodies that possess murine or rat (or other species) variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient.
• murine or rat derived antibodies In order to avoid the utilization of murine or rat derived antibodies, one can develop humanized antibodies or generate fully human antibodies through the introduction of human antibody function into a rodent so that the rodent would produce antibodies having fully human sequences.
• chimeric antibody refers to a genetically engineered fusion of parts of an animal antibody, typically a mouse antibody, with parts of a human antibody. Generally, chimeric antibodies contain approximately 33% mouse protein and 67% human protein. Developed to reduce the Human Anti-animal Antibodies response elicited by animal antibodies, they combine the specificity of the animal antibody with the efficient human immune system interaction of a human antibody.
• Humanized antibodies and antibody fragments therefrom can also be prepared according to known techniques.
• “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non- human immunoglobulin.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• the human chimeric antibody of the invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell.
• the CH domain of a human chimeric antibody it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used.
• the CL of a human chimeric antibody may be any region which belongs to Ig, and those of kappa class or lambda class can be used.
• Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See patent documents US5,202,238; and US5,204,244).
• the humanized antibody of the invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell.
• the humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type).
• humanized antibody expression vector of the tandem type In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred.
• tandem type humanized antibody expression vector include pKANTEX93 (WO 97/10354), pEE18 and the like.
• the antibodies are fully human monoclonal antibodies.
• Such fully human monoclonal antibodies directed against human CD3 or human TCR can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.
• transgenic and transchromosomic mice include mice referred to herein as the HuMAb Mouse® and KM Mouse®, respectively, and referred as "human Ig mice.”
• the HuMAb Mouse® (Medarex®, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy ( ⁇ and ⁇ ) and ⁇ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and ⁇ chain loci (see e.g., Lonberg et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or ⁇ , and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg et al. (1994), supra; reviewed in Lonberg
• Fully human antibodies of the invention can also be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome.
• This mouse is referred to herein as a "KM mouse” and is described in detail in PCT Publication WO 02/43478.
• a modified form of this mouse, which further comprises a homozygous disruption of the endogenous FcyRIIB receptor gene, is also described in PCT Publication WO 02/43478 and referred to herein as a "KM/FCGR2D mouse®.”
• mice with either the HCo7 or HCol2 heavy chain transgenes or both can be used.
• transgenic animal embodiments include the Xenomouse (Abgenix, Inc., U.S. Patent Nos. 5,939,598; 6,075, 181; 6,114,598; 6,150,584 and 6,162,963). Further embodiments include "TC mice” (Tomizuka et al. (2000) Proc. Natl. Acad. Set USA 97:722-727) and cows carrying human heavy and light chain transchromo somes (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894; PCT Publication WO 02/092812).
• a number of anti-CD3 antibodies are known, including but not limited to, OKT3 (muromonab/Orthoclone OKT3TM, Ortho Biotech, Raritan, NJ; U. S. Patent No. 4,361, 549); hOKT3Yl (teplizumab) (MGA031) (Herald et al, NEJM 346 (22): 1692-1698 (2002); TRX4 (otelixizumab); HuM291 (NuvionTM, Protein Design Labs, Fremont, CA); gOKT3-5 (Alegre et al. , J. Immunol. 148 (11): 3461-8 (1992); 1F4 (Tanaka et al, J. Immunol.
• the anti-CD3 antibody is a non-mitogenic anti-CD3 antibody or a fragment thereof.
• the non-mitogenic anti-CD3 antibody is a non- mitogenic anti-CD3 monoclonal antibody.
• the present invention relates to a non- mitogenic anti-CD3 antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• the non-mitogenic anti-CD3 antibody is used in a method for inducing cell death of T-ALL cells, in particular TCR-expressing T-ALL cells or blasts (also referred herein as CD3 -positive primary T-ALL cells).
• non-mitogenic has its general meaning in the art and has properties to bind to the CD3 antigen but without inducing cytokine production, as described in US7041289 Bl .
• a non-mitogenic anti-CD3 antibody delivers for instance a partial T cell signal that renders activated T cells unresponsive.
• the non-mitogenic anti-CD3 antibody is TRX4 (otelixizumab) or MGA031 (teplizumab).
• the present invention relates to an anti-TCR antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• the anti-TCR antibody is used in a method for inducing cell death of T-ALL cells, in particular TCR- expressing T-ALL cells or blasts (also referred herein as CD3-positive primary T-ALL cells).
• the antibody of the invention recognizes TCR a chains, and, thus the ⁇ / ⁇ TCR, generally (i.e., is a pan-specific TCR antibody, and, in particular, a pan-specific TCR a chain antibody).
• the antibody of the invention recognizes TCR ⁇ chains, and, thus the ⁇ / ⁇ TCR, generally (i.e., is a pan-specific TCR antibody, and, in particular, a pan-specific TCR ⁇ chain antibody).
• anti-TCR antibodies in particular anti-c$-TCR antibodies
• anti-c$-TCR antibodies are known, including but not limited to those described in the international patent applications n° WO2013037484 and WO2015066379 which disclose humanized anti-c ⁇ TCR antibodies, derived from the murine monoclonal antibody BMA031 (see European patent application EP0403156) such as GL1BM VH31, GL1BM VH28, HEBE1 H66, and HEBE1 H71.
• 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 suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
• the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject 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 intervals, 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.]).
• T-cell acute lymphoblastic leukemia refers a malignancy accounting for about 15% and 25% of ALL in pediatric and adult cohorts respectively.
