JP2005518785A - Internal medicine - Google Patents

Abstract

In the manufacture of a medicament for use as an immunostimulant, such as a vaccine adjuvant, inhibitors of the Notch signaling pathway are used.

Description

  The present invention relates to the modulation of immune function, particularly through the use of modulators of the Notch signaling pathway.

  International Patent Publication WO 98/20142 describes how manipulation of the Notch signaling pathway can be used in immunotherapy and in the prevention and / or treatment of T cell mediated diseases. In particular, this document covers allergies, autoimmunity, transplant rejection, tumor-induced abnormalities of the T cell lineage, as well as eg malaria parasite species, microfilariae, helminths, mycobacteria, HIV, cytomegalovirus, Pseudomonas, Toxoplasma, Echinococcus, B Discusses methods that can target infections caused by influenza A, measles, hepatitis C, or toxic.

  It has also been shown that it is possible to produce certain regulatory T cells that can transmit antigen-specific tolerance to other T cells, a process termed infection tolerance (WO 98/20142). The functional activity of these cells can be mimicked by overexpression of Notch ligand protein on their cell surface or on the surface of antigen presenting cells. In particular, regulatory T cells can be generated by overexpression of members of the Delta or Serrate family of Notch ligand proteins. Delta- or Serrate-induced T cells specific for one antigenic epitope can also convey tolerance to T cells that recognize other epitopes of the same or related antigens, a phenomenon called “epitope spreading”. is there.

  Notch ligand expression also plays a role in cancer. Indeed, up-regulated Notch ligand expression has been observed in several tumor cells. These tumor cells can confer T cell unresponsiveness to re-stimulation with specific antigens and thus provide an explanation of how tumor cells interfere with normal T cell responses. Down-regulation of Notch signaling in T cells in vivo can be used to prevent tumor cells from inducing immune tolerance with those T cells that recognize tumor-specific antigens. This in turn allows T cells to initiate an immune response against tumor cells (WO 00/135990).

A description of the Notch signaling pathway and conditions affected thereby can be found in Applicant's published PCT application PCT / GB97 / 03058 (filed Nov. 6, 1997 and GB 9623236.8 filed Nov. 7, 1996). Claims GB97156674.9 filed July 24, 1997, and GB9719350.2 filed September 11, 1997, published as WO 98/20142), PCT / GB99 / 04233 (December 15, 1999) Claims the priority of GB9827604.1 filed December 15, 1999, published as WO 00/36089, and PCT / GB00 / 04391 (filed November 17, 2000, November 18, 1999) Claims priority of GB9927328.6 of Japanese application, WO013 Can be found in public as 990). Each of PCT / GB97 / 03058 (WO98 / 20142), PCT / GB99 / 04233 (WO00 / 36089), and PCT / GB00 / 04391 (WO0135990) are hereby incorporated by reference.
WO98 / 20142 WO00 / 135990 PCT / GB97 / 03058 (filed on November 6, 1997, GB9623236.8 filed on November 7, 1996, GB97156674.9 filed on July 24, 1997, and GB9719350 filed on September 11, 1997) .2 claim priority, published as WO 98/20142) PCT / GB99 / 04233 (filed on December 15, 1999, claiming priority of GB9827604.1 filed on December 15, 1999, published as WO00 / 36089) PCT / GB00 / 04391 (filed on November 17, 2000, claims priority of GB9927328.6 filed on November 18, 1999, published as WO0135990) US5428130 (Genentech) US6121045 (Millennium) WO97 / 01571 WO96 / 27610 WO92 / 19734 Geysen, European Patent No. 0138855, published September 13, 1984 US6281344 (Phylos) US-A-20020119540 US5648464 US5849869 US 6004924 WO0020576 US5859205 EP-A-0120694 (Celltech Limited) EP-A-0125023 (Genentech Inc. and City of Hope) EP-A-0171496 (Res. Dev. Corp. 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  The present invention aims to provide further methods of modulating the immune system by modifying the Notch signaling pathway, particularly for treating infections.

According to a first aspect of the invention,
i) an inhibitor of the Notch signaling pathway, or a polynucleotide encoding such an inhibitor, and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant,
Products are provided that include simultaneous, concomitant, individual, or sequential use combination preparations for modulating the immune system.

  Preferably, this factor does not act by down-regulating Notch or Notch ligand expression.

According to another aspect of the invention,
i) Notch antagonist factor, or an inhibitor of Notch signaling in the form of a polynucleotide encoding such factor, and ii) encoding a pathogen antigen or antigenic determinant, or a pathogen antigen or antigenic determinant Polynucleotides,
Products are provided that include simultaneous, concomitant, individual, or sequential use combination preparations for modulating the immune system.

According to another aspect of the invention,
i) an agent that inhibits Notch-Notch ligand interaction, or an inhibitor of Notch signaling in the form of a polynucleotide encoding such an agent, and ii) a pathogen antigen or antigenic determinant, or a pathogen antigen or antigen A polynucleotide encoding the determinant,
Products are provided that include simultaneous, simultaneous, separate, or sequential use combination preparations to modulate the immune system.

  Suitably, such a product may take the form of a pharmaceutical composition or kit.

  Suitably, such products may take the form of therapeutic vaccine compositions or kits (including so-called “pharmaccine”) for treating infections.

  Alternatively, such a product can take the form of a prophylactic vaccine composition or kit for preventing infection.

  According to another aspect of the invention, there is provided the use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use as an immunostimulant. Preferably, the agent is not used to reverse bacterial, infection, or tumor-induced immunosuppression, or to treat a tumor.

  As used herein, the term “immunostimulatory agent” refers to an agent that can restore reduced immune function or enhance normal immune function, or both. This factor can boost the subject's immune system in general or with respect to a particular antigen or antigenic determinant. Immunostimulants may be used for the treatment of conditions requiring systemic immune stimulation, including immune deficiency conditions such as acquired immune deficiency syndrome (AIDS) and severe combined immunodeficiency (SCID), and antigen specific stimulation such as vaccination It can be used in situations where it is desired.

  According to another aspect of the invention, there is provided the use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use in vaccination against pathogens.

  According to another aspect of the invention, there is provided the use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use as a vaccination adjuvant against pathogens.

  As used herein, the term “pathogen” refers to a parasite that causes disease, usually a microorganism. This term includes, for example, viruses, bacteria, protozoa, and fungi.

  As used herein, the term “pathogen antigen” includes an antigen found in a pathogen, or an antigenic determinant (epitope, preferably an immunodominant epitope) or an epitope region (preferably an immunodominant epitope region) of such antigen. It refers to a fragment, variant, or derivative of such an antigen. Preferably, the antigen is immunogenic (immunogen). Suitably the antigen is a microbial pathogen antigen.

  According to another aspect of the invention, a method of stimulating the immune system by administering an inhibitor of the Notch signaling pathway, preferably a reversal of bacterial, infection, or tumor-induced immunosuppression, or of a tumor Methods are provided that do not include treatment.

  As used herein, the terms “inhibitor of Notch signaling” and “inhibitor of Notch signaling pathway” are any one that results in (or includes) activation of Notch receptors. Or any factor that can reduce multiple upstream or downstream events. Preferably, inhibitors of Notch signaling do not act by down-regulating Notch or Notch ligand expression.

  According to another aspect of the invention, a method of stimulating the immune system by administering an inhibitor of a Notch signaling pathway, wherein the inhibitor down-regulates expression of Notch or a Notch ligand. A method that does not work is provided.

  According to another aspect of the present invention, there is provided a method of vaccinating against a pathogen by administering an inhibitor of the Notch signaling pathway.

  According to another aspect of the invention, a method is provided for enhancing vaccination against pathogens by administering an inhibitor of the Notch signaling pathway.

  According to another aspect of the invention, a method of stimulating the immune system to treat or prevent an infection by administering an inhibitor of Notch signaling pathway, wherein the method inhibits bacterial, infection or tumor-induced immunosuppression. Methods are provided that do not involve reversal or tumor treatment.

  According to another aspect of the invention, a method of stimulating the immune system to treat or prevent an infection by administering an inhibitor of Notch signaling pathway, wherein the inhibitor of Notch signaling pathway is Notch or Methods are provided that do not work by downregulating Notch ligand expression.

  According to another aspect of the invention, a method of treating acute pathogen infection is provided by administering an inhibitor of Notch signaling pathway.

  According to another aspect of the invention, a method of treating a chronic pathogen infection is provided by administering an inhibitor of the Notch signaling pathway.

  According to another aspect of the present invention, a method for increasing a subject's immune response to a vaccine antigen or antigenic determinant, comprising simultaneously, separately or sequentially with said vaccine antigen or antigenic determinant, or said vaccine antigen or There is provided a method comprising administering to said subject an effective amount of an inhibitor of Notch signaling pathway simultaneously, separately or sequentially with a polynucleotide encoding an antigenic determinant.

  Preferably, the inhibitor of Notch signaling inhibits Notch signaling in immune cells such as APC, B cells, or T cells.

  Suitably, the inhibitor of the Notch signaling pathway can be a Notch signaling repressor, or a factor that increases the expression or activity of the Notch signaling repressor.

  Preferably, the inhibitor of the Notch signaling pathway is a factor capable of inhibiting the activity of the Notch receptor or Notch ligand.

  Alternatively, or in addition, an inhibitor of the Notch signaling pathway can be a factor that can inhibit the activity of a downstream component of the Notch signaling pathway or down-regulate expression.

  Preferably, the inhibitor of the Notch signaling pathway interacts with a Notch receptor or Notch ligand to prevent endogenous Notch ligand-receptor interaction (also referred to as “Notch-Notch ligand interaction”). Preferably, it can be a factor that binds to a Notch receptor or Notch ligand. Such factors can be referred to as “Notch antagonists”. Preferably, the inhibitor inhibits Notch ligand-receptor interaction in immune cells such as lymphocytes and APCs, preferably in lymphocytes, preferably T cells.

  Suitably, the inhibitor of Notch signaling can be a protein or polypeptide, or a polynucleotide encoding such a protein or polypeptide.

  In one embodiment, for example, an inhibitor of Notch signaling can comprise or encode an extracellular domain of Delta, or a fragment, derivative, or homologue thereof.

  Suitably, for example, an inhibitor of Notch signaling comprises or encodes a Serrate or Jagged extracellular domain, or a fragment, derivative or homologue thereof.

  Suitably, for example, an inhibitor of Notch signaling comprises or encodes the extracellular domain of Notch, or a fragment, derivative or homologue thereof.

Suitably, for example, an inhibitor of Notch signaling is
i) a protein or polypeptide comprising a Notch ligand DSL domain, and optionally a Notch ligand N-terminal domain, or a heterologous amino acid sequence, but substantially free of a Notch ligand EGF-like domain;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least one Notch ligand EGF-like domain;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least two Notch ligand EGF-like domains;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) a protein or polypeptide comprising a Notch ligand DSL domain and 0, 1, or 2 Notch ligand EGF-like domains, but no more than two;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch ligand DSL domain with at least 30%, preferably at least 50% amino acid sequence similarity or identity to the DSL domain of human Delta1, Delta3, or Delta4, and EGF of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity to the like domain;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch ligand DSL domain with at least 30%, preferably at least 50% amino acid sequence similarity or identity to the DSL domain of human Delta1, Delta3, or Delta4, and EGF of human Delta1, Delta3, or Delta4 A protein comprising 0, 1, or 2 Notch ligand EGF-like domains with at least 30%, preferably at least 50% amino acid sequence similarity or identity to the like domain, but no more than two Polypeptide,
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch EGF-like domain with at least 30%, preferably at least 50% amino acid sequence similarity or identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising a NotchEGF-like domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity to EGF12 of
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch ligand DSL domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity to the human Jagged 1 or Jagged 2 DSL domain, and to the human Jagged 1 or Jagged 2 EGF-like domain, A protein or polypeptide comprising at least one Notch ligand EGF-like domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch ligand DSL domain having at least 30%, preferably at least 50% amino acid sequence similarity or identity to the human Jagged 1 or Jagged 2 DSL domain, and to the human Jagged 1 or Jagged 2 EGF-like domain, A protein or polypeptide comprising a Notch ligand EGF-like domain of 0, 1, or 2 but not more than 2 having at least 30%, preferably at least 50% amino acid sequence similarity or identity;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch ligand DSL domain with at least 70% amino acid sequence similarity or identity to the DSL domain of human Delta1, Delta3, or Delta4, and to the EGF-like domain of human Delta1, Delta3, or Delta4, A protein or polypeptide comprising at least one Notch ligand EGF-like domain having at least 70% amino acid sequence similarity or identity;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

Suitably, for example, an inhibitor of Notch signaling is
i) Notch ligand DSL domain with at least 70% amino acid sequence similarity or identity to the DSL domain of human Delta1, Delta3, or Delta4, and to the EGF-like domain of human Delta1, Delta3, or Delta4, A protein or polypeptide comprising a Notch ligand EGF-like domain of 0, 1, or 2 but not more than 2 with at least 70% amino acid sequence similarity or identity;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

  The advantage of using a protein or polypeptide having preferably not more than two Notch ligand EGF-like domains provides effective inhibition of Notch signaling with little or no competitive agonist activity and thus more selective inhibition. Is to provide. Such proteins and polypeptides can also occur more easily, for example in bacterial expression systems.

  However, it is understood that Notch signaling inhibition is also demonstrated by constructs having more than two such EGF-like repeats.

Suitably, for example, an inhibitor of Notch signaling is
i) an EGF domain having at least 70% amino acid sequence similarity or identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least to EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising an EGF domain having 70% amino acid sequence similarity or identity;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides.

  Suitably, the protein or polypeptide can be fused to a heterologous amino acid sequence such as an immunoglobulin Fc (IgFc) domain, eg, a human IgG1 or IgG4 Fc domain.

  Suitably the protein or polypeptide further comprises a Notch ligand N-terminal domain.

  Alternatively, for example, an inhibitor of Notch signaling can include an antibody, antibody fragment, or antibody derivative, or a polypeptide encoding an antibody, antibody fragment, or antibody derivative. Suitably, the antibody, antibody fragment, or antibody derivative binds to a Notch receptor or Notch ligand so as to prevent Notch ligand-receptor interaction.

Suitably, for example, the inhibitor of Notch signaling is less than about 1000 μM, preferably less than about 100 μM, preferably less than about 10 μM, preferably less than about 1000 nM, preferably less than about 100 nM, suitably from about 0.1 to about It can have an IC _{50 of} 100 nM (preferably measured using the Dynabeads assay of Example 12, preferably in the assay described herein).