• T-ALLs are a heterogeneous group of diseases with regard to immunophenotype, cytogenetics, molecular genetic abnormalities and clinical features, including response to therapy as described in Graux et al. 2006.
• EGIL European Group for the Immunological Characterization of Leukemias
• the presence of cytoplasmic or membrane expression of CD3 defines T-ALL.
• T-ALL the immature subgroup or pro-T-ALL is defined by the expression of only CD7;
• pre-T-ALL also expresses CD2 and/or CD5 and/or CD8;
• Till or cortical T-ALL shows CD la positivity;
• TIV finally, mature T-ALL is characterized by the presence of surface CD3 and CD la negativity.
• T-ALL present genetic abnormalities (involving the T- cell receptor (TCR), involving known oncogenes such as Notchl, HOXA cluster, TLXl(HOXl l) and TLX3 (HOX11L2), TALI and LYL1, involving tyrosine kinases such as ABL1, JAK3 and JAKl).
• TCR T- cell receptor
• the patients are refractory to other treatments.
• refractory represents previously treated patients which were either non responsive to treatment with the agent or had a response to treatment and then relapsed.
• sequential polychemotherapy are used for treating T-ALL according to different protocols.
• said sequential polychemotherapy comprises sequentially administering to said patients corticosteroids (e.g. prednisone) and L-asparaginase (GRAALL regimen).
• corticosteroids e.g. prednisone
• GAAALL regimen L-asparaginase
• the invention relates to an in vitro method for predicting the responsiveness of a patient affected with a T-ALL to a treatment with a TCR-activating agent.
• the invention relates to an in vitro method for predicting the responsiveness of a patient affected with a T-ALL to a treatment with a TCR-activating agent comprising a step of determining the expression of TCR/CD3 complex at the surface of T-ALL cells obtained from a patient, wherein the absence of the TCR/CD3 complex is indicative of non-responsiveness to said treatment.
• the term "predicting” refers to a probability or likelihood for a patient to respond to the treatment with a TCR-activating agent.
• the term “responsiveness” refers to ability to assess the likelihood that treatment will or will not be clinically effective.
• a patient denotes a mammal, typically a human.
• a patient refers to any subject (typically human) afflicted with T-ALL.
• TCR-activating agent is intended to encompass any refers to a natural or synthetic compound that has a biological effect which stimulate TCR/CD3 complex intracellular signaling activity.
• the TCR activating agent stimulates TCR/CD3 complex presents on T-ALL cells.
• stimulation of the TCR/CD3 complex is associated with a series of biochemical events initiated by TCR engagement (e.g., by antigen-MHC complex or anti-CD3 cross-linking) and that typically triggers (or contributes to triggering) one or more pathways (by, for example, activating transcription of certain genes such as, e.g., BCL2L1-BIM, GIMAP4, LiPA4 or Nur77 also referred as NR4A1).
• the TCR-activating agents include but are not limited to antibodies, synthetic or native sequence peptides and small molecules which bind to the TCR/CD3 complex.
• the TCR-activating agent is an anti-CD3 antibody as above- described.
• the anti-CD3 antibody is a non-mitogenic anti-CD3 antibody such as for instance TRX4 (otelixizumab) or MGA031 (teplizumab).
• the TCR-activating agent is an anti-TCR antibody as above- described.
• the anti-TCR antibody is an anti-aP-TCR antibody such as for instance GL1BM VH31, GL1BM VH28, HEBE1 H66, and HEBE1 H71.
• the TCR-activating agent is an epitope obtained from a T-ALL cells of the patient, more particularly TCR idiotypes which may function as antigen and activate T cells.
• the TCR-activating agent is an inhibitor of TCR regulating tyrosine phosphatases, e.g. CD45, SHPl, SHP2 and PTPN22.
• the term "inhibitor of TCR regulating tyrosine phosphatases” refers to any compound, natural or synthetic, which results in a decreased phosphorylation of the tyrosine present on the intracellular domain of TCR.
• TCR regulating tyrosine phosphatases inhibitors such as inhibitors of CD45 and/or SHPl are described in the international patent applications n° W09946268 and W09946236.
• the inhibitor of TCR regulating tyrosine phosphatases is an inhibitor of CD45, SHPl, SHP2 or PTPN22 gene expression.
• inhibitor of gene expression refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene is meant a CD45, SHP1, SHP2 or PTPN22 mRNA, protein, peptide, or polypeptide.
• Inhibitors of gene expression for use in the invention may be based on antisense oligonucleotide constructs.
• Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein (i.e. CD45), and thus activity, in a cell.
• antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the target protein can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
• RNA interference small double stranded RNA
• dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).
• Short hairpin RNAs can also function as inhibitors of gene expression for use in the invention.
• determining encompasses detecting or quantifying. Indeed, an expression level can be qualitative or quantitative with or without reference to a control or a predetermined value. Thus, a determination of whether a polypeptide is present or absent (e.g., detectable or undetectable) constitutes determining its expression level in various embodiments while in other embodiments, a quantitative level is determined.
• detecting means determining if TCR/CD3 complex is present or not at the surface of T-ALL cells obtained from a patient and "quantifying" means determining the amount of TCR/CD3 complex present at the surface of T-ALL cells.