  In one embodiment, the modulator of the Notch signaling pathway is a fusion protein comprising a Notch ligand extracellular domain and a domain of an immunoglobulin Fc segment (eg, IgG1Fc or IgG4Fc, preferably human IgG1Fc or human IgG4Fc), or such a fusion protein A polynucleotide encoding can be included. A suitable method for the preparation of such a fusion protein is described, for example, in Example 2 of WO 98/20142. IgG fusion proteins can be prepared as is well known in the art, for example, as described in US5428130 (Genentech).

  Suitably, the modulator of the Notch signaling pathway can be multimerized, preferably dimerized, by chemical cross-linking or disulfide bond formation between protein or polypeptide pairs. For example, if the protein or polypeptide contains heterologous amino acid sequences in the form of an immunoglobulin Fc domain, these can be collected into dimers joined by disulfide bonds formed between the Fc domains (eg, as shown in the accompanying figures). (See schematic diagram of dimer construct).

  When a protein or polypeptide is thus multimerized or dimerized, its multimerized / dimerized form will have more DSL and EGF domains than those described for the individual monomers. Can be contained. However, the ratio of DSL to EGF preferably does not change, for example, so that the ratio of DSL to EGF-like domains is 1: 0, 1: 1, or 1: 2 for the entire multimerized assembly.

Suitably, for example, an inhibitor of Notch signaling consists of a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or a multimer of such proteins or polypeptides, or a polynucleotide encoding such a Notch ligand protein or polypeptide.

Suitably, for example, an inhibitor of Notch signaling consists of a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
Or a multimer of such proteins or polypeptides, or a polynucleotide encoding such a Notch ligand protein or polypeptide.

Suitably, for example, an inhibitor of Notch signaling consists of a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) one Notch ligand EGF domain,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or a multimer of such proteins or polypeptides, or a polynucleotide encoding such a Notch ligand protein or polypeptide.

Suitably, for example, an inhibitor of Notch signaling consists of a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) two Notch ligand EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or a multimer of such proteins or polypeptides, or a polynucleotide encoding such a Notch ligand protein or polypeptide.

  According to another aspect of the present invention, in the manufacture of a medicament for use as an immunostimulant, a binding agent that binds to a Notch ligand so as to prevent binding of the ligand to the Notch receptor, or a poly that encodes such a binding agent. The use of nucleotides is provided.

  According to another aspect of the present invention, the use of an antibody or antibody derivative that binds to a Notch receptor or Notch ligand, or a polynucleotide encoding such an antibody or antibody derivative, in the manufacture of a medicament for use as an immunostimulatory agent. Provided.

  According to another aspect of the present invention, a method for increasing a subject's immune response to a vaccine antigen or antigenic determinant, wherein the subject has an effective amount of Notch signaling pathway simultaneously, separately or sequentially with said vaccine antigen. There is provided a method comprising administering an inhibitor of:

  According to another aspect of the invention, administering a binding agent that binds to a Notch receptor or Notch ligand so as to prevent ligand-receptor interaction, or administering a polynucleotide encoding such a binding agent. By doing so, a method of stimulating the immune system is provided. The binding agent can include, for example, one or more extracellular domains of Notch or its ligand.

  According to another aspect of the invention, the immune system is administered by administering an antibody or antibody derivative that binds to a Notch receptor or Notch ligand, or by administering a polynucleotide encoding such an antibody or antibody derivative. A method of stimulating is provided.

  According to another aspect of the invention, there is provided an adjuvant composition comprising an inhibitor of the Notch signaling pathway.

  According to another aspect of the present invention, there is provided a vaccine composition comprising the above-described adjuvant composition and an antigen. Suitably, the antigen can be a viral, fungal, parasite, or bacterial antigen.

According to another aspect of the invention, a method for modulating the immune system in a mammal comprising the steps of:
i) administering an effective amount of an inhibitor of the Notch signaling pathway, and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding the pathogen antigen or antigenic determinant, simultaneously, simultaneously, separately or sequentially. Is provided.

According to another aspect of the invention, for simultaneous, simultaneous, separate or sequential use in the regulation of the immune system,
Combinations of i) inhibitors of the Notch signaling pathway, and ii) pathogen antigens or antigenic determinants, or polynucleotides encoding pathogen antigens or antigenic determinants are provided.

  According to another aspect of the present invention, it is used to modulate the immune system simultaneously, simultaneously, individually or sequentially with a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant. Inhibitors of the Notch signaling pathway are provided.

According to another aspect of the invention, in the manufacture of a medicament that modulates the immune system,
There is provided the use of i) an inhibitor of the Notch signaling pathway, and ii) a pathogen antigen or antigenic determinant, or a combination of polynucleotides encoding the pathogen antigen or antigenic determinant.

  According to another aspect of the invention, the immune system is modulated in a simultaneous, contemporaneous, separate or sequential combination with a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant. Use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament is provided.

  According to another aspect of the present invention, there is provided a pharmaceutical kit comprising an inhibitor of Notch signaling pathway and a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant.

  According to another aspect of the invention, a conjugate comprising first and second sequences, wherein the first sequence encodes a pathogen antigen or antigenic determinant, or a pathogen antigen or antigenic determinant A conjugate comprising a polynucleotide, wherein the second sequence comprises a polypeptide or polynucleotide for modulation of Notch signaling is provided.

  According to another aspect of the invention, a conjugate comprising first and second sequences, wherein the first sequence encodes a pathogen antigen or antigenic determinant, or a pathogen antigen or antigenic determinant A conjugate comprising a polynucleotide, wherein the second sequence encodes an inhibitor of Notch signaling is provided.

  Preferably, the conjugate is in the form of a vector comprising a first polynucleotide sequence encoding a modulator of the Notch signaling pathway and a second polynucleotide sequence encoding a pathogen antigen or antigenic determinant.

  Preferably, the conjugate is in the form of an expression vector.

  Preferably, in such a conjugate, the first polynucleotide sequence encodes a Notch ligand, or a fragment, derivative, homologue, analogue or allelic variant thereof.

  Suitably, the first polynucleotide sequence of the conjugate encodes a Delta or Serrate / Jagged protein, or a fragment, derivative, homologue, analogue or allelic variant thereof.

  Suitably, the first polynucleotide sequence of the conjugate encodes a protein or polypeptide comprising a Notch ligand DSL domain and optionally at least one Notch ligand EGF-like domain.

  Suitably, the first polynucleotide sequence of the conjugate encodes a protein or polypeptide comprising a Notch ligand DSL domain and at least two Notch ligand EGF-like domains.

  Suitably, the first polynucleotide sequence of the conjugate encodes a protein or polypeptide comprising a Notch ligand DSL domain and one or two, but not more than two Notch ligand EGF-like domains.

  Suitably, the first and second polynucleotide sequences of the conjugate are each operably linked to one or more promoters.

  According to another aspect of the invention, a method is provided for increasing a TH2 immune response by administering a modulator of Notch signaling.

  According to another aspect of the invention, a method is provided for increasing a TH1 immune response by administering a modulator of Notch signaling.

  According to another aspect of the invention, a method is provided for increasing IFN-γ expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, there is provided a method of increasing IL-2 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, there is provided a method of increasing TNFα expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, there is provided a method of increasing IL-4 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, a method is provided for increasing IL-5 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, there is provided a method of increasing IL-13 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, a method is provided for reducing IL-10 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the invention, a method is provided for increasing IL-5 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the present invention, there is provided a method for producing an immunostimulatory cytokine profile having reduced IL-10 expression and increased IL-5 expression by administering an inhibitor of Notch signaling.

  According to another aspect of the present invention, a method of producing an immunostimulatory cytokine profile having increased IL-2, IFNγ, IL-5, IL-13, and TNFα expression by administering an inhibitor of Notch signaling Is provided. Suitably, this cytokine profile further indicates reduced IL-10 expression.

  In one embodiment of the invention, the inhibitor of Notch signaling is administered to a patient in vivo. Alternatively, an inhibitor of Notch signaling can be administered to cells ex vivo, and then the cells can be administered to the patient.

  Suitably, modulators of Notch signaling alter cytokine expression in leukocytes, fibroblasts or epithelial cells. Preferably, the modulator of Notch signaling alters cytokine expression in dendritic cells, lymphocytes or macrophages, or precursors or tissue specific derivatives thereof.

  Preferably, the inhibitor of Notch signaling or Notch signaling pathway used in the present invention is an inhibitor of Notch-Notch ligand interaction. Suitably, such inhibitors of Notch-Notch ligand interaction prevent endogenous Notch-Notch ligand interactions and at the same time lower Notch receptor activation than results from endogenous Notch-Notch ligand interactions. An agent that binds to a Notch receptor or a Notch ligand so as to effect or preferably not cause significant activation. For example, the inhibitor may be an EGF-like domain 11 and / or EGF-like domain 12 of a Notch receptor, or a DSL domain and / or an EGF-like domain 1 and / or an EGF-like domain 2 of a Notch ligand such as Delta, Serrate, or Jagged. Can be combined. Thus, for example, the inhibitor can comprise EGF-like domains 11 and 12 of the Notch receptor. Alternatively, the inhibitor can comprise a Notch ligand DSL domain and at least one EGF-like domain of a Notch ligand, such as Delta, Serrate, or Jagged. Suitably, for example, it may comprise the extracellular domain of a Notch receptor, such as the extracellular domain of Notch1, Notch2, Notch3, or Notch4. Alternatively, the inhibitor can comprise the extracellular domain of a Notch ligand, such as Delta (eg, mammalian Delta1, Delta3, or Delta4), Serrate, or Jagged (eg, mammalian Jagged1 or Jagged2).

If the inhibitor binds to a Notch receptor, the inhibitor can selectively bind to one Notch receptor, such as Notch1, or suitably by their similar structure, a series of Notch receptors, Or it can have some affinity for substantially all of them. Similarly, when an inhibitor binds to a Notch ligand, the inhibitor can selectively bind to one Notch ligand, such as Delta1, or suitably by their similar structure, a series of Notch ligands, Or it can have some affinity for substantially all of them, or the inhibitor can comprise an antibody that specifically binds to one or more Notch receptors. Preferably, the antibody binds to the antibody, but reduces or substantially prevents binding of the native Notch ligand, or at least reduces or substantially prevents Notch receptor activation. Join the body. Suitably, for example, such an antibody can bind to EGF11 and / or 12 of a Notch receptor (eg, Notch1, Notch2, Notch3, and / or Notch4). The antibodies can be selective for one Notch receptor, such as Notch1, or, suitably, by their similar structure, some affinity for a series of Notch receptors, or substantially all of them. Can have sex.

  Alternatively, the inhibitor can comprise an antibody that specifically binds to one or more Notch ligands. Preferably, the antibody binds to the antibody but reduces or substantially prevents binding of the ligand to the native Notch receptor, or at least reduces or substantially prevents Notch receptor activation. Bind to the Notch ligand. Suitably, for example, such antibodies can bind to the DSL domain and / or EGF-like domains 1 and / or 2 of a Notch ligand (eg, mammalian Delta1, Delta3, Delta4, Jagged1 or Jagged2). . The antibodies can be selective for one Notch ligand, such as Delta1, or, suitably, by their similar structure, provide some affinity for a series of Notch ligands, or substantially all of them. Can have.

  It will be appreciated that combinations of antibodies with complementary specificities may be used.

  In other embodiments, for example, the inhibitor of Notch signaling can be an inhibitor of NotchIC protease.

  As used herein, the term “NotchIC protease” refers to all of the intracellular (IC) domains from a Notch receptor, proteolytically acting to cleave the Notch receptor to activate the Notch signaling pathway. Or an enzyme or enzyme complex that results in a partial release. Enzymes thought to be involved in such cleavage include presenilin and gamma secretase enzymes, and presenilin dependent gamma secretase enzymes, or complexes.

  As used herein, the term “presenilin-dependent gamma secretase” means an enzyme having gamma secretase proteolytic activity that requires presenilin for activity or activation. Presenilin may be required, for example, as a coactivator or as part of an enzyme complex.

  Examples of presenilin proteins that can be modulated in the present invention include presenilin-1 (PS1) and presenilin-2 (PS2).

  Modulators of NotchIC protease activity are preferably selected from polypeptides and fragments thereof, linear peptides, cyclic peptides, and nucleic acids encoding them, synthetic and natural compounds, including low molecular weight organic or inorganic compounds, and antibodies. The modulator can be, for example, an agonist or antagonist of presenilin or presenilin-dependent gamma secretase, optionally in combination with factors that can up-regulate or down-regulate the Notch signaling pathway, respectively.

  An example of a presenilin antagonist that can be used in the present invention is the 26S proteasome, or the nucleic acid sequence that encodes it. Examples of the synthesis inhibitor include difluoroketone inhibitors described in Citron et al. And Wolfe et al.

Inhibitors described in Sinha and Liederburg (2-naphthoyl-VF-CHO, N- (2-naphthoyl) -Val-phenylalaninal, and N-benzyloxycarbonyl-Leu-phenylalaninal Z-LF-CHO), An inhibitor described in Esler et al., An inhibitor described in Figueiredo-Pereira et al. (N-benzyloxycarbonyl-Leu-Leucinal Z-LL-CHO), an inhibitor described in Higaki et al. (N-trans-3,5- Dimethoxycinnamoyl) -Ile-leucinal t-3,5-DMC-IL-CHO), inhibitors described by Murphy et al. (Boc-GVV-CHO N-tert-butyloxycarbonyl-Gly-Val-valinal), and Inhibitors described in Riston et al. (1- ( S) -endo-N- (1,3,3) -trimethylbicyclo [2.2.1] hept-2-yl) -4-fluorophenylsulfonamide).

In other embodiments, the inhibitor of Notch signaling is not an inhibitor of NotchIC protease (ie, preferably not a presenilin and gamma secretase enzyme inhibitor, preferably a presenilin dependent gamma secretase enzyme or complex inhibitor). Not)

According to another aspect of the invention, by administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or by administering a multimer of such proteins or polypeptides (each monomer may be the same or different)
Alternatively, a method is provided for altering an immune response by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.

According to another aspect of the invention, by administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or by administering a multimer of such proteins or polypeptides (each monomer may be the same or different)
Alternatively, a method is provided for increasing an immune response by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.

According to another aspect of the invention, by administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or by administering a multimer of such proteins or polypeptides (each monomer may be the same or different)
Alternatively, a method of reducing immune tolerance is provided by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.

According to another aspect of the invention, by administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or by administering a multimer of such proteins or polypeptides (each monomer may be the same or different)
Alternatively, a method of altering T cell activity is provided by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.

According to another aspect of the invention, by administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or by administering a multimer of such proteins or polypeptides (each monomer may be the same or different)
Alternatively, methods are provided for increasing helper (T _H ) or cytotoxic (T _C ) T cell activity by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.

According to another aspect of the invention, by administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or by administering a multimer of such proteins or polypeptides (each monomer may be the same or different)
Alternatively, methods are provided for reducing the activity of regulatory T cells by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.