• TCR/CD3 T cell receptor-CD3 complex
• MHC major histocompatibility complex
• the method of the invention comprises a step of determining the expression TCR/CD3 complex at the surface of T-ALL cells obtained from a patient, wherein the absence of TCR/CD3 complex is indicative of non-responsiveness to said treatment.
• Determination of the expression level of TCR/CD3 complex at the surface of T-ALL cells may be performed by a variety of techniques.
• the expression level as determined is a relative expression level.
• the determination comprises contacting the biological sample with selective reagents ligands, and thereby detecting the presence, or measuring the amount, of polypeptides of interest originally in said biological sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a specific ligand array.
• the substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
• the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
• the contacting may be made under any condition suitable for a detectable complex, such as an antibody-antigen complex, to be formed between the reagent and the polypeptides of the biological sample.
• Such methods comprise contacting the biological sample of a patient containing T-ALL cells with a binding partner capable of selectively interacting with the protein present in said sample.
• the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
• the binding partners of the invention such as antibodies, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art.
• Labels are known in the art that generally provide (either directly or indirectly) a signal.
• the term "labelled" with regard to the antibody or aptamer is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g.
• FITC fluorescein isothiocyanate
• PE phycoerythrin
• Cy5 lndocyanine
• the aforementioned assays generally involve the coating of the binding partner (ie. antibody) in a solid support.
• Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e.
• polystyrene latex e.g., beads or microtiter plates
• polyvinylidine fluoride e.g., diazotized paper
• nylon membranes e.g., nylon membranes
• activated beads e.g., magnetically responsive beads, and the like.
• the step of determining the expression of TCR/CD3 complex at the surface of T-ALL cells is performed by immunoassays such as for instance immunophenotyping as described in Asnafi et al., 2004 or by immunohistochemistry (IHC).
• immunoassays such as for instance immunophenotyping as described in Asnafi et al., 2004 or by immunohistochemistry (IHC).
• the step of determining the expression of a target protein such as TCR/CD3 complex by a cell population such as T-ALL cells may be carried out by a variety of methods for detecting a particular immune cell population available for a skilled artisan, including immunoselection techniques, such as high-throughput cell sorting using flow cytometric methods, affinity methods with antibodies labeled to magnetic beads, biodegradable beads, non-biodegradable beads, and combination of such methods.
• immunoselection techniques such as high-throughput cell sorting using flow cytometric methods, affinity methods with antibodies labeled to magnetic beads, biodegradable beads, non-biodegradable beads, and combination of such methods.
• flow cytometric methods refers to a technique for counting cells of interest, by suspending them in a stream of fluid and passing them through an electronic detection apparatus.
• Flow cytometric methods allow simultaneous multiparametric analysis of the physical and/or chemical parameters of up to thousands of particles per second, such as fluorescent parameters.
• Modern flow cytometric instruments usually have multiple lasers and fluorescence detectors.
• a common variation of flow cytometric techniques is to physically sort particles based on their properties, so as to purify or detect populations of interest, using "fluorescence-activated cell sorting".
• FACS fluorescence-activated cell sorting
• T-ALL cells may be stained with a FITC-labeled anti-CD38 mAb (such as the clone 145-2C11 purchased by Pharmingen).
• a FITC-labeled anti-CD38 mAb such as the clone 145-2C11 purchased by Pharmingen.
• the invention relates to an in vitro method for predicting the responsiveness of a patient affected with a T-ALL to a treatment with a TCR-activating agent comprising a step of determining the presence of TCR- expressing T-ALL cells (also referred as CD3-positive T-ALL cells) in a biological sample obtained from a patient, wherein the absence of the TCR-expressing T-ALL cells (or CD3- positive T-ALL cells) is indicative of non-responsiveness to said treatment.
• TCR-expressing T-ALL cells also referred as CD3-positive T-ALL cells
• IHC specifically provides a method of determining the expression of a target protein in a biological sample or tissue specimen in situ.
• the overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the target of interest.
• a biological sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy.
• Current methods of IHC use either direct labelling or secondary antibody-based or hapten-based labelling. Examples of known IHC systems include, for example, EnVision TM (DakoCytomation), Powervision® (Immunovision, Springdale, AZ), the NBATM kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine ® (Nichirei Corp, Tokyo, Japan).
• the method further comprises a step of analysing TCR clustering by performing flow cytometry analysis.
• the invention relates to a TCR-activating agent for use in a method for treating T-ALL in a patient classified as responder according to the method above-described.
• the TCR-activating agent is a non-mitogenic anti-CD3 antibody.
• the invention also relates to a method for treating T-ALL in a patient in need thereof and identified as responder according to a method of the invention comprising a step of administrating to said patient a therapeutically amount of a TCR-activating agent.
• responder refers to a patient who may respond to a treatment with a TCR-activating agent such as an anti-CD3 antibody, i.e. least one of his symptoms is expected to be alleviated, or the development of the disease is stopped, or slowed down.
• the invention also relates to pharmaceutical compositions comprising a TCR-activating agent for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• the invention also relates to pharmaceutical compositions comprising an anti-CD3 antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• said anti-CD3 antibody is a non-mitogenic anti-CD3 antibody such as for instance TRX4 (otelixizumab) or MGA031 (teplizumab).
• the invention also relates to pharmaceutical compositions comprising an anti-TCR antibody for use in a method for treating T-cell acute lymphoblastic leukemia (T-ALL).
• T-ALL T-cell acute lymphoblastic leukemia
• the anti-TCR antibody is an anti-aP-TCR antibody such as for instance GL1BM VH31, GL1BM VH28, HEBE1 H66, and HEBE1 H71.