  Suitably, the regulatory T cells are Tr1 or Th3 regulatory T cells.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components for use in the treatment of a disease:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the present invention, a Notch ligand protein or polypeptide, or polynucleotide for use as described in claim 22, wherein the Notch ligand protein or polypeptide consists essentially of the following components: Become
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
The polynucleotide is provided as a Notch ligand protein or polypeptide or a polynucleotide encoding such a Notch ligand protein or polypeptide.

According to another aspect of the present invention, a Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that modifies the immune response:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the present invention, a Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that modifies the immune response:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains, and iii) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, in the manufacture of a medicament for increasing an immune response, a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, in the manufacture of a medicament for reducing immune tolerance, a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the present invention, a Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that modifies T cell activity:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the present invention, a Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that increases helper (T _H ) or cytotoxic (T _C ) T cell activity:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the present invention, in the manufacture of a medicament for reducing the activity of regulatory T cells, a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) one EGF domain,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally all or part of the Notch ligand N-terminal domain,
iii) an immunoglobulin Fc domain, and iv) optionally one or more additional heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) one EGF domain iii) optionally all or part of the Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or multimers of such proteins or polypeptides (each monomer may be the same or different),
Alternatively, a polynucleotide encoding such a Notch ligand protein or polypeptide is provided.

According to another aspect of the invention, a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) two EGF domains, and iii) optionally one or more heterologous amino acid sequences,
Alternatively, a polynucleotide sequence encoding such a Notch ligand protein or polypeptide is provided.

  As used herein, the term “consisting essentially of” or “consisting essentially of” includes the identified sequence and domain, but is substantially free of other sequences or domains, particularly substantially Means a construct that does not contain any other Notch or Notch ligand sequence or domain.

  For the avoidance of doubt, the term “comprising” means that any additional features or components may be present.

  According to another aspect of the present invention, there is provided a vector comprising a polynucleotide encoding the aforementioned Notch ligand protein or polypeptide. The invention further provides host cells transformed or transfected with such vectors. According to another aspect of the present invention, there is provided a cell that displays the above Notch ligand protein or polypeptide on its surface and / or is transfected with a polynucleotide encoding such a protein or polypeptide. Is done.

  Suitably the protein or polypeptide is not bound to the cell. Alternatively, the protein or polypeptide can be cell-bound.

  In one embodiment, the protein or polypeptide can be fused to a heterologous amino acid sequence corresponding to all or part of an immunoglobulin Fc segment. In one embodiment, the heterologous amino acid sequence is not a TSST sequence, and preferably is not a superantigen sequence, particularly where the Notch ligand protein or polypeptide contains only two EGF repeat domains.

  Preferably, the protein or polypeptide comprises at least part of a mammalian, preferably human Notch ligand sequence.

  Suitably, the protein or polypeptide comprises a Delta, Serrate, or Jagged Notch ligand domain, or a domain having at least 30% amino acid sequence similarity or identity thereto.

  Suitably, the protein or polypeptide comprises a Delta1, Delta3, Delta4, Jagged1 or Jagged2 Notch ligand domain, or a domain having at least 30% amino acid sequence similarity thereto.

  Preferably the protein or polypeptide inhibits the Notch receptor. Suitably the protein or polypeptide is a Notch signaling antagonist.

  According to another aspect of the present invention, a polynucleotide encoding the protein or polypeptide described above is provided. In accordance with other aspects of the invention, there are provided vectors comprising such polynucleotides, and host cells transformed or transfected with such vectors.

  According to another aspect of the present invention, there is provided a cell that displays the above Notch ligand protein or polypeptide on its surface and / or is transfected with a polynucleotide encoding such a protein or polypeptide. Is done.

  In one embodiment, the modulator of the Notch signaling pathway comprises a fusion protein comprising a Notch ligand extracellular domain and a domain of an immunoglobulin Fc segment (eg, IgG1 Fc or IgG4 Fc), or a polynucleotide encoding such a fusion protein. Can be included. A suitable method for the preparation of such a fusion protein is described, for example, in Example 2 of WO 98/20142. IgG fusion proteins can be prepared as is well known in the art, for example, as described in US5428130 (Genentech).

  According to another aspect of the invention, there is provided a method of increasing TNFα expression by administering a protein, polypeptide or polynucleotide as described above.

  According to another aspect of the invention, a method is provided for reducing IL-10 expression by administering a protein, polypeptide or polynucleotide as described above.

  According to another aspect of the invention, a method is provided for increasing IL-5 expression by administering a protein, polypeptide or polynucleotide as described above.

  According to another aspect of the invention, there is provided a method of increasing IL-13 expression by administering a protein, polypeptide or polynucleotide as described above.

  Suitably, the protein, polypeptide, or polynucleotide alters cytokine expression in leukocytes (such as lymphocytes or macrophages), fibroblasts, or epithelial cells, or precursors or tissue specific derivatives thereof.

  According to another aspect of the present invention, there is provided a method for producing an immunostimulatory cytokine profile having reduced IL-10 expression and increased TNFα expression by administering a protein, polypeptide or polynucleotide as described above. Is done.

  According to another aspect of the present invention, a method of producing an immunostimulatory cytokine profile having reduced IL-10 expression and increased IL-5 expression by administering the protein, polypeptide or polynucleotide described above. Is provided.

  According to another aspect of the invention, a method of producing an immunostimulatory cytokine profile having reduced IL-10 expression and increased IL-13 expression by administering a protein, polypeptide or polynucleotide as described above. Is provided.

  According to another aspect of the invention, there is provided a method for producing an immunostimulatory cytokine profile having increased IL-5, IL-13, and TNFα expression by administering a protein, polypeptide or polynucleotide as described above. Provided.

  According to another aspect of the present invention, immunostimulatory activity having increased IL-2, IFNγ, IL-5, IL-13, and TNFα expression by administering a protein, polypeptide or polynucleotide as described above. Methods are provided for generating a cytokine profile. Suitably, this cytokine profile further indicates reduced IL-10 expression.

  According to another aspect of the invention, there is provided a method for increasing a TH2 immune response by administering a protein, polypeptide or polynucleotide as described above.

  According to another aspect of the invention, there is provided a method for increasing a TH1 immune response by administering a protein, polypeptide or polynucleotide as described above.

  Various preferred features and embodiments of the present invention will now be described in more detail by way of non-limiting example and with reference to the accompanying drawings.

  The practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, unless otherwise indicated, and are within the capabilities of those skilled in the art. . Such techniques are explained in the literature. For example, J. et al. Sambrook, E .; F. Fritsch, and T.W. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, Ausubel, F.M. M.M. Et al. (1995 and Periodic Addendum, Current Protocols in Molecular Biology, Chapters 9, 13, and 16; John Wiley & Sons, New York, NY), B.C. Roe, J. et al. Crabtree, and A.M. Kahn, 1996, DNA Isolation and Sequencing: Essential Technologies, John Wiley & Sons, J. Am. M.M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice, Oxford University Press, M.M. J. et al. Gait (ed.), 1984, Oligonucleotide Synthesis: A Practical Appropriate Ill Press, D.C. M.M. J. et al. Lilley and J.M. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic, Academic. E. Coligan, A.M. M.M. Kruisbeek, D.W. H. Margulies, E.M. M.M. Shevach, and W.W. See Strober (1992 and Periodic Addendum, Current Protocols in Immunology, John Wiley & Sons, New York, NY). The general text of each is hereby incorporated by reference.

  For the avoidance of doubt, Drosophila and vertebrate names are used interchangeably and all homologs are within the scope of the present invention.

Notch signaling As used herein, the expression “Notch signaling” is synonymous with the expression “Notch signaling pathway”, and any not resulting from (or including) activation of Notch receptors. Refers to one or more upstream or downstream events.

  Preferably, “Notch signaling” allows us to activate or inhibit Notch / Notch ligand interaction, up- or down-regulation of Notch or Notch ligand expression or activity, and for example proteolytic cleavage of Notch, And any event directly upstream or downstream of Notch receptor activation or inhibition, including activation or inhibition of Notch signaling transduction, including up-regulation or down-regulation of the Ras-Jnk signaling pathway.

  Thus, by “Notch signaling”, Applicants refer to the Notch signaling pathway as a signal transduction pathway that includes factors that interact genetically and / or molecularly with Notch receptor proteins. For example, factors that interact with Notch proteins based on both molecules and genes are, by way of example only, Delta, Serrate, and Deltax. Factors that interact genetically with Notch proteins are, by way of example only, Mastermind, Hairless, Su (H), and presenilin.

  In one aspect, Notch signaling includes signaling events that occur extracellularly or at the cell membrane. In other embodiments, Notch signaling includes signaling events that occur within a cell, eg, within the cytoplasm, or inside the cell nucleus.

Modulators of Notch signaling As used herein, the term “modulate” refers to changes and alterations in the biological activity of the Notch signaling pathway, or its target signaling pathway. The term “modulator” preferably refers to an antagonist or inhibitor of Notch signaling, ie, a compound that blocks, at least in part, the normal biological activity of the Notch signaling pathway. Conveniently, such compounds may be referred to herein as inhibitors or antagonists. Preferably, the modulator is an antagonist of Notch signaling, preferably an antagonist of Notch receptor (eg, an antagonist of Notch1, Notch2, Notch3, and / or Notch4 receptor).

  An antagonist of the Notch receptor is preferably an agent that binds to the extracellular domain of Notch and reduces or inhibits the activation of signaling. Preferably, the antagonist of Notch receptor binds Notch in immune cells such as APC, B cells, or T cells.

  Alternatively, inhibitors of Notch signaling can bind to Notch ligands and reduce their ability to bind to and / or activate Notch receptors. Preferably, such inhibitors bind to Notch ligand in immune cells such as APC, B cells, or T cells.

  The active agent of the present invention can be an organic compound or other chemical. In one embodiment, the modulator is an organic compound that includes two or more hydrocarbyl groups. As used herein, the term “hydrocarbyl group” means a group comprising at least C and H and optionally including one or more other suitable substituents. Examples of such substituents include halo, alkoxy, nitro, alkyl groups, cyclic groups and the like. In addition to the possibility that the substituents are cyclic groups, combinations of substituents can also form cyclic groups. If the hydrocarbyl group contains more than one C, the carbons need not necessarily be bonded to each other. For example, at least two of the carbons can be bonded via a suitable element or group. Thus, the hydrocarbyl group can contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur, nitrogen, and oxygen. Candidate modulators can include at least one cyclic group. The cyclic group can be a polycyclic group such as a non-fused polycyclic group. For some applications, this factor comprises at least one said cyclic group bonded to another hydrocarbyl group.

  In a preferred embodiment, the modulator is an amino acid sequence, or a chemical derivative thereof, or a combination thereof. In other preferred embodiments, the modulator is a nucleotide sequence, which can be a sense sequence or an antisense sequence. The modulator can also be an antibody.

  The modulator can be a synthetic compound or a naturally isolated compound.

  A very important component of the Notch signaling pathway is the Notch receptor / Notch ligand interaction. Thus, Notch signaling can include a change in expression, property, amount, or activity of a Notch ligand or receptor, or resulting cleavage product. In addition, Notch signaling may be a Notch signaling pathway membrane protein or G protein, or Notch signaling pathway enzyme, such as a protease, kinase (eg, serine / threonine kinase), phosphatase, ligase (eg, ubiquitin ligase), or glycosyltransferase, A change in amount or activity may be included. Alternatively, signaling can include changes in expression, properties, amounts, or activities of DNA binding factors such as transcription factors.

  In a preferred form of the invention, Notch signaling is a specific signaling and the detected signal is substantially or at least primarily Notch signaling pathway, preferably Notch, not other significant interference or competing factors such as, for example, cytokine signaling. / Notch means due to ligand interaction. Thus, in a preferred embodiment, the term “Notch signaling” herein excludes cytokine signaling. Therefore, preferably the modulator or inhibitor of Notch signaling is not a cytokine, preferably not a mitogen.

  Preferably, the modulator of Notch signaling is not a factor that acts primarily by inhibiting or downregulating the expression of Notch signaling, such as Delta and / or Serrate. Thus, it is understood that such inhibition or down-regulation can occur as a result of the main mode of action of the Notch signaling modulator, but is preferably not the main mode of action of the modulator. Preferably, the main mode of action of the Notch signaling modulator is to modulate (preferably inhibit) the interaction between Notch and Notch ligand already expressed in immune cells.

  Preferably, therefore, the modulator of Notch signaling is not a Toll protein or BMP, preferably Noggin, Chordin, Follistatin, Xnr3, FGF, Or it is not a factor that reduces or prevents the production of Fringe.

  The Notch signaling path is described in more detail below.

  An important target for Notch-dependent transcriptional activation is the gene for the enhancer of split complex (E [spl]). Furthermore, these genes are direct targets for binding by Su (H) proteins and have been shown to be transcriptionally activated in response to Notch signaling. By analogy with EBNA2, a viral coactivator that interacts with the mammalian Su (H) homolog CBF1 and possibly coordinates it with other proteins to convert it from a transcriptional repressor to a transcriptional activator, Notch intracellular domain Proteins can bind to Su (H) and contribute to the activation domain, thereby allowing Su (H) to activate transcription of E (spl) as well as other target genes. Su (H) is not required for all Notch-dependent determinations, in part because Notch works with other DNA-binding transcription factors or using other mechanisms that transduce extracellular signals. It should also be noted that this suggests mediating cell fate selection.

  In one embodiment, the active agent can be a Notch ligand or a polynucleotide encoding a Notch ligand. Notch ligands useful in the present invention include endogenous Notch ligands that can bind to Notch receptor polypeptides that are typically present in the membranes of various mammalian cells, eg, hematopoietic stem cells.

  As used herein, the term “Notch ligand” means an agent that can interact with a Notch receptor to produce a biological effect. The term includes natural protein ligands such as Delta and Serrate, and artificial / modified constructs with equivalent activity.

  Specific examples of mammalian Notch ligands identified to date include Delta families such as Delta or Delta-like 1 (Genbank accession number AF003522, Homo sapiens), Delta-3 (Genbank accession number AF084576, Rattus norvegicus), And Delta-like 3 (Mus musculus) (Genbank accession number NM — 016941) and US6121045 (Millennium), Delta-4 (Genbank accession numbers AB043894 and AF253468, Homo sapiens) and Serrate family such as Serrate / 01571, WO 96/27610, and W O92 / 19734), Jagged-1 (Genbank accession number U73936, Homo sapiens) and Jagged-2 (Genbank accession number AF029778, Homo sapiens), and LAG-2. Homology between family members is extensive.