• an antibody of the 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, aminoethylcellulose, aminoethyl cellulose, aminoethyl cellulose, aminoethyl cellulose, aminoethyl sulfate, a pharmaceutically acceptable graft copolymer, or adiluent, encapsulating material or formulation auxiliary of any type.
• compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
• 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 doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
• an effective amount of the antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• 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 must be sterile and must be fluid to the extent that easy syringability 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.
• Dosages to be administered depend on individual needs, on the desired effect and the chosen route of administration. It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• the total dose required for each treatment may be administered by multiple doses or in a single dose.
• the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
• the daily dosage of the antibodies may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
• the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
• a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
• An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 10 mg/kg of body weight per day.
• the pharmaceutical composition of the invention may further comprise an additional drug, in particular a chemotherapeutic drug useful in T-ALL.
• chemotherapeutic drugs useful in T-ALL include but not limited to, corticosteroids (e.g. prednisone), L-asparaginase, non-selective inhibitors of NOTCH 1, ABLl inhibitors and JAK3 inhibitors and PBK-AKT-mTOR pathway inhibitors as described in Roti et al, 2014.
• Non-selective inhibitors of NOTCH1 include soluble or cell-associated Notch decoys, [gamma] -secretase inhibitors, intracellular MAML1 decoys, and Ras signaling inhibitors.
• [gamma]-Secretase inhibitors (GSI) have the most immediate therapeutic potential.
• Blocking specific E3 ligases responsible for ubiquitination of Notch ligands or mono-ubiquitination of Notch receptors can also be used.
• Still other NOTCH 1 inhibitors that can be used are described in WO 2003/013527 and paragraphs 0016-0019 of US 2006/205666.
• Non-limiting examples of ABLl inhibitors include imatinib (STI571) (Novartis), nilotinib AMN107 (Novartis), dasatinib (BMS-354825) (BMS), ponatinib (AP24534), bosutinib (SKI-606) (Pfizer) and bafetinib.
• Exemplary ABLl inhibitors include but are not limited to, those as described in the following patent applications: N-phenyl-2-pyrimidine- amine derivatives (EP0564409), pyrimidinylaminobenzamide derivatives (WO2004/005281), cyclic compounds (WO00/62778), bicyclic heteroaryl compounds (WO2007/075869), substituted 3-cyano quinoline derivatives (US6002008), 4-anilo-3-quinolinecarbonitrile derivatives (WO2005/04669) and amide derivatives (US7728131 and WO2005/063709).
• Non- limiting examples of PI3K inhibitors include: NVP-BEZ235 (BEZ235) (Novartis); LY294002 (Cell Signaling #9901); GDC-0941 (Genentech/Roche); GDC-0980 (Genentech); PI- 103 (Piramed); XL147 (Exilixis/Sanofi-Aventis); XL418 (Exilixis); XL665 (Exelixis); LY29002 (Eli Lilly); ZSTK474 (Zenyaku Kogyo); BGT226 (Novartis); wortmannin; quercetin; tetrodotoxin citrate (Wex Pharmaceuticals); thioperamide maleate; IC87114; PIK93; TGX-115; deguelin; NU 7026; OSU03012; tandutinib (Millennium Pharmaceuticals); MK-2206 (Merck); OSU-03012;
• Exemplary PI3K inhibitors include but are not limited to those as described in the following patent applications: WO2008/027584, WO2008070150, 2,3-dihydroimidazo[l,2-c]quinazolines (WO2008/125833), 2-morpholin-4- yl-pyrimidines (WO2008/125835), pyrimidines (WO2008/125839), bicyclic heteroaryls (WO2009/010530), thiazolidinones (WO2009/026345), pyrrolothiazoles (WO2009/071888), tricyclic thiazole and thiophene derivatives (WO2009/071890), fused bicyclic thiazole and thiophene derivatives (WO2009/071895) and oxazole substituted indazoles (WO2010/125082).
• the invention also relates to a method for treating T-cell acute lymphoblastic leukemia (T-ALL) in a patient in need thereof, comprising a step of administering to said patient a therapeutically effective amount of an anti-CD3 antibody or an anti TCR-antibody.
• T-ALL T-cell acute lymphoblastic leukemia
• terapéuticaally effective amount is meant an amount sufficient to achieve a concentration of antibodies which is capable of preventing, treating or slowing down the disease to be treated. Such concentrations can be routinely determined by those of skilled in the art.
• the amount of the polypeptide actually administered will typically be determined by a physician or a veterinarian, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the patient, the severity of the subject's symptoms, and the like. It will also be appreciated by those of skilled in the art that the dosage may be dependent on the stability of the administered polypeptide.
• the treatment with an anti-CD3 antibody is administered in more than one cycle, i.e. the administration of an anti-CD3 antibody (such as an OKT3, TRX4 or MGA031 monoclonal antibodies) is repeated at least once.
• the intervals i.e. the time between the start of two subsequent cycles, are typically several days.
• Kit-of-parts compositions
• the anti-CD3 antibody such as the OKT3 or the anti-TCR antibody as above-described and the chemotherapeutic agent may be combined within one formulation and administered simultaneously. However, they may also be administered separately, using separate compositions. It is further noted that they may be administered at different times.
• the invention relates to a kit-of-parts composition
• a kit-of-parts composition comprising an anti-CD3 antibody as above-defined and a chemotherapeutic agent.