  Additional homologues of known mammalian Notch ligands can be identified using standard techniques. By “homologue” is meant a gene product that exhibits sequence homology, amino acid or nucleic acid sequence homology to, for example, any one of the known Notch ligands described above. Typically, a homologue of a known Notch ligand will have at least 10, preferably at least 20, preferably at least 50, suitably at least 100 of that Notch ligand relative to the corresponding known Notch ligand. It is at least 20%, preferably at least 30% identical at the amino acid level over the amino acid sequence or over the entire length. Techniques and software for calculating sequence homology between two or more amino acid or nucleic acid sequences are well known in the art (eg, http://www.ncbi.nlm.nih.gov, Ausubel et al. , Current Protocols in Molecular Biology (1995) John Wiley & Sons, Inc.).

Notch ligands identified to date, diagnostic DSL domain contains 22 amino acids from the 20 to the amino terminus of the protein (D .Delta, S .Serrate, L .Lag2), and up to 14 on the extracellular surface Or more EGF-like repeats. Thus, homologues of Notch ligands preferably also contain a DSL domain at the N-terminus and up to 14 or more EGF-like repeats on the extracellular surface.

  In addition, a suitable homologue can bind to the Notch receptor. Binding can be assessed by a variety of known techniques including in vitro binding assays.

  Homologs of Notch ligands can be identified in several ways, for example, under moderate to high stringency conditions (eg, 0.03 M sodium chloride and 0.03 M quenching at about 50 ° C. to about 60 ° C. (Sodium acid) by probing a genomic or cDNA library with a probe containing all or part of a nucleic acid encoding a Notch ligand. Alternatively, homologues can be obtained using degenerate PCR using primers that are designed to target variants that generally encode conserved amino acid sequences and sequences within the homologue. This primer is used under lower stringency conditions used for cloning sequences that contain one or more degenerate positions and have a single sequence primer for a known sequence.

  Inhibition of Notch signaling can also be achieved by mimicking or enhancing the activity or expression of an inhibitor of the Notch signaling pathway. Thus, a polypeptide for inhibiting Notch signaling includes molecules that can mimic or enhance the activity or expression of any Notch signaling inhibitor. Preferably, the molecule comprises a polypeptide that increases the production or activity of a compound capable of causing a reduction in the expression or activity of Notch, a Notch ligand, or any downstream component of the Notch signaling pathway, or such a polypeptide. The encoding polynucleotide. Such molecules include the Toll-like receptor protein family and derivatives, fragments, variants, and homologues thereof such as growth factors such as bone morphogenetic protein (BMP), BMP receptor, and activin.

  By a protein for inhibiting Notch signaling, or a polynucleotide encoding such a protein, Applicants inhibit Notch, Notch signaling pathway, or any one or more components of Notch signaling pathway Means a molecule that can.

  In one embodiment, the molecule can reduce or prevent Notch or Notch ligand expression. Such molecules can be nucleic acid sequences that can reduce or prevent Notch or Notch ligand expression.

  Suitably, the nucleic acid sequence encodes a polypeptide selected from a Toll-like receptor protein family or growth factors such as bone morphogenetic protein (BMP), BMP receptor, and activin. Preferably, this factor produces compounds capable of causing an increase in the expression of Notch ligands such as Noggin, chodin, follistatin, Xnr3, fibroblast growth factor, and derivatives, fragments, variants, and homologues thereof. Or a polynucleotide encoding such a polypeptide.

  Alternatively, the nucleic acid sequence comprises Notch ligand and a polypeptide capable of upregulating Notch ligand expression, such as Noggin, chodin, follistatin, Xnr3, fibroblast growth factor, and derivatives, fragments, variants thereof, And an antisense construct derived from a sense nucleotide sequence encoding a polypeptide selected from homologues.

  Preferably, however, the inhibitor of Notch signaling is a molecule capable of inhibiting Notch-Notch ligand interaction. A molecule can be considered to modulate a Notch-Notch ligand interaction if it can inhibit the interaction between Notch and its natural ligand, preferably to a sufficient extent to provide a therapeutic effect.

  Factors that modulate Notch-Notch ligand interaction can be, for example, antibodies, antibody fragments or derivatives, peptides, small organic molecules, peptidomimetics, and the like. An antibody is a preferred factor. Such antibodies can be polyclonal or monoclonal, complete or truncated and can be, for example, heterologous, homologous, or syngeneic.

  For example, an antibody capable of binding to a Notch receptor or Notch ligand can be used to inhibit normal Notch-Notch ligand interactions according to the present invention.

  As used herein, the expression “Notch-Notch ligand interaction” (which can be used interchangeably with the term “Notch ligand-receptor interaction”) refers to a Notch family member and one or more such Means an interaction between a member of the ligand and a ligand capable of binding.

  An agent can be considered to inhibit Notch-Notch ligand interaction if it can inhibit the interaction between Notch and its ligand, preferably to a sufficient extent to provide a therapeutic effect.

  While oligopeptides and peptides can be preferred factors, other sources such as combinatorial libraries provide compounds other than oligopeptides that have the necessary binding properties.

  Non-peptidic factors include a number of chemical species, but typically they are organic molecules, preferably small organic molecules having a molecular weight of about 50 to about 2500 daltons. Suitable factors include functional groups necessary for structural interaction with the protein, in particular hydrogen bonding, often at least one group selected from, for example, amino, carbonyl, carboxyl, hydroxyl, or sulfhydryl groups, preferably at least Contains two such functional chemical groups. The compounds can be, for example, cyclic or heterocyclic structures and / or aromatic or polycyclic aromatic structures substituted with one or more such functional groups.

  Suitably, the factor causes at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% binding of human Notch to human Delta and / or Serrate. , 99% or 100% blocked.

  Preferably, the receptor is activated when the inhibitor is a receptor or a nucleic acid sequence encoding the receptor. Thus, for example, when the factor is a nucleic acid sequence, the receptor is preferably constitutively active upon expression.

  Inhibitors of Notch signaling further include downstream inhibitors of the Notch signaling pathway, compounds that prevent expression of the Notch target gene or induce gene expression that is suppressed by the Notch signaling pathway. Examples of such proteins include dominant-negative forms of Dsh or Num, and NotchIC or Deltax. Proteins for inhibiting Notch signaling further include variants of the wild-type component of the Notch signaling pathway that have been modified so that their presence blocks instead of transducing the signaling pathway. An example of such a compound is a Notch receptor that has been modified such that proteolytic cleavage of its intracellular domain is no longer possible.

  Notch signaling can also be inhibited by inhibiting Notch signaling conversion.

Notch signaling conversion The Notch signaling pathway directs binary cell fate decisions in the embryo. Notch was first described in Drosophila as a transmembrane protein that functions as a receptor for two different ligands, Delta and Serrate. Vertebrates express multiple Notch receptors and ligands (discussed below). To date, at least four Notch receptors (Notch-1, Notch-2, Notch-3, and Notch-4) have been identified in human cells (eg, GenBank accession numbers AF308602, AF308601, and U95299, Homo). see sapiens).

  The Notch protein is synthesized as a single polypeptide precursor that is cleaved by a furin-like convertase to yield two polypeptide chains that are further processed to form the mature receptor. The Notch receptor present in the plasma membrane contains a heterodimer of two Notch proteolytic cleavage products, one containing a C-terminal fragment consisting of a portion of the extracellular domain, a transmembrane domain, and an intracellular domain. The other contains the majority of the extracellular domain. The proteolytic cleavage step of Notch that activates the receptor occurs in the Golgi apparatus and is mediated by a furin-like convertase.

The Notch receptor is an extracellular domain containing up to 36 epidermal growth factor (EGF) -like repeats [Notch1 / 2 = 36, Notch3 = 34, Notch4 = 29], three cysteine-rich repeats (Lin-Notch (L / N) repeats), and inserted into the membrane as heterodimeric molecules consisting of transmembrane subunits containing cytoplasmic domains. The Notch cytoplasmic domain contains six ankyrin-like repeats, a polyglutamine stretch (OPA), and a PEST sequence. An additional domain, called RAM23, is located in close proximity to the ankyrin repeat and is involved in binding to the transcription factor known as Drosophila hairless suppressor [Su (H)], and vertebrate CBF1 (Tamura K etc. (1995) Curr.Biol 5:. 1416~1423 (Tamura)). Notch ligand addition are characteristic of all Notch ligands, with cysteine-rich DSL (D elta- S errate L ag2 ) domain, shows a plurality of EGF-like repeats in the extracellular domain (Artavanis-Tsakomas etc. (1995) Science268: 225-232, Artavanis-Tsakomas et al. (1999) Science 284: 770-776).

  The Notch receptor is activated by the binding of extracellular ligands such as Delta, Serrate, and Scabrous to EGF-like repeats in the Notch extracellular domain. Delta requires cleavage for activation. It is cleaved at the cell surface by the ADAM disintegrin metalloprotease Kuzbanian, and this cleavage event releases a soluble and active form of Delta. An oncogenic variant of the human Notch-1 protein with a truncated extracellular domain, also known as TAN-1, has been found to be constitutively active and involved in T cell lymphoblastic leukemia Yes.

  The cdc10 / ankyrin intracellular domain repeat mediates physical interactions with intracellular signal transduction proteins. Most notably, the cdc10 / ankyrin repeat interacts with the hairless suppressor [Su (H)]. Su (H) is a Drosophila homologue of the C promoter binding factor 1 [CBF-1], a mammalian DNA binding protein involved in Epstein-Barr virus-induced B cell immortalization. At least in cultured cells, Su (H) binds to cytoplasmic cdc10 / ankyrin repeats and has been demonstrated to translocate to the nucleus by Notch receptor interaction with the ligand Delta of neighboring cells. Su (H) contains response elements found in the promoters of several genes and is known to be an important downstream protein of the Notch signaling pathway. The involvement of Su (H) in transcription is thought to be regulated by hairless.

The intracellular domain of Notch (NotchIC) also has direct nuclear function (Lieber et al. (1993) Genes Dev7 (10): 1949-65 (Lieber)). Recent studies have indeed shown that Notch activation requires the six cdc10 / ankyrin repeats of the Notch intracellular domain to reach the nucleus and participate in transcriptional activation. The site of proteolytic cleavage in Notch's intracellular tail has been identified to be between gly 1743 and val 1744 (referred to as site 3 or S3) (Schroeter EH et al. (1998) Nature 393 ). (6683) : 382-6 (Schroeter)). The proteolytic cleavage step that releases cdc10 / ankyrin repeats for nuclear translocation is believed to depend on presenilin activity.

The intracellular domain accumulates in the nucleus where it is shown to form a transcriptional activator complex with the CSL family protein CBF1 (hairless suppressor, Drosophila Su (H), C. elegans Lag-2). (Schroeter, Struhl G et al. (1998) Cell 93 (4) : 649-60 (Struhl)). This NotchIC-CBF1 complex then activates target genes such as bHLH protein HES (split-like hairy enhancer) 1 and 5 (Weinmaster G. (2000) Curr. Opin. Genet. Dev. 10 : 363-369. (Weinmaster)). This nuclear function of Notch has also been shown for mammalian Notch homologues (Lu FM et al. (1996) Proc Natl Acad Sci 93 (11) : 5663-7 (Lu)).

S3 processing occurs only in response to binding of Notch ligands Delta or Serrate / Jagged. Golgi's early translational modification of the Notch receptor (Munro S, Freeman M. (2000) Curr. Biol. 10 : 813-820 (Munro), Ju BJ et al. (2000) Nature 405 : 191-195 (Ju)) At least to some extent, it is thought to control the expression of the two ligands on the cell surface. The Notch receptor is modified in the extracellular domain by the glycosyltransferase enzyme Ringe that binds to the Lin / Notch motif. Fringe modifies Notch by adding O-linked fucose groups to EGF-like repeats (Moloney DJ et al. (2000) Nature 406 : 369-375 (Moloney), Brucker K et al. (2000) Nature 406 : 411-415). Brucker)). This modification by Fringe does not interfere with ligand binding but may affect ligand-induced conformational changes in Notch. More recent studies suggest that Fringe's action prevents functional interaction with Notch's Serrate / Jagged ligand, but allows selective binding to Delta (Panin VM et al. (1997). Nature 387 : 908-912 (Panin), Hicks C et al. (2000) Nat. Cell. Biol. 2 : 515-520 (Hicks)). Drosophila has a single Fringe gene, but vertebrates are known to express multiple genes (Radical, Manic, and Lunatic Rings) (Irvine KD (1999) Curr. Opin. Genet. Devel. 9 ). : 434-441 (Irvine)).

  Signal transduction from the Notch receptor can occur by two different pathways (Figure 1). A clearer pathway is Notch intracellular, which translocates to the nucleus and forms a transcriptional activator complex with the CSL family protein CBF1 (Haires suppressor, Drosophila Su (H), C. elegans Lag-2). Includes proteolytic cleavage of the domain (NotchIC). The NotchIC-CBF1 complex then activates target genes such as bHLH protein HES (split-like hairy enhancer) 1 and 5. Notch can also transmit signals in a CBF-1-independent manner, including the cytoplasmic zinc finger-containing protein Deltax. Unlike CBF1, Deltax does not translocate to the nucleus after Notch activation, but instead interacts with Grb2 and regulates the Ras-Jnk signaling pathway.

  Target genes for the Notch signaling pathway include Deltex, Hes family genes (especially Hes-1), split enhancer [E (spl)] complex genes, IL-10, CD-23, CD-4, and Dll- 1 is included.

Deltex, an intracellular docking protein, replaces Su (H), leaving a site of interaction with Notch's intracellular tail. Deltex is a cytoplasmic protein containing a zinc finger (Artavanis-Tsakomas etc. (1995) Science268: 225~232, etc. Artavanis-Tsakomas (1999) Science284: 770~776, Osborne B, Miele L. (1999) Immunity 11 : 653-663 (Osborne)). Deltax interacts with an Ankyrin repeat of the Notch intracellular domain. Studies have shown that Deltax promotes Notch pathway activation by interacting with Grb2 and modulating the Ras-JNK signaling pathway (Matsuno et al. (1995) Development 121 (8): 2633-44. Matsuno K et al. (1998) Nat. Genet. 19 : 74-78). Deltax also acts as a docking protein that prevents Su (H) from binding to Notch's intracellular tail (Matsuno). Thus, Su (H) is released into the nucleus where it acts as a transcriptional modulator. Recent evidence further suggests that Deltatex, but not the Su (H) homolog CBF1, is responsible for E47 function inhibition in vertebrate B cell lines (Ordantrich et al. (1998) Mol. Cell. Biol. 18 : 2230-2239 (Ordrich)). Deltax expression is upregulated as a result of Notch activation in the positive feedback loop. The sequence of the Homo sapiens Delta (DTX1) mRNA can be found in GenBank accession number AF053700.

Hes-1 (hairy enhancer of split-1) (Takebayashi K. et al. (1994) J Biol Chem 269 (7) : 150-6 (Takebayashi)) is a transcription factor having a basic helix-loop-helix structure. It is. Hes-1 binds to an important functional site of the CD4 silencer, resulting in suppression of CD4 gene expression. Therefore, Hes-1 is strongly involved in T cell fate decisions. Other genes in the Hes family include Hes-5 (enhancement homolog of mammalian splits) whose expression is also upregulated by Notch activation, and Hes-3. Hes-1 expression is upregulated as a result of Notch activation. The sequence of Mus musculus Hes-1 can be found in GenBank accession number D16464.