• the invention relates to a kit-of-parts composition
• a kit-of-parts composition comprising an anti-TCR antibody as above-defined and a chemotherapeutic agent.
• the invention relates to a kit-of-parts composition
• a kit-of-parts composition comprising an anti-CD3 antibody as above-defined and a chemotherapeutic agent for use in a method for treating T-ALL.
• the invention relates to a kit-of-parts composition
• a kit-of-parts composition comprising an anti-TCR antibody as above-defined and a chemotherapeutic agent for use in a method for treating T-ALL.
• kit means especially a "kit-of-parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously or at different time points.
• the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
• the ratio of the total amounts of the combination partners to be administered in the combined preparation can be varied.
• the combination partners can be administered by the same route or by different routes. When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 7, days before the second partner.
• FIGURES are a diagrammatic representation of FIGURES.
• Figure 1 In vivo TCR stimulation with an agonistic monoclonal antibody inhibits T-cell leukemogenesis in the mouse TEL-JAK2 model.
• A Percentage of leukemic cells within peripheral blood leukocytes of female mice treated with 5 daily doses of 20 ⁇ g 145- 2C11 mAb or control hamster IgG (arrows), starting 1 day after female TJM leukemic cell inoculation.
• B Kaplan-Meier leukemia- free survival curves of mice transplanted with TJM leukemic cells and treated with antibody before leukemia onset (arrows) (Log-rank (Mantel- Cox) test, P value ⁇ 0.01).
• C Percentage of leukemic cells within peripheral blood leukocytes of mice treated with 5 daily doses of 20 ⁇ g 145-2C11 mAb or control hamster IgG, starting 2 days after TEL-JAK2 leukemic cell inoculation (arrows).
• D Kaplan-Meier leukemia-free survival curves for mice transplanted with TEL-JAK2 leukemic cells and treated with antibody before leukemia onset (P value ⁇ 0.01).
• E Percentage of leukemic cells within peripheral blood leukocytes of female TEL-JAK2:Marylin double transgenic mice (TJM) secondary leukemia mice treated with 5 daily doses of 20 ⁇ g 145-2C11 mAb or control hamster IgG (arrows), after leukemia detection.
• F Kaplan-Meier leukemia- free survival curves for mice transplanted with TEL-JAK2 leukemic cells and treated with antibody after leukemia onset (arrows) (P value ⁇ 0.05).
• FIG. 2 In vivo TCR stimulation with an agonistic monoclonal antibody inhibits T-cell leukemogenesis in human T-ALL cells xenografted in NSG mice.
• B NSG mice were injected with 10 6 T-ALL cells.
• Figure 3 Anti-CD3 stimulation of ALL-SIL TCR-HY cells induces TCR signaling and cell death.
• A Percentage of annexin V-positive ALL-SIL or ALL-SIL TCR-HY cells either left unstimulated or stimulated by CD3/CD28 coated-beads for the indicated time.
• B Western Blot analysis of caspase3 and caspase7 cleavage in ALL-SIL and ALL-SIL TCR-HY cells either left unstimulatedor stimulated by CD3/CD28 coated-beads for 48 and 72 h. Actin was used as a loading control.
• C Growth curve of unstimulated and CD3/CD28-stimulated ALL-SIL and ALL-SIL TCR-HY cells.
• E Phospho-ERK, phospho-AKT and phospho- STAT3 expression detected by flow cytometry in non- stimulated (top line in each panel) or 30 min anti-CD3/CD28-stimulated (bottom line) ALL-SIL TCR-HY cells.
• FIG. 4 Anti-CD3 induces human primary T-ALL cell death in vitro and mimics thymic negative selection. Comparison of cell death induction in CD3-positive primary, diagnostic T-ALL cells (9 cases) versus CD3 -negative primary T-ALL cells (4 cases) after 3 days of co-culture on OP9-DL1 in the presence or absence of CD3/CD28 stimulation.
• mice The E ⁇ SR ⁇ -7 ⁇ X-J4AT2 transgenic mice (line 71) (Carron et al, 2000), Marylin transgenic mice (Lantz et al, 2000), and Rag2 knock-out, all on the C57BL/6 background, and Swiss Nude were maintained under specific-pathogen- free conditions in the animal facilities of the Institut Curie (Orsay, France) and University of Algarve (Faro, Portugal). All experimental procedures were performed in strict accordance with the recommendations of the European Commission (Directive 86/609/EEC), French National Committee (87/848) and Portuguese authorities (Decreto-Lei n°129/92) for the care and use of laboratory animals.
• European Commission Directive 86/609/EEC
• French National Committee 87/848
• Portuguese authorities Decreto-Lei n°129/92
• mice were euthanized by CO2 inhalation when terminally ill, due to either severe dyspnea caused by massive expansion of leukemic cells in the thymus, or weakness caused by leukemic dissemination to vital organs such as bone marrow, lung, and liver. Blood was collected from the submandibular vein.
• leukemia transplantation assays 0.5-2 x 10 6 leukemic T cells collected from diseased female TEL-JAK2: Marylin double transgenic mice (TJM) mice were intravenously injected in the tail vein of recipient mice of the indicated gender and strain and regularly monitored for external signs of disease development or through blood analysis.
• TJM Marylin double transgenic mice
• Anti- CD3s 145-2C11 monoclonal antibody or control ChromPureSyrian Hamster IgG (Jackson Immunoresearch) was diluted in sterile PBS and intravenously administered on a regimen of 5 daily doses of 20 ⁇ g for C57BL6 mice or 50 ⁇ g for SwissNudemice). Statistical analyses and survival curves were calculated using Prism 5 (GraphPad).
• the anti-CD3 OKT3 MAb BioXCell; 2 rounds of treatment with 40 ⁇ g/mice/day for 5 consecutive days separated by a 2 days interval
• IgG2a iso type-matched
• mice were treated when leukemic cells reached 1-4% of blood nucleated cells. Mice were monitored weekly by flow cytometry for leukemic load in peripheral blood, T-ALL cells being identified as FSC hl , hCD7+, hCD45+ cells.
• T-ALL patient samples and cell lines Immunophenotypic and oncogenic features of T-ALL patients were identified as described (6, 47, 48). Fresh or thawed primary T-ALL samples (peripheral blood or bone marrow) obtained at diagnosis from adult and pediatric patients were used, as well as T-ALL cells xenografted in NSG mice. Informed consent was obtained according to the Declaration of Helsinki. All samples used contained >80% blasts. CD4+CD8+ (DP) human thymocytes were obtained and processed as described in Supplementary methods. The ALL-SIL cell line (DSMZ, Braunschweig, Germany, ACC511) were grown in vitro and transduced as described in Supplementary methods.
• Flow cytometry Single cell suspensions prepared from lymphoid organs, bone marrow flushed from tibias or blood were stained with fluorochrome-labeled antibodies, and detected on a FACSCalibur cytometer (BD Biosciences), as previously described (Carron et al, 2000). FITC-, PE-, PE-Cy5-, PerCP/Cy5.5- or APC-conjugated antibodies specific for CD4 (H129.19), CD8aD(53-6.7), CD3s (145-2C11), TCRp (H57-597), V 6 TCR (RR4-7), (GL3), CD25 (7D4), CD45.1 (A20), and CD45.2 (104) (BD Biosciences) were used.
• Antibodies used to identify human T-ALL cells were FITC-conjugated anti- human CD7 cl 4H9 and APC- conjugated anti-human CD45 (eBiosciences). Data analysis was performed using CellQuest (BD Biosciences) software.
• Splenocytes were obtained from C57BL/6 female mice, subsequently irradiated (2000 rad) and preincubated with antigenic peptides at 37°C for 1 h (peptide pulsing).
• Peptides used were DBY, the male antigen epitope specifically recognized by Marilyn transgenic TCR-HY, at 10, 1, 0.1, 0.01, 0.001 and 0.0001 ⁇ and OVA, a non-specific peptide, at 10 ⁇ .
• Splenocytes were then plated out in 48-well plates, without removal of peptides. ALL-SIL TCR-HY cells were added to the co-culture system at a 1 : 100 ratio.
• Antibodies used for human cells CD3, TCRVb6, Antibodies against phosphorylated ERK1/2, phosphorylated p38, phosphorylatedS6, were from BD, phosphorylated AKT from Cell Signaling.
• Anti CD3/CD28 cell stimulation Short-term stimulation was performed on cells cultured in serum- free medium for at least 15min at 37°C prior to stimulation.
• Murine anti- hCD3 mAb (OKT3, Bio legend) (20 ⁇ g/ml) was added to the cell culture on ice for 10 min and mAbs were then cross-linked by addition of goat-anti-mouse antibody to a final concentration of 50 ⁇ g/ml for 15 min.
• Stimulation (0-10 min) was triggered by warming cells at 37°C and terminated either by cooling cells in cold PBS or fixing cells for Phosphokinase array and intracellular phosphoprotein (Phosflow) assays, respectively.
• a bead-based assay was used in which cell lines were cultured in 96- or 24-well-plates and exposed to 4.5 ⁇ diameter superparamagnetic beads covalently coupled to anti-CD3 and anti- CD28 antibodies (Dynabeads® Human T-Activator CD3/CD28, Invitrogen) (duration of stimulation from 1 to 12 days).
• Prolonged stimulation of primary T-ALL cells was performed in a co-culture assay with confluent OP9-DL1 in a a-MEM media supplemented with 20% FBS (Hyclone; ThermoFisherScientific), 50 ⁇ g/ml streptomycin and 50 IU penicillin and recombinant human cytokines hFLT3-L (5 ng/mL), hIL-7 (2 ng/mL) and hSCF (10 ng/mL) (Miltenyi).
• FBS Hyclone; ThermoFisherScientific
• 50 ⁇ g/ml streptomycin 50 ⁇ g/ml streptomycin and 50 IU penicillin and recombinant human cytokines hFLT3-L (5 ng/mL), hIL-7 (2 ng/mL) and hSCF (10 ng/mL) (Miltenyi).
• CD3/CD28 coated-beads were
• Cell stimulation and intracellular flow cytometry with phosphorylation specific antibodies Before being stimulated, cells were deprived of serum for at least 15min at 37°C. Cells were stimulated in serum-free RPMI medium at 37°C. For bead-based stimulation, 4.5 ⁇ diameter, superparamagnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies (Dynabeads® Human T-Activator CD3/CD28, Invitrogen) were used. The stimulation was terminated by addition of prewarmed formaldehyde (Cytofix Buffer, BD Biosciences) at 37°C for lOmin.