The E (spl) gene complex [E (spl) -C] (Leimester C. et al. (1999) Mech Dev 85 (1-2) : 173-7 (Leimeister)) contains seven genes, and E (spl) And only Groucho show a visible phenotype upon mutation. E (spl) is named for its ability to enhance Split mutations, and Split is an alias for Notch. Indeed, the E (spl) -C gene represses Delta by regulating the expression of the achaete-scute complex gene. E (spl) expression is upregulated as a result of Notch activation.

  Interleukin-10 (IL-10) was first characterized in mice as a factor produced by Th2 cells that can suppress cytokine production by Th1 cells. IL-10 has since been shown to be produced by many other cell types including macrophages, keratinocytes, B cells, Th0 and Th1 cells. It exhibits extensive homology with the Epstein-Barr bcrf1 gene now called viral IL-10. A small number of immunostimulatory effects have been reported, but are considered primarily immunosuppressive cytokines. Inhibition of T cell responses by IL-10 is mediated primarily by a reduction in the accessory function of antigen presenting cells. IL-10 is specifically reported to suppress the production of numerous inflammatory cytokines by macrophages and to inhibit costimulatory molecules and MHC class II expression. IL-10 also has anti-inflammatory effects on other myeloid cells such as neutrophils and eosinophils. In B cells, IL-10 affects isotype switching and proliferation. More recently, it has been reported that IL-10 plays a role in the induction of regulatory T cells and is a possible mediator of their inhibitory action. Although it is not clear whether IL-10 is a direct downstream target of the Notch signaling pathway, its expression has been observed to be strongly upregulated upon Notch activation. The mRNA sequence of IL-10 can be found in GenBank reference number GI1041812.

  CD-23 is human leukocyte differentiation antigen CD23 (FCE2), which is a key molecule for B cell activation and proliferation. This is a low affinity receptor for IgE. In addition, truncated molecules can be secreted and function as effective mitogen growth factors. The sequence of CD-23 can be found in GenBank reference number GI1783344.

  CTLA4 (cytotoxic T lymphocyte activating protein 4) is an auxiliary molecule found on the surface of T cells and is thought to play a role in the regulation of airway inflammatory cell recruitment and T helper cell differentiation after allergen inhalation. Yes. The promoter region of the gene encoding CTLA4 has a CBF1 responsive element whose expression is upregulated as a result of Notch activation. The CTLA4 sequence can be found in GenBank accession number L1506.

  Dlx-1 (distalless-1) (McGuinness T. et al. (1996) Genomics 35 (3): 473-85 (McGuinness)) expression is downregulated as a result of Notch activation. The sequence of Dlx can be found in GenBank accession number U51000-3.

  CD-4 expression is downregulated as a result of Notch activation. The sequence of the CD-4 antigen can be found in GenBank accession number XM006966.

  Other genes involved in the Notch signaling pathway, such as Numb, Mastermind, and Dsh, and all genes whose expression is regulated by Notch activation are within the scope of the present invention.

As noted above, the Notch receptor family is involved in cell-cell signaling events that affect T cell fate decisions. In this signaling, NotchIC is localized in the nucleus and functions as an activating receptor. Mammalian NotchIC interacts with the transcriptional repressor CBF1. It has been suggested that NotchICcdc10 / ankyrin repeat is essential for this interaction. Hsieh et al. (Hsieh et al. (1996) Molecular & Cell Biology 16 (3) : 952-959) further suggests that the N-terminal 114 amino acid region of mouse NotchIC contains a CBF1 interaction domain. Furthermore, NotchIC has been proposed to target DNA-bound CBF1 in the nucleus and act by stopping CBF1-mediated repression by masking the repression domain. The Epstein-Barr virus (EBV) immortalizing protein EBNA ″ also utilizes CBF1 tethering and repression masking to upregulate the expression of CBF1-suppressed B cell genes. Therefore, mimicking Notch signal transduction is involved in EBV driven immortalization. Strobl et al. (Strobl et al. (2000) J Virol 74 (4) : 1727-35) similarly reports that “EBNA2 can therefore be regarded as a functional equivalent of an activated Notch receptor”. Other EBV proteins that fall into this category include BARF0 (Kusano and Raab-Traab (2001) J Virol 75 (1) : 384-395 (Kusano and Raab-Trub)) and LMP2A.

  Any one or more suitable targets, such as amino acid sequences and / or nucleotide sequences, can be used to identify compounds and / or target molecules that can modulate the Notch signaling pathway in any of a variety of drug screening techniques. Can be used to identify. The target used in such a test can be free in solution, attached to a solid support, supported on the cell surface, or located intracellularly.

  Drug screening techniques can be based on the method described in Geysen, European Patent No. 0138855, published September 13, 1984. In summary, a number of different small peptide candidate modulators or target molecules are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compound is reacted with an appropriate target or fragment thereof and washed. The bound entity is then detected, for example, by an appropriately adapted method well known in the art. The purified target can also be coated directly onto the plate for use in drug screening techniques. The plate used for high throughput screening (HTS) is preferably a multi-well plate having 96384, or more than 384 wells / plates. Cells can also be spread like a “lawn”. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. The high throughput screening described above for synthetic compounds can also be used to identify organic candidate modulators and target molecules.

  The present invention further contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the target compete with the test compound for binding to the target.

  Techniques used to screen and develop factors such as antibodies, peptidomimetics, and small organic molecules that can bind to components of the Notch signaling pathway are well known in the art. These techniques include the use of a phage display system to express signaling proteins, and E. coli transfected to produce proteins for binding studies of potential binding compounds. using cultures of E. coli or other microorganisms (eg, G. Cesarini, FEBS Letters, 307 (1): 66-70 (July 1992), H. Gram et al., J. Immunol. Meth. 161. 169-176 (1993) and C. Summer et al., Proc. Natl. Acad. Sci. USA, 89: 3756-3760 (May 1992)). Further library and screening techniques are described for example in US Pat. No. 6,281,344 (Phylos).

Polypeptide, Protein, and Amino Acid Sequence As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “protein”.

  In general, “peptide” refers to a short amino acid sequence that is 10 to 40 amino acids long, preferably 10 to 35 amino acids.

  Amino acid sequences can be prepared and isolated from appropriate sources, can be produced synthetically, or can be prepared using recombinant DNA techniques.

Nucleotide Sequence As used herein, the term “nucleotide sequence” is synonymous with the term “polynucleotide”.

  The nucleotide sequence is genomic or synthetic or recombinantly derived DNA or RNA. These can also be cloned by standard techniques. The nucleotide sequence can be double-stranded or single-stranded, whether in the sense or antisense strand, or a combination thereof.

Long nucleotide sequences are generally produced using recombinant means, such as PCR (polymerase chain reaction) cloning techniques. This involves creating a pair of primers (eg, about 15 to 30 nucleotides) adjacent to the region of the targeting sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, desired Performing a polymerase chain reaction (PCR) under conditions that result in amplification of the region, isolating the amplified fragment (eg, by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. This primer can be designed to contain an appropriate restriction enzyme recognition site so that the amplified DNA can be cloned into an appropriate cloning vector. In general, primers are produced by synthetic means and include producing a desired nucleic acid sequence step by step, one nucleotide at a time. Techniques that accomplish this using automated techniques are readily available in the art.
“Polynucleotide” refers to a polymeric form of nucleotides of at least 10 bases to 10,000 bases in length, or more, which are ribonucleotides or deoxyribonucleotides, or a modified form of either nucleotide type. The term also includes single and double stranded forms of DNA, as well as derivative forms such as protein nucleic acids (PNA).

  These can be constructed using standard recombinant DNA methods. The nucleic acid can be RNA or DNA, preferably DNA. If the nucleic acid is RNA, the manipulation can be performed via a cDNA intermediate. In general, a nucleic acid sequence encoding the first region is prepared and provided with appropriate restriction sites at the 5 'and / or 3' ends. Conveniently, this sequence is manipulated in standard laboratory vectors, such as plasmid vectors based on pBR322 or pUC19 (see below). For precise details of suitable techniques, mention may be made of Molecular Cloning (Cold Spring Harbor, 1989) by Sambrook et al., Or similar standard references.

  The source of nucleic acid can be confirmed by referring to published literature or a data bank such as GenBank. Nucleic acid encoding the desired first or second sequence can be obtained from an academic or commercial source if such source is willing to provide the material, or sequence data Can be obtained by synthesis or cloning of appropriate sequences. In general, this can be done by referring to the literature describing the cloning of the gene.

  Alternatively, representative nucleic acids are known in the art if limited sequence data is available, or if it is desired to express nucleic acids that are homologous or related to known nucleic acids. It can be regarded as a nucleotide sequence that hybridizes to a nucleic acid sequence.

  For some applications, preferably the nucleotide sequence is DNA. For some applications, preferably the nucleotide sequence is prepared using recombinant DNA techniques (eg, recombinant DNA). For some applications, preferably the nucleotide sequence is cDNA. For some applications, preferably the nucleotide sequence can be the same as the native form.

  Alternatively, representative nucleic acids are known in the art if limited sequence data is available, or if it is desired to express nucleic acids that are homologous or related to known nucleic acids. It can be regarded as a nucleotide sequence that hybridizes to a nucleic acid sequence.

  Those skilled in the art will appreciate that many different nucleotide sequences can encode the same protein used in the present invention as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art will employ the present invention to reflect the codon usage of any particular host organism in which the target protein of the invention or the protein for modulating Notch signaling is expressed using conventional techniques. It is understood that nucleotide substitutes can be made that do not affect the protein encoded by the nucleotide sequence.

Variants, derivatives, analogs, homologues, and fragments In addition to the specific amino acid sequences and nucleotide sequences described herein, the present invention further includes variants, derivatives, analogs, homologues, and fragments thereof. Includes use.

  In the present invention, a variant of any given sequence is a specific sequence of residues (amino acids or nucleic acid residues) in such a way that the polypeptide or polynucleotide maintains at least one endogenous function. Is a modified sequence. Variant sequences can be altered by addition, deletion, substitution, modification, replacement, and / or alteration of at least one residue present in the native protein.

  As used herein, the term “derivative” with respect to a protein or polypeptide of the invention refers to or from that sequence, provided that the resulting protein or polypeptide maintains at least one endogenous function, Including substitution, alteration, modification, substitution, deletion, and / or addition of one (or more) amino acid residues.

  As used herein, the term “analog” with respect to a polypeptide or polynucleotide includes any mimetic, ie, a chemical compound having at least one endogenous function of the polypeptide or polynucleotide that it mimics.

  Within the definition of “protein” and “polypeptide” useful in the present invention, certain amino acid residues can be modified in such a way that the protein maintains at least one endogenous function; Such modified proteins are referred to as “variants”. Variant proteins can be modified by the addition, deletion and / or substitution of at least one amino acid present in the native protein.

  Typically, amino acid substitutions can consist of, for example, 1, 2, or 3 to 10 or 20 substitutions provided that the modified sequence maintains the required target activity or ability to modulate Notch signaling. Amino acid substitutions can include the use of non-natural analogs.

  Proteins useful in the present invention can have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Intentional amino acid substitutions can be made based on residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic similarity so long as the target or regulatory function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine or arginine, and have uncharged polar head groups with similar hydrophilicity values. Amino acids possessed include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

  For simplicity of reference, the 1-letter and 3-letter codes (and associated codons) of the major natural amino acids are shown below.

Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column, preferably the same row in the third column, can be substituted for each other.

As used herein, the term “protein” includes single-chain polypeptide molecules as well as multiple polypeptide complexes in which individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms “polypeptide” and “peptide” refer to a polymer in which the monomers are amino acids and are joined by peptide or disulfide bonds. The terms subunit and domain can also refer to polypeptides and peptides having biological functions.

  Peptides useful in the present invention have at least target or signaling modulation capability. “Fragments” are also variants, and the term typically refers to a selected region of a protein that is of interest in a binding assay and whose binding pair is known or determinable. Thus, a “fragment” is a portion of a full-length polypeptide, eg, from about 8 to about 1500 amino acids long, preferably from about 8 to about 745 amino acids long, preferably from about 8 to about 300, more preferably from about 8 It refers to an amino acid sequence that is about 200 amino acids, more preferably about 10 to about 50 or 100 amino acids long. “Peptide” refers to a short amino acid sequence of 10 to 40 amino acids in length, preferably 10 to 35 amino acids.

  Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. When insertion is performed, synthetic DNA encoding the insert is inserted with 5 'and 3' flanking regions corresponding to the native sequence on either side of the insertion site. The flanking region contains convenient restriction sites corresponding to the sites of the native sequence so that the sequence can be cleaved with the appropriate enzyme and the synthetic DNA can be ligated to the cleavage site. The DNA is then expressed according to the present invention to produce the encoded protein. These methods are merely illustrative of many standard techniques known in the art for manipulating DNA sequences, and other known techniques can be used.

  Nucleotide sequence variants can also be made. Such variants preferably include codon optimized sequences. Codon optimization is known in the art as a method to enhance RNA stability and thus gene expression. Genetic code duplication means that several different codons may encode the same amino acid. For example, leucine, arginine, and serine are each encoded by six different codons. Different organisms prefer the use of different codons. For example, viruses such as HIV use a number of rare codons. By altering the nucleotide sequence so that the rare mammalian codon is replaced by the corresponding mammalian codon commonly used, the expression of the sequence can be increased in mammalian target cells. Codon usage tables are well known in the art for mammalian cells and are similar for various other organisms.

  Where the active agent is a nucleotide sequence, it can suitably be a codon optimized for expression in mammalian cells. Preferably at least a portion of the sequence is codon optimized. More preferably, the entire sequence is codon optimized.

Sequence homology, similarity, identity As used herein, the term “homology” can be synonymous with “identity”. Homologous sequences are understood to include amino acid sequences that can be at least 75, 85, or 90% identical, preferably at least 95 or 98% identical. In particular, homology should typically be considered with respect to regions of the sequence known to be essential for activity (eg, amino acids at positions 51, 56, and 57). Although homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties / functions), in the present invention, it is preferable to represent homology in terms of sequence identity.

  Homology comparison can be done visually, but more generally can be done using readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences.

  The percent homology can be calculated for a contiguous sequence, that is, one sequence is aligned with the other, and each amino acid in one sequence is one residue at a time with the corresponding amino acid in the other sequence. Compare directly. This is referred to as an “ungapped” alignment. Typically, such ungapped alignments are made only for a relatively short number of residues.