• Cell death analysis Cells were incubated with CD3/CD28 beads according to the manufacturer's instruction, in a 96-well plate. A different time of culture, cell death was analysed by flow cytometry using AnnexinV-APC and propidium iodide (BD Biosciences) Western Blot: Cells were stimulated as previously describe with OKT3 or beads.
• Proteins were separated on tris-glycine gels under reducing conditions and transferred to nitrocellulose membranes (Biorad). Immunob lotting was performed with following antibodies: phosphor-ERKl/2 (ref, cell Signaling), ERK1/2 (cell Signaling), NFAT1 (cell Signaling), Caspase3 (BD), Caspase7 (BD). Flow cytometry analysis of TCR clustering.
• ALL-SIL TCR-HY cells were stained with TCR V 6-PE mAb (BD Biosciences) for 30 min at 4°C and DAPI for 5 min at room temperature prior stimulation and split into two samples; one sample remained unstimulated while the other one was stimulated by styrene beads (Polyscience) pre-coated with human anti- CD3s antibody (OKT3, Biolegend) for 10 min at 37°C. Cells were fixed with 1% formaldehyde. Staining was analyzed usinglmageStream X mkll (Amnis Merck-Millipore). Data analysis was performed using the IDEAS image analysis software (Amnis).
• ALL-SIL TCR-HY cells and human DP thymocytes were either left unstimulated or were stimulated with CD3/CD28 mAbs for 5 min. Each stimulated condition was performed in replicate.
• Cell lysates and Proteome Profiler Human phosphokinase array were performed according to the manufacturer's protocol. Chemiluminescence was detected by ChemiDoc XRS+ (Bio-Rad). The average signal (pixel density) of duplicate spots representing each phosphorylated kinase protein was normalized with the average signal of reference positive duplicate spots of each membrane using Image Lab software (Bio-Rad). Signal ratios for selected phosphoproteins were displayed in a heat-map using Treeview software. Cyclosporine A (Novartis) and PD184352 (Selleckchem) were used at ⁇ M. Effects were determined by analysis of cell death (Annexin V/PI assay) and expression of CD25 and CD69 activation markers.
• RNA extraction was performed for the following cells: ALL-SIL-TCRa -GFP co-cultured on OP9-DL1, ALL-SIL-TCRa -GFP without co-culture, ALL-SIL transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. These samples were obtained as previously described (14) in duplicate, and RNA extraction was performed at an early time-point before cell death onset (48 h of co- culture for ALL-SIL-TCRa -GFP and 48h after puromycin selection for TLX shRNA/sh- control ALL-SIL). RNA hybridization was performed on Affymetrix U133 plus 2.0 microarrays.
• the statistical data analysis was performed with R version2.9.0 using the "Affy" package from Bioconductor.
• the probe intensities were log2 transformed and normalized using RMA.
• Identification of differentially expressed genes was performed by Significance Analysis of Microarrays (SAM), using 500 permutations and a false discovery rate threshold of 5%.
• Functional analysis of differentially expressed genes was performed using the Ingenuity Pathway Analysis (IP A; www.ingenuity.com) and the Database for Annotation, Visualization, and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov)softwares.
• Transgenic Marylin mice express in thymocytes an MHC class II -restricted TCR ( ⁇ . ⁇ ) specific for the H-Y male antigen (DBY), which in males induces thymocyte negative selection and maturation arrest (Lantz et al, 2000).
• DBY H-Y male antigen
• TEL-JAK2 transgenic mice were crossed with Marilyn mice to generate TEL-JAK2; Marylin (TJM) double transgenic mice.
• TJM males developed leukemia with significantly delayed latency.
• Flow cytometry analysis showed that of five diseased mice analyzed, one developed an IgM-positive immature-type B-cell lymphoma affecting the thymus and spleen, while four mice developed Thyl .2 + CD4 ⁇ CD8 + CD25 + T-cell leukemia infiltrating the thymus, spleen, lymph nodes and bone marrow.
• transgenic TCR- ⁇ cell surface expression was low or absent.
• mice expressing the transgenic Marilyn TCR present reduced thymocyte cellularity due to self-antigen-induced negative selection (Lantz et al, 2000).
• female TJM CD45.1+ leukemic cells were transplanted to either female or male syngenic CD45.2 recipients.
• the majority of recipient males presented on average much lower numbers of donor malignant cells in blood and survived considerably longer.
• female recipients presented higher infiltration of CD45.1+ leukemic cells in spleen, lymph nodes, bone marrow and other organs than males sacrificed simultaneously.
• TJM leukemic mice treated TJM leukemic mice with an anti-CD3smAb (145-2C11).
• anti-CD3smAb 145-2C11
• mice were transplanted to female mice, to avoid Marilyn TCR antigenic stimulation, and then treated 24 hours later and for 5 consecutive days with anti-CD3smAb, peripheral blood leukemia and fatal disease was prevented, as compared to control antibody- treated mice ( Figure 1 A,B).
• anti-CD3streatment delayed the increase in peripheral blood leukemia and prolonged the survival of mice transplanted with either TJM ( Figure IE) or TEL-JAK2 ( Figure IF) leukemic cells.
• the CD3santibody also delayed peripheral blood leukemia in Swiss Nude mice transplanted with TEL-JAK2 leukemic cells, indicating that the anti-leukemogenic effect of this antibody is due to a direct action on TCR/CD3 -positive leukemic T cells and not on host-derived T cells.