  This is a very simple and consistent method, but, for example, in a sequence pair that is otherwise identical, one insertion or deletion pushes the subsequent amino acid residue out of alignment, and thus global alignment. It is not possible to consider that a large reduction in% homology potentially occurs when performing. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account the possibility of insertions and deletions without unduly penalizing the overall homology score. This is accomplished by inserting “gaps” in the sequence alignment to maximize local homology.

  However, a more complex method is that sequence alignments that have as few gaps as possible (reflecting a high degree of association between two comparative sequences) when the same number of identical amino acids are compared to those that have many gaps Assign a “gap penalty” to each gap that occurs in the alignment. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a small penalty for each successive residue in the gap. This is the most commonly used gap score system. High gap penalties will of course result in optimized alignments with fewer gaps. Most alignment programs allow you to change the gap penalty. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

  Therefore, to calculate the maximum homology%, it is necessary to first create an optimal alignment taking into account the gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S. Devereux). Examples of other software that can perform sequence comparison include, but are not limited to, the BLAST package, FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410 (Atschul)), And a set of GENEWORKS comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, ibid, pages 7-58 to 7-60). However, the use of the GCG Bestfit program is preferred.

http: // www. ncbi. nlm. nih. The five BLAST programs available with gov perform the following tasks:
The blastp-amino acid query sequence is compared to a protein sequence database.
The blastn-nucleotide query sequence is compared to a nucleotide sequence database.
The six-frame conceptual translation product of the blastx-nucleotide query sequence (both strands) is compared to the protein sequence database.
The tblastn-protein query sequence is compared to a nucleotide sequence database dynamically translated (both strands) in all six reading frames.
The 6-frame translation of the tblastx-nucleotide query sequence is compared to the 6-frame translation of the nucleotide sequence database.

  BLAST uses the following search parameters:

  HISTOGRAM—Displays a histogram of scores for each search. Default is yes (see parameter H in the BLAST Manual).

  DESCRIPTIONS-Limit the number of short descriptions of matching sequences reported to the specified number. The default limit is 100 descriptions (see parameter V on the manual page).

  EXPECT-Statistical significance threshold for reported matches against database sequences. According to the Karlin and Altschul (1990) probabilistic model, the default value is 10, so that 10 matches are found only by chance. If the statistical significance attributed to the match is greater than the EXPECT threshold, no match is reported. A lower EXPECT threshold is more stringent and the likelihood of a match being reported is lower. A small number is allowed (see parameter E in the BLAST Manual).

  CUTOFF—Score cutoff for reporting high score segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for database sequences only if the statistical significance attributed to them is at least as high as that attributed to a single HSP with a score equal to the CUTOFF value. A high CUTOFF value is more stringent and the likelihood of a reported match is low (see parameter S in the BLAST Manual). Typically, significance thresholds can be managed more intuitively using EXPECT.

  ALIGNMENTS-Limit the database sequence to the number specified to report high score segment pairs. The default limit is 50. Unexpectedly, if a larger database sequence meets the reported statistical significance threshold (see EXPECT and CUTOFF), only the match with the highest statistical significance is reported (parameter B of the BLAST Manual). checking).

  Specifies an alternative scoring matrix for MATRIX-BLASTP, BLASTX, TBLASTN, and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). Valid alternative choices include PAM40, PAM120, PAM250, and IDENTITY. There is no alternative scoring matrix available for BLASTN and an error response is returned if a MATRIX instruction is specified in the BLASTIN request.

  Restrict the STRAND-TBLASTN search to the top or bottom strand of the database sequence, or limit the BLASTN, BLASTX, or TBLASTX search to the reading frame of the top or bottom strand of the query sequence.

  FILTER-Woughton and Federhen (1993) Computers and Chemistry 17: Segments of low-complexity query sequences determined by the SEG program of 149-163, or Claverie and States (1993) Computers and Chemistry 17N: 201-201 Or, in the case of BLASTN, masks segments consisting of short period internal repeats determined by Tatusov and Lipman's DUST program (see http://www.ncbi.nlm.nih.gov). Filtering removes reports that are statistically significant but biologically insignificant from the output (eg, acidic, basic, or proline rich regions) and can be used for specific matching with database sequences Biologically more interesting regions of the query sequence can be left.

  The low complexity sequences found by the filter program are replaced using the letter “N” (eg “NNNNNNNNNNNNN” for nucleotide sequences and the letter “X” (eg “XXXXXXXXXX”) for protein sequences.

  Filtering applies only to the query sequence (or its translation product) and not to the database sequence. Default filtering is DUST for BLASTN and SEG for other programs.

  When applied to a SWISS-PROT array, it is not uncommon to be masked at all by SEG, XNU, or both, and so it should not be expected that filtering will always be effective. Furthermore, in some cases the entire sequence is masked, suggesting that the statistical significance of any matches reported against the unfiltered query sequence should be questioned.

  In addition to the NCBI-gi-deposit and / or locus name, the NCBI gi identifier is shown in the output.

  More preferably, the sequence comparison is performed at http: // www. ncbi. nlm. nih. This is done using a simple BLAST search algorithm provided in gov / BLAST.

  In some embodiments of the invention, no gap penalty is used when determining sequence identity.

  Although the final% homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, an adjusted similarity score matrix is generally used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix, the default matrix for a series of BLAST programs. The GCG Wisconsin program generally uses public default values, or custom symbol comparison tables if supplied (see user manual for further details). For GCG packages, the use of public default values is preferred, and for other software, the use of a default matrix such as BLOSUM62 is preferred.

  After creating an optimal alignment by software, it is possible to calculate% homology, preferably% sequence identity. The software typically does this as part of the sequence comparison and presents the results numerically.

  Nucleotide sequences that are homologous to the sequences used in the present invention or that are variants of the sequences can be obtained by several methods, for example by probing DNA libraries made from various sources. . In addition, other virus / bacteria, or cell homologues, particularly those found in mammalian cells (eg, rat, mouse, bovine, and primate cells) can be obtained, and generally such Homologues and fragments thereof can selectively hybridize to the sequences shown in the sequence listing herein. Such sequences are probes that probe cDNA libraries made from other animal species, or genomic DNA libraries of other animal species, and contain all or part of the reference nucleotide sequence under moderate to high stringency conditions. It can be obtained by probing such a library. Similar considerations apply to obtaining species homologues and allelic variants of amino acid and / or nucleotide sequences useful in the present invention.

  Mutants, and strain / species homologs, are also subjected to degenerate PCR using primers designed to target variants and homologue sequences encoding conserved amino acid sequences within sequences useful in the present invention. Can be obtained. Conserved sequences can be predicted, for example, by aligning the amino acid sequences of several variants / homologs. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used. Primers used in degenerate PCR contain one or more degenerate positions and are used under stringency conditions lower than sequence cloning using single sequence primers against known sequences.

  Mutants, and strain / species homologs, are also subjected to degenerate PCR using primers designed to target variants and homologue sequences encoding conserved amino acid sequences within sequences useful in the present invention. Can be obtained. Conserved sequences can be predicted, for example, by aligning the amino acid sequences of several variants / homologs. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used. Primers used in degenerate PCR contain one or more degenerate positions and are used under stringency conditions lower than sequence cloning using single sequence primers against known sequences.

  For example, as described in Sambrook et al., 1989, Section 14, PCR technology requires the use of oligonucleotide probes that hybridize to nucleic acids. A method for selecting oligonucleotides is described below.

  As used herein, a probe comprises a sequence of nucleotides containing, for example, 10 to 50, preferably 15 to 30, most preferably at least about 20 contiguous bases that are identical (or complementary) to the same or more adjacent bases. It has single-stranded DNA or RNA. The nucleic acid sequence selected as the probe should be long enough and well defined so that false positive results are minimized. This nucleotide sequence is generally based on a conserved or highly homologous nucleotide sequence or region of the polypeptide. Nucleic acids used as probes can be degenerate at one or more positions.

Preferred regions in which the probe is constructed include 5 ′ and / or 3 ′ coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, the full-length cDNA clone disclosed herein or a fragment thereof can be used as a probe. Preferably, the nucleic acid probe of the present invention is labeled with a suitable labeling means that is easily detected during hybridization. For example, a suitable labeling means is a radioactive label. A preferred method of labeling DNA fragments is by combining α ³² PdATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are generally end-labeled with γ ³² P-labeled ATP and polynucleotide kinase. However, other methods (eg, non-radioactive) can be used to label fragments or oligonucleotides, including, for example, enzyme labels, fluorescent labels with appropriate fluorophores, and biotin labels.

  Preferred is a sequence probe that hybridizes under high stringency conditions.

  Alternatively, such a nucleotide sequence can be obtained by site-directed mutagenesis of the characterized sequence. This may be useful when the sequence requires silent codon changes, for example to optimize the codon selection of the particular host cell in which the nucleotide sequence is expressed. Other sequence changes may be desirable to introduce restriction enzyme recognition sites or to alter the activity of the polynucleotide or encoded polypeptide.

  In general, with respect to nucleotide sequences used in the present invention, the term “variant”, “homologue” or “derivative” refers to the target nucleotide or protein that modulates T cell signaling. Including, but not limited to, substitution, alteration, modification, substitution, deletion, and / or addition of one (or more) nucleic acids from or to that sequence.

  As noted above, with respect to sequence homology, there is preferably at least 75% homology to the reference sequence, more preferably at least 85%, and even more preferably at least 90%. More preferably, there is at least 95% homology, more preferably at least 98%. Nucleotide homology comparisons can be performed as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has 10 match values for each identical nucleotide, with each mismatch being -9. For each nucleotide, the default gap start penalty is -50 and the default gap extension penalty is -3.

Hybridization The present invention further encompasses nucleotide sequences that can selectively hybridize to a reference sequence, or any variant, fragment, or derivative thereof, or any complement described above. The nucleotide sequence is preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40, or 50 nucleotides in length.

  As used herein, the term “hybridization” includes “a process in which a nucleic acid strand binds to a complementary strand through base pairing” as well as an amplification process performed in polymerase chain reaction (PCR) technology.

  Nucleotide sequences useful in the present invention that can selectively hybridize to the nucleotide sequences set forth herein or their complements are generally at least 20, preferably at least 25 or 30, such as at least 40, 60, or Over at least 75 contiguous nucleotides is at least 75%, preferably at least 85% or 90%, more preferably at least 95% or 98% homologous to the corresponding nucleotide sequence set forth herein. Preferred nucleotide sequences of the present invention comprise a region homologous to the nucleotide sequence, preferably at least 80 or 90%, more preferably at least 95% homologous to the nucleotide sequence.

The term “selectively hybridize” means that the nucleotide sequence used as a probe is used under conditions where the target nucleotide sequence of the invention is found to hybridize to the probe at a level well above background. Means. Background hybridization can occur, for example, due to the presence of other nucleotide sequences in the cDNA or genomic DNA library being screened. In this event, the background indicates the level of signal produced by the interaction of the probe with non-specific DNA members of the library, and is less than 10 times, preferably less than 100 times the specific interaction observed with the target DNA. It is strength. The strength of the interaction can be measured, for example, by radiolabelling the probe, for example with ³² P.

  Hybridization conditions are as described in Berger and Kimmel (1987, Guide to Molecular Cloning Technologies, Methods in Enzymology, Vol 152, Academic Press, San Diego, CA). To give a clear “stringency” as explained below.

  The highest stringency is typically about Tm-5 ° C. (5 ° C. below the Tm of the probe), the high stringency is about 5 ° C. to 10 ° C. below the Tm, and the medium stringency is about 10 ° C. from the Tm. At between 20 ° C. and 20 ° C., low stringency occurs at about 20 ° C. to 25 ° C. below Tm. As will be appreciated by those skilled in the art, the highest stringency hybridization can be used to identify or detect the same nucleotide sequence, while the moderate (or lower) stringency hybridization is similar or It can be used to identify or detect related polynucleotide sequences.

In a preferred embodiment, the present invention, under stringent conditions (e.g. 65 ° C. and 1 × SSC {1 × SSC = 0.15M of NaCl, _Na 3 citrate of 0.015 M, pH 7.0), the present invention It includes a nucleotide sequence that can hybridize to a nucleotide sequence. Where the nucleotide sequence of the present invention is double stranded, both strands of the double strand are encompassed by the present invention either individually or in combination. When the nucleotide sequence is single-stranded, it is understood that the complementary sequence of the nucleotide sequence is also included within the scope of the present invention.

  Hybridization stringency refers to conditions under which a polynucleic acid hybrid is stable. Such conditions are obvious to those skilled in the art. As known to those skilled in the art, the stability of a hybrid is reflected in the melting temperature (Tm) of the hybrid, with a decrease of about 1 to 1.5 ° C. for every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under high stringency conditions followed by various stringency washes.

  As used herein, high stringency preferably refers to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 65-68 ° C., 1M Na +. High stringency conditions include, for example, 6 × SSC, 5 × Denhardt, 1% SDS (sodium dodecyl sulfate), 0.1Na + pyrophosphate, and 0.1 mg / ml denatured salmon sperm DNA as a non-specific competitor. It can be provided by hybridization in aqueous solution. Following hybridization, several high stringency washes are performed, and the final wash (approximately 30 minutes) can be performed at the hybridization temperature, 0.2-0.1 × SSC, 0.1% SDS.

  It is understood that these conditions can be adapted and reproduced using a variety of buffers, eg, formamide based buffers, and temperatures. Denhardt's solution and SSC are well known to those skilled in the art, as are other suitable hybridization buffers (eg, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press). , New York or Ausubel et al., (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Since the length of the hybridizing pair and the GC content also affect, the optimal hybridization conditions must be determined experimentally.

Cloning and Expression Nucleotide sequences that are not 100% homologous to the sequences of the invention, but are within the scope of the invention, can be obtained by several methods. Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries made from various sources. In addition, other virus / bacteria, or cell homologues, particularly those found in mammalian cells (eg, rat, mouse, bovine, and primate cells) can be obtained, generally such Homologues and fragments thereof can selectively hybridize to the sequences shown in the sequence listing herein. Such sequences may be probed with cDNA libraries made from other animal species, or genomic DNA libraries of other animal species, and including all or part of a reference nucleotide sequence under moderate to high stringency conditions. It can be obtained by probing such a library. Similar considerations apply to obtaining species homologues and allelic variants of amino acid and / or nucleotide sequences useful in the present invention.

  Mutants, and strain / species homologues, also use degenerate PCR with primers designed to target the sequences of the conserved amino acid sequences within the sequences of the invention and homologous sequences. Can be obtained. Conserved sequences can be predicted, for example, by aligning the amino acid sequences of several variants / homologs. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used. Primers used in degenerate PCR contain one or more degenerate positions and are used under stringency conditions lower than sequence cloning using single sequence primers against known sequences.

  Alternatively, such a nucleotide sequence can be obtained by site-directed mutagenesis of the characterized sequence. This may be useful when the sequence requires silent codon changes, for example to optimize the codon selection of the particular host cell in which the nucleotide sequence is expressed. Other sequence changes may be desirable to introduce restriction enzyme recognition sites or to alter the activity of proteins that modulate T cell signaling encoded by the target protein or nucleotide sequence.

  Nucleotide sequences such as DNA polynucleotides useful in the present invention can be produced recombinantly, synthetically, or by any means available to those skilled in the art. These can also be cloned by standard techniques.

  In general, primers are produced by synthetic means and include producing a desired nucleic acid sequence step by step, one nucleotide at a time. Techniques that accomplish this using automated techniques are readily available in the art.

  Long nucleotide sequences are generally produced using recombinant means, such as PCR (polymerase chain reaction) cloning techniques. This involves creating a pair of primers (eg, about 15 to 30 nucleotides) adjacent to the region of the targeting sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, desired Performing a polymerase chain reaction (PCR) under conditions that result in amplification of the region, isolating the amplified fragment (eg, by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. This primer can be designed to contain an appropriate restriction enzyme recognition site so that the amplified DNA can be cloned into an appropriate cloning vector.

  The invention further relates to vectors comprising polynucleotides useful in the invention, host cells that have been genetically engineered using the vectors of the invention, and production of polypeptides useful in the invention by such techniques.

  For recombinant production, the host cell can be genetically engineered to incorporate an expression system or a polynucleotide of the invention. Introduction of polynucleotides into host cells can be accomplished by methods described in many standard laboratory manuals, such as Davis et al. And Sambrook et al., Eg, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, positive injection. It can be performed by ion lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection and the like. It will be appreciated that such methods can be used in vitro or in vivo as drug delivery systems.

  Representative examples of suitable hosts include streptococci, staphylococci, E. coli. bacterial cells such as E. coli, Streptomyces, and Bacillus subtilis cells, fungal cells such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, CHO, COS, NSO, HeLa, C127, 3T3, BHK, 293, and Animal cells such as Bowes melanoma cells, as well as plant cells are included.

  A wide variety of expression systems can be used to produce polypeptides useful in the present invention. Such vectors include in particular chromosomal, episomal and viral vectors such as bacterial plasmid-derived, bacteriophage-derived, transposon-derived, yeast episome-derived, insert element-derived, yeast chromosomal element-derived, papovaviruses such as baculovirus, SV40, etc. Vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retrovirus, and combinations thereof, such as those derived from plasmid and bacteriophage genetic elements such as cosmids and phagemids Etc. are included. Expression system constructs can include control regions that not only produce expression but also regulate expression. In general, in this regard, any system or vector suitable for maintaining, propagating, or expressing a polynucleotide in a host and / or expressing a polypeptide can be used for expression. Appropriate DNA sequences can be inserted into the expression system by any of a variety of well-known conventional techniques, such as those described in Sambrook et al.

  In order to secrete the translated protein into the endoplasmic reticulum lumen, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. These signals can be endogenous to the polypeptide or they can be heterologous signals.

  The protein or polypeptide can be in the form of a “mature” protein or can be part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include additional amino acid sequences containing secretory or leader sequences or presequences (HIS oligomers, immunoglobulin Fc, glutathione S transferase, FLAG, etc.) to aid in purification. Similarly, it may be desirable for such additional sequences to provide additional stability during recombinant production. In such cases, additional sequences can be cleaved (eg, chemically or enzymatically) to produce the final product. However, in some cases, the additional sequence may confer a desirable pharmacological profile (such as in the case of an IgFc fusion protein), in which case the additional sequence may not be removed such that the additional sequence is present in the final product upon administration. It may be preferable.

  The protein or polypeptide can be in the form of a “mature” protein or can be part of a larger protein such as a fusion protein or precursor. For example, it is often advantageous to include additional amino acid sequences containing secretory or leader sequences or presequences (HIS oligomers, immunoglobulin Fc, glutathione S transferase, FLAG, etc.) to aid in purification. Similarly, it may be desirable for such additional sequences to provide additional stability during recombinant production. In such cases, additional sequences can be cleaved (eg, chemically or enzymatically) to produce the final product. However, in some cases, the additional sequence may confer a desirable pharmacological profile (such as in the case of an IgFc fusion protein), in which case the additional sequence may not be removed such that the additional sequence is present in the final product upon administration. It may be preferable.

  In addition, mammalian and microbial host cells containing other polynucleotides encoding such vectors or fusion proteins, and their production and use are also included in the present invention.

  Active factors used in the present invention are well known, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Can be recovered and purified from recombinant cell cultures by established methods. Most preferably, high performance liquid chromatography is used for purification. When the polypeptide is denatured during isolation and / or purification, known techniques for refolding the protein can be used to regenerate the active conformation.

  Various preferred features and embodiments of the invention will now be described in more detail by way of non-limiting examples.

  Agents that can be used to modulate Notch signaling by inhibiting Notch ligand expression include nucleic acids encoding polypeptides that affect the expression of genes encoding Notch ligands. For example, in the case of Delta expression, the binding of extracellular BMPs (bone morphogenetic proteins, Wilson and Hemmati-Brivanlou, Hemmati-Brivanlou and Melton) to its receptor is due to inhibition of the expression of transcription factors in the achaete / scute complex. Causes down-regulation of transcription. This complex is thought to be directly involved in the regulation of Delta expression. Thus, any polypeptide that upregulates BMP expression and / or stimulates binding of BMP to its receptor may result in a reduction in the expression of Notch ligands such as Delta and / or Serrate. There is. Examples include nucleic acids encoding BMP itself. In addition, any substance that inhibits expression of the transcription factor of the achaete / scute complex may down-regulate Notch ligand expression.

  BMP family members include BMP1 to BMP6, BMP7, also referred to as OP1, OP2 (BMP8), and the like. BMP belongs to the transforming growth factor beta (TGFbeta) superfamily, which includes, in addition to TGFbeta, activin / inhibin (eg, alpha inhibin), Mueller inhibitor, and glial cell line-derived neurotrophic factor. .

  Other examples of polypeptides that inhibit the expression of Delta and / or Serrate include Toll-like receptors (Medzitov) or any other receptor that binds to the innate immune system (eg, CD14, complement receptors, scavenger receptors) Body, or defensin protein), and other polypeptides that reduce or prevent production of Valenzuela, Sasai, Follistatin (Iemura), Xnr3, and derivatives and variants thereof. Noggin and chodin bind to BMP, thereby preventing activation of their signaling cascade, resulting in reduced Delta transcription. Thus, a reduction in noggin and chodin levels may result in a reduction in Notch ligand, especially Delta expression.

  More specifically, in Drosophila, Toll transmembrane receptors play a central role in signaling pathways that specifically control innate nonspecific immune responses. This Toll-mediated immune response reflects an ancestral conserved signaling system with homologous components in a wide range of organisms. Human Toll homologues have been identified among Toll-like receptor (TLR) genes and Toll / interleukin-1 receptor-like (TIL) genes, and are characterized by the characteristic Toll motif extracellular leucine-rich repeat domain and Contains a cytoplasmic interleukin-1 receptor-like region. Toll-like receptor genes (including TIL genes) currently include TLR4, TIL3, TIL4, and four other identified TLR genes.

  Other suitable sequences that can be used to down-regulate Notch ligand expression include immune costimulatory molecules (eg, CD80, CD86, ICOS, SLAM) and other accessory molecules associated with immune synergy (eg, CD2 , Sequences encoding LFA-1) are included.

  Other suitable agents that can be used to down-regulate Notch ligand expression include nucleic acids that inhibit the action of transforming growth factors such as members of the fibroblast growth factor (FGF) family. FGF is mammalian basic FGF, acidic FGF, or other member of the FGF family, such as FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7. be able to. Preferably, the FGF is not acidic FGF (FGF-1, Zhao et al., 1995). Most preferably, the FGF is a member of the FGF family that acts by stimulating upregulation of Serrate polypeptide expression with APC. It has been shown that members of the FGF family can upregulate Serrate-1 gene expression with APC.

Inhibition of Notch signaling by use of antisense constructs Suitable nucleic acid sequences include nucleic acid sequences encoding antisense constructs, such as antisense Notch ligand constructs or antisense sequences corresponding to other components of the Notch signaling pathway described above. Can be included. The antisense nucleic acid can be an oligonucleotide such as a synthetic single stranded DNA. More preferably, however, the antisense is an antisense RNA produced in the patient's own cells as a result of the gene vector introduction. This vector results in the production of antisense RNA with the desired specificity in introducing the vector into the host cell.

  Antisense nucleic acids can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA, or modifications or derivatives thereof, which can be administered directly to cells, or of exogenous transduction sequences. It can be produced intracellularly by transcription.

  For example, as described in US-A-20020119540, an inhibitory antisense or double-stranded oligonucleotide can further comprise at least one modified base moiety, including but not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5 -Carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosilk eosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- Methylgua , 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl eosin, 5'-methoxy Carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wivetoxosin, pseudouracil, queocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2- Carboxypropyl) uracil, (acp3) w, and , It is selected from the group consisting of 6-diaminopurine.

  Antisense oligonucleotides can also include one or more modified sugar moieties such as, for example, arabinose, 2-fluoroarabinose, xylulose, or hexose.

  In other embodiments, the antisense oligonucleotide can be, for example, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphordiamidate, methylphosphonate, alkylphosphotriester, form, if desired. It can comprise at least one modified phosphate backbone, such as an acetal, or an analog thereof. Alternatively, other polymer backbones such as modified polypeptide backbones can also be used (eg, protein nucleic acid: PNA).

  In other embodiments, the antisense oligonucleotide can be an alpha anomeric oligonucleotide. Alpha anomeric oligonucleotides form specific double-stranded hybrids containing complementary RNAs that are parallel to each other unlike normal beta units (Gautier et al., 1987, Nucl. Acids Res. 15: 6625-6641). . This oligonucleotide may be, for example, a 2′-0-methyl ribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15: 6131-6148), or a chimeric RNA-DNA analog (Inoue et al., 1987, FEBS Lett. 215: 327-330). Oligonucleotides can be synthesized by standard methods known in the art using, for example, an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). By way of example only, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16: 3209), and methylphosphonate oligonucleotides are prepared using a porous glass polymer support. (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451).

Preferably, the nucleic acid sequence used in the present invention can inhibit the expression of Serrate and Delta, preferably Serrate1 and Serrate2, and Delta1, Delta3, and Delta4 in APCs such as dendritic cells. In particular, the nucleic acid sequence can inhibit Serrate expression in APC or T cells, but does not inhibit Delta expression, or can inhibit Delta expression, but may not inhibit Serrate expression. Alternatively, the nucleic acid sequences used in the present invention inhibit Delta expression in T cells such as CD4 ⁺ helper T cells, or other cells of the immune system that express Delta (eg in response to stimulation of cell surface receptors). can do. In particular, the nucleic acid sequence can inhibit Delta expression in T cells, but may not inhibit Serrate expression. In one particularly preferred embodiment, the nucleic acid sequence can inhibit Notch ligand expression in both T cells and APCs, eg, inhibit Serrate expression with APC and inhibit Delta expression with T cells.

  Preferred suitable substances that can be used to down-regulate Notch ligand expression include growth factors and cytokines. More preferably, soluble protein growth factors can be used to inhibit Notch or Notch ligand expression. For example, Notch ligand expression can be reduced or inhibited by adding BMP or activin (a member of the TGF-β superfamily). Furthermore, T cells, APC, or tumor cells can be cultured alone or in combination with BMP in the presence of inflammatory cytokines including IL-12, IFN-γ, IL-18, TNF-α.

  Molecules for inhibiting Notch signaling further include polypeptides, or polynucleotides encoding them, that can alter Notch-protein expression or presentation in the cell membrane or signaling pathway. Molecules that reduce or prevent presentation as a fully functional cell membrane protein can include MMP inhibitors, such as hydroxymate-based inhibitors.

  Other substances that can be used to reduce the interaction between Notch and Notch ligand are exogenous Notch or Notch ligands, or functional derivatives thereof. For example, a Notch ligand derivative preferably has a DSL domain at the N-terminus and 1-8, suitably 2-5, EGF-like repeats on the extracellular surface. A peptide corresponding to the Delta / Serrate / LAG-2 domain of the supernatant of COS cells expressing hJagged1 and a soluble form of the extracellular portion of hJagged1 was found to mimic the effects of Jagged1 in inhibiting Notch1. (Li).

  In one embodiment, the Notch ligand derivative can be a fusion protein, eg, a fusion protein comprising a segment of the Notch ligand extracellular domain and an immunoglobulin Fc segment such as IgGFc or IgMFc.

  Alternatively, the modulator is an extracellular of a Notch receptor (eg, Notch1, Notch2, Notch3, Notch4, or homologs thereof) that can bind to Notch ligand and reduce interaction with an endogenous Notch receptor. It can include all or part of the domain. Preferably, such modulators can include at least the 11th and 12th domains of Notch (EGF11 and EGF12), since Notch's 11th and 12th domains are believed to be important for Notch ligand interaction. .

  For example, the rat Notch-1 / Fc fusion protein is available from R & D Systems Inc (Minneapolis, USA and Abingdon, Oxon, UK, catalog number 1057-TK). This includes the 12 amino terminal EGF domain of rat Notch-1 (amino acid residues Met1 to Glu488) fused to the Fc region of human IgG (Pro100 to Lys330) via a polypeptide linker (IEGRMD).

  Other Notch signaling pathway antagonists include antibodies that inhibit interactions between components of the Notch signaling pathway, such as antibodies of Notch or Notch ligands.

The term “antibody” includes complete molecules and fragments thereof, such as Fab, Fab ′, F (ab ′) ₂ , Fv, and scFv, that can bind to epitope determinants. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:
(I) Fab, this fragment containing a monovalent antigen-binding fragment of an antibody molecule, is produced by digesting the whole antibody with the enzyme papain to produce one complete light chain and part of one heavy chain it can.
(Ii) Fab ′, this fragment of the antibody molecule can be obtained by treating the whole antibody with pepsin and then reducing to give one complete light chain and part of the heavy chain, Two Fab ′ fragments are obtained.
(Iii) (Fab ′) ₂ , a fragment of an antibody obtained by treating the whole antibody with pepsin without subsequent reduction, F (ab ′) ₂ being two Fab ′ linked by two disulfide bonds It is a dimer of fragments.
(Iv) Fv, defined as a genetically engineered fragment containing a variable gene fusion single chain molecule.
(V) A fragment consisting essentially of only the variable (V _H or V _L ) antigen-binding domain of an antibody (so-called “domain antibody”).

  General methods of producing antibodies are known in the art (eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New, the original of which is hereby incorporated by reference). See York (1988)). The antibody can be monoclonal or polyclonal, but is preferably monoclonal.