• TALL cells obtained from a TCR+ T-ALL diagnostic cases xenotransplanted into NOD/SCID/y c -/- (NSG) mice.
• NSG mice were injected with T-ALL cells and, 24 hours later, divided into 2 groups. The first was treated (2 rounds of 5 consecutive days) with the CD3-specific OKT3 monoclonal antibody and the second with an isotype control antibody.
• Leukemic cells hCD45 + hCD7 + ) expansion was followed over time in the blood of recipient mice.
• Anti-CD3 stimulation of ALL-SIL TCR-HY cells induces TCR signaling and cell death.
• TCR-expressing T-ALL To test whether anti-CD3 stimulation of TCR-expressing T-ALL diagnostic samples also induced cell death, primary human T-ALL cells belonging to the major molecular oncogenic T-ALL subtypes(10, 18) were treated with anti-CD3/anti-CD28. Massive T-ALL cell death and TCR signaling activation were observed in TCR-expressing T-ALL but not in TCR-negative cases, regardless of their molecular oncogenic subtype (Fig. 4). As silencing of the TLX1 driving oncogene in ALL-SIL cells was previously shown to induce both differentiation and cell death(14), we compared the transcriptional profiles of ALL-SIL cells in response to either TCR stimulation or small hairpin RNA (shRNA)-mediated knock-down of TLX1.
• shRNA small hairpin RNA
• T-ALL therapies involve complex, often toxic chemotherapeutic regimens. Although T-ALL outcome has improved with current therapy, survival rates remain only around 50 and 70% at 5 years in adult and pediatric T-ALL, respectively(20, 21).
• the genetic bases of T-ALL progression and maintenance are well characterized but have not translated so far into targeted therapies(lO). There is thus unmet need for new treatments to offer therapeutic options for refractory disease and to prevent relapse.
• chronic/strong TCR signalling causes massive T-ALL cell death and shows potent tumor suppressive function in vivo.
• Muromonab-CD3 (OKT3) was approved by the U.S. Food and Drug Administration (FDA) in 1985 for therapy of acute, glucocorticoid-resistant rejection of allogeneic renal, heart and liver transplants (22) and was in fact the first monoclonal antibody introduced in the clinic. Since then, a number of other monoclonal antibodies to CD3 were developed (23) that may prove superior to OKT3 in T-ALL treatment. Of note, encouraging response to OKT3 therapy was reported in an adult patient with an aggressive and chemotherapy-resistant T-ALL, but the basis of this response was not studied (24). A major drawback of current chemotherapeutic regimens in T-ALL is the frequent resistance to treatment and relapse.
• FDA U.S. Food and Drug Administration
• LICs Leukemia initiating cells from residual disease are thought to be responsible for relapsing cases (25, 26).
• resistance of T-ALL to chemotherapy is in part linked to the recurrent genetic abnormalities selected during disease progression, e.g. inactivation of the PTEN tumor suppressor gene and the resulting activation of PI3kinase/AKT signaling (27, 28).
• Whether administration of anti-CD3 therapy during the remission phase or its association with conventional chemotherapy regimens could target LICs and/or bypass molecular mechanisms of primitive resistance represent promising directions to be explored in future prospective studies.
• T cells mature in the thymus following a highly orchestrated process controlled both by cell intrinsic (e.g. transcription factors) and extrinsic (e.g. stroma-derived cytokines/chemokines) molecular cues (29, 30).
• Cell surface TCRD ⁇ expression in DP thymocytes allows recognition of specific self-MHC/peptide to transduce a positive selection signal and maturation into SP thymocytes.
• DP thymocytes not receiving this signal die through lack of stimulation; whereas those whose TCR binds too strongly to self-MHC/peptide undergo activation- induced cell death and negative selection (5, 31).
• TCR binding to pMHC is the triggering event but how TCR engagement leads to such divergent outcomes (survival and proliferation versus death) remains unclear (32).
• these two contrasting processes are driven by a TCR signaling machinery of qualitatively similar composition (31).
• the difference lies in the molecular interpretation of signals of different strength, which may rely on compartmentalization of key signaling players. Indeed, it has been shown that a small increase in ligand affinity for the TCR leads to a marked change in the subcellular localization (plasma membrane for negative selecting ligands versus Golgi complex for positively selecting ones) of essential adaptors of the Ras signaling pathway.
• TCR B-cell receptor
• pre-BCR B-cell malignancies
• T-ALL often arises from immature T-cell precursors before the stage of negative selection (6, 42). Consequently, contingent expression of a non-negatively selected TCR will render T-ALL cells sensitive to TCR-activating apoptotic signals that mimic negative selection, as demonstrated here by anti- CD3 treatment in vitro and in vivo.
• selective events enable cells to escape post-malignancy negative selection, such as loss of TCR surface expression, as found in a subset of T-ALL patients (6) and as demonstrated here in TJM double transgenic male mice.
• Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer cell. 2002 Feb;l(l):75-87.
• TEL-JAK2 transgenic mice develop T-cell leukemia. Blood. 2000 Jun 15;95(12):3891-9.
• Yui MA Rothenberg EV. Developmental gene networks: a triathlon on the course to T cell identity. Nature reviews. 2014 Aug;14(8):529-45.
• Pre-TCR expression cooperates with TEL-JAK2 to transform immature thymocytes and induce T-cell leukemia. Blood. 2007 May 1;109(9):3972-81.
• T-ALL T-cell acute lymphoblastic leukemia
• GAAALL Ratio ALL

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