Suitably, the binding affinity (equilibrium binding constant (Ka)) is at least about 10 ⁶ M ⁻¹ , at least about 10 ⁷ M ⁻¹ , at least about 10 ⁸ M ⁻¹ , or at least about 10 ⁹ M ⁻¹ . Can be.

  Suitably, the antibody, derivative or fragment is one or more DSL, EGF, or N-terminal domains of a Notch ligand, or one or more EGF or Lin / Notch (L / N) domains of Notch (eg, It binds to Notch's EGF repeats 11 and 12).

  In one embodiment, the agent can be an antibody, derivative, or fragment that binds Notch.

  In other embodiments, the agent can be an antibody, derivative, or fragment that binds to Delta.

  In other embodiments, the agent can be an antibody, derivative, or fragment that binds to Serrate or Jagged.

  Suitable antibodies for use as blocking agents are obtained by immunizing host animals with Notch or peptides containing all or part of Notch ligands such as Delta or Serrate / Jagged.

  The peptides used can include the complete protein, or a fragment or derivative thereof. Preferred immunogens include all or part of the extracellular domain of human Notch, Delta, or Serrate / Jagged, and these residues contain any post-translational modifications found in natural proteins such as glycosylation. Immunogens containing extracellular domains are well known in the art, such as expression of cloned genes using conventional recombinant methods and / or isolation from T cells or cell populations that express high levels of Notch or Notch ligands. Can be produced by a number of techniques.

  Monoclonal antibodies can be produced by means well known in the art. In general, the spleen and / or lymph nodes of the immunized host animal provide a source of plasma cells. Plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells. Individual hybridoma culture supernatants are screened using standard techniques to identify those that produce antibodies with the desired specificity. This antibody can be purified from hybridoma cell supernatant or ascites by conventional techniques such as affinity chromatography using Notch, Notch ligand, or fragments thereof conjugated to insoluble carrier protein A sepharose or the like.

  For example, antibodies against Notch or Notch ligands are described in US Pat. No. 5,648,464, US5849869, and US600004924 (Yale University / Imperial Cancer Technology), the originals of which are hereby incorporated by reference.

  Antibodies raised against the Notch receptor are also described in WO0020576 (this original is also incorporated herein by reference). For example, this document discloses the production of antibodies against human Notch-1 EGF-like repeats 11 and 12. For example, in certain embodiments, WO0020576 is a monoclonal antibody secreted by a hybridoma having ATCC deposit number HB12654, designated A6, a monoclonal antibody secreted by a hybridoma having ATCC deposit number HB12656, designated A1. And a monoclonal antibody secreted by a hybridoma having ATCC deposit number HB12655, designated F3.

  Preferably, the antibody used for the treatment of human patients is a chimeric or humanized antibody. Antibody “humanization” techniques are well known in the art. These techniques typically involve the use of recombinant DNA technology to manipulate the DNA sequence encoding the polypeptide chain of the antibody molecule.

  As described in US Pat. No. 5,859,205, an early method of humanizing monoclonal antibodies (Mabs) involves the production of chimeric antibodies, where the antigen binding site comprising the complete variable region of one antibody is a constant derived from another antibody. It is bound to the area. Such chimerization procedures are described in EP-A-0120694 (Celltech Limited), EP-A-0125023 (Genentech Inc. and City of Hope), EP-A-0171496 (Res. Dev. Corp. Japan), EP- A-0173494 (Stanford University) and WO86 / 01533 (Celltech Limited). For example, WO86 / 01533 discloses a method for preparing antibody molecules having the variable region of mouse MAb and the constant region of human immunoglobulin.

  In another approach described in EP-A-0239400 (Winter), the complementarity-determining region (CDR) of the murine MAb can be obtained by frame-directing the variable region of a human immunoglobulin by site-directed mutagenesis using long oligonucleotides. Grafted into the work area. Such CDR-grafted humanized antibodies are less likely to produce an anti-antibody response compared to humanized chimeric antibodies due to the much lower percentage of non-human amino acid sequences they contain. Examples of mouse MAbs recognizing lysozyme and rat MAbs recognizing human T cell antigens humanized by CDR grafting include Verhoeyen et al. (Science, 239, 1534-1536, 1988) and Riechmann et al. (Nature, 332, 323-324, 1988). The preparation of CDR-grafted antibodies against human T cell antigens is also described in WO 89/07452 (Medical Research Council).

  In WO 90/07861, Queen et al. Present four criteria for designing humanized immunoglobulins. The first criterion is to use a specific human immunoglobulin framework that is significantly homologous to the humanized non-human donor immunoglobulin, or the common framework of many human antibodies, as human acceptors. The second criterion is to use the donor amino acid rather than the acceptor when the human acceptor residue is exceptional and the donor residue is typical as a human sequence at a particular residue of the framework . The third criterion is to use donor framework amino acid residues rather than acceptors at positions immediately adjacent to the CDRs. The fourth criterion is to use donor amino acid residues at framework positions, where the amino acid has a side chain atom within about 3A of the CDR and interacts with the antigen or humanized CDR in a three-dimensional immunoglobulin model. Expected to be able to. It is suggested that criteria 2, 3, or 4 can be applied in addition to or instead of criterion 1, and can be applied alone or in combination.

The choice of isotype depends on the desired effector function, such as complement binding or the activity of antibody-dependent cytotoxicity. Suitable isotypes include IgG1
, IgG3, and IgG4. Suitably a human light chain constant region, kappa or lambda may be used.

Chemical binding Chemically linked sequences can be prepared from individual protein sequences (if necessary) and linked using known chemical coupling techniques. This conjugate can be collected using conventional liquid phase or solid phase peptide synthesis methods to yield a fully protected precursor in which only the terminal amino group is in a deprotected reactive form. This functional group can then be reacted directly with a protein for modulating T cell signaling, or an appropriate reactive derivative thereof. Alternatively, the amino group can be converted to a different functional group suitable for reaction with a cargo moiety or linker. Thus, for example, reaction of an amino group with succinic anhydride provides a selectively addressable carboxyl group, and further extension of the peptide chain by a cysteine derivative yields a selectively addressable thiol group. Once a suitable selectively addressable functional group is obtained for the delivery vector precursor, a protein or derivative thereof for modulating T cell signaling can be coupled, for example, by amide, ester, or disulfide bond formation. . Cross-linking reagents that can be used are, for example, Neans G. et al. E. And Feney R .; E. , Chemical Modification of Proteins, Holden-Day, 1974, pages 39-43.

  As mentioned above, the target protein and the protein for modulating T cell signaling can be linked directly or indirectly via a cleavable linker moiety. Direct binding occurs through any common functional group, such as a hydroxy, carboxy, or amino group, in a protein that regulates T cell signaling. Preferred indirect binding occurs through the binding moiety. Suitable linking moieties include difunctional and polyfunctional alkyl, aryl, aralkyl, or peptide moieties, alkyl, aryl, or aralkyl aldehydes, acids, esters, and anhydrides, sulfhydryl or carboxyl groups, such as maleimide benzoic acid derivatives, maleimides Propionic acid derivatives, succinimide derivatives, and the like are included, or may be derived from bromide or cyanuric chloride, carbonyldiimidazole, succinimidyl ester, sulfonic acid halide, and the like. The functional group of this linker moiety used on the one hand to form a covalent bond between the linker and the T cell signaling regulatory protein, and on the other hand, a covalent bond between the linker and the target protein can contain two or more, eg amino, hydrazino. , Hydroxyl, thiol, maleimide, carbonyl, carboxyl group, and the like. The linker moiety can comprise a short sequence of 1 to 4 amino acid residues, optionally including a cysteine residue that allows the linker moiety to bind to the target protein.

Notch Ligand Domain As noted above, a natural Notch ligand typically includes several different domains. Some predicted / potential domain positions (based on the amino acid number of the precursor protein) of various natural human Notch ligands are shown below.

DSL Domain A typical DSL domain can include most or all of the following common amino acid sequences.

Preferably, the DSL domain can comprise most or all of the following common amino acid sequences:

In the array,
ARO is an aromatic amino acid residue such as tyrosine, phenylalanine, tryptophan, or histidine;
NOP is a nonpolar amino acid residue such as glycine, alanine, proline, leucine, isoleucine, or valine;
BAS is a basic amino acid residue such as arginine or lysine,
ACM is an acid or amide amino acid such as aspartic acid, glutamic acid, asparagine, or glutamine.

  Preferably, the DSL domain can comprise most or all of the following common amino acid sequences:

(In the sequence, Xaa can be any amino acid and Asx is aspartic acid or asparagine).

  An alignment of the DSL domains of Notch ligands from various sources is shown in FIG.

  The DSL domain used can be derived from any suitable species including, for example, Drosophila, Xenopus, rat, mouse, or human. Preferably, the DSL domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.

  As used herein, the term “DSL domain” is understood to include sequence variants, fragments, derivatives, and mimetics that have activity corresponding to the natural domain.

  Suitably, for example, the DSL domain used in the present invention is at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80% relative to the DSL domain of human Jagged1. %, Preferably at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the DSL domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably relative to the DSL domain of human Jagged2 Can have at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the DSL domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably relative to the DSL domain of human Delta1 Can have at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the DSL domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably relative to the DSL domain of human Delta3 Can have at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the DSL domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably relative to the DSL domain of human Delta4 Can have at least 90%, preferably at least 95% amino acid sequence identity.

EGF-like domains EGF-like motifs have been found in various proteins including not only EGF, Notch and Notch ligands but also those involved in the blood coagulation cascade (Furie and Furie, 1988, Cell 53: 505-518). For example, this motif can be found in extracellular proteins such as blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7: 2053-2061, Furie and Furie, 1988, Cell 53: 505-518), other Drosophila genes ( Knust et al., 1987, EMBO J. 761-766, Rothberg et al., 1988, Cell 55: 1047-1059), as well as some cell surface receptor proteins such as thrombomodulin (Suzuki et al., 1987, EMBO J. 6: 1891-1897). ) And the LDL receptor (Sudhof et al., 1985, Science 228: 815-822). The protein binding site has been mapped to the thrombomodulin and urokinase EGF repeat domains (Kurosawa et al., 1988, J. Biol. Chem 263: 5993-5996, Appella et al., 1987, J. Biol. Chem. 262: 4437-4440). .

  As reported by PROSITE, a typical EGF domain can contain six cysteine residues that have been shown to be involved in disulfide bonds (in EGF). Its main structure is not necessarily essential, but is reported to be a double-stranded β sheet, followed by a loop to a C-terminal short double-stranded sheet. The subdomains between conserved cysteines are very diverse in length, as shown in the schematic diagram of the typical EGF-like domain below.

In the figure,
C: Conserved cysteine involved in disulfide bond G: Often conserved glycine a: Conserved aromatic amino acid *: Position of both patterns x: Arbitrary residue Region between 5th and 6th cysteines Contains two conserved glycines, at least one of which is generally present in most EGF-like domains.

  The EGF-like domain used can be derived from any suitable species including, for example, Drosophila, Xenopus, rat, mouse, or human. Preferably, the EGF-like domain is derived from a vertebrate, preferably a mammalian, preferably a human Notch ligand sequence.

  As used herein, the term “EGF domain” is understood to include sequence variants, fragments, derivatives, and mimetics that have activity corresponding to the natural domain.

  Suitably, for example, the EGF-like domain used in the present invention is at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably relative to the EGF-like domain of human Jagged1 It can have at least 80%, preferably at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the EGF-like domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, relative to the EGF-like domain of human Jagged2 Preferably having at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the EGF-like domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80% relative to the EGF-like domain of human Delta1 Preferably having at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the EGF-like domain used in the present invention is at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80% relative to the EGF-like domain of human Delta3, for example Preferably having at least 90%, preferably at least 95% amino acid sequence identity.

  Alternatively, the EGF-like domain used in the present invention is, for example, at least 30%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, relative to the EGF-like domain of human Delta4 Preferably having at least 90%, preferably at least 95% amino acid sequence identity.

  As a practical matter, whether any particular amino acid sequence is at least X% identical to other sequences can be routinely determined using known computer programs. For example, the overall maximum match between a query sequence and a subject sequence, also referred to as global sequence alignment, is the FASTDB based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6: 237-245). It can be obtained using a program such as a computer program. In some sequence alignments, the query sequence and the subject sequence are both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is given as a percent identity.

  The term “Notch ligand N-terminal domain” means the portion of the Notch ligand sequence from the N-terminus to the start of the DSL domain. This term is understood to include sequence variants, fragments, derivatives, and mimetics that have activity corresponding to the natural domain.

  As used herein, the term “heterologous amino acid sequence” or “heterologous nucleotide sequence” refers to a native sequence (eg, if a Notch ligand sequence is not found in the native Notch ligand sequence) or a sequence that is not found in its coding sequence. Means. Preferably, any such heterologous amino acid sequence is not a TSST sequence, preferably not a superantigen sequence.

  Whether a substance can be used to activate Notch is described in, for example, Applicants' co-pending international patent application claiming priority of GB0118153.6, and the examples described herein. Can be determined using various screening assays.

Screening Assays Whether a substance can be used to modulate Notch signaling can be determined using an appropriate screening assay (see, eg, the Examples herein).

  Notch signaling can be monitored by protein assays or nucleic acid assays. Activation of the Notch receptor results in proteolytic cleavage of its cytoplasmic domain and translocation to the cell nucleus. As used herein, “detectable signal” refers to any detectable sign resulting from the presence of a cleaved intracellular domain of Notch. Thus, increased Notch signaling can be assessed at the protein level by measuring the intracellular concentration of the cleaved Notch domain. Notch receptor activation also catalyzes a series of downstream reactions, leading to changes in the expression levels of certain well-defined genes. Thus, increased Notch signaling can be assessed at the nucleic acid level by measuring the intracellular concentration of a particular mRNA. In a preferred embodiment of the invention, the assay is a protein assay. In another preferred embodiment of the invention, the assay is a nucleic acid assay.

  The advantage of using a nucleic acid assay is that it is sensitive and small samples can be analyzed.

  The intracellular concentration of a particular mRNA measured at any given time reflects the expression level of the corresponding gene at that time. Accordingly, mRNA levels of downstream target genes in the Notch signaling pathway can be measured in indirect assays of immune system T cells. In particular, increased Deltex, Hes-1, and / or IL-10 mRNA levels may indicate, for example, induction of anergy, such as Dll-1 or IFN-γ mRNA levels, or IL-2, IL-5, and IL Increased levels of mRNA encoding cytokines such as -13 may indicate improved responsiveness.

  Various nucleic acid assays are known. Any conventional technique known or later disclosed can be used. Examples of suitable nucleic acid assays are described below and include amplification, PCR, RT-PCR, RNase protection, blotting, spectrometry, reporter gene assays, gene chip arrays, and other hybridization methods.

  In particular, the presence, amplification, and / or expression of a gene may be determined using, for example, a conventional Southern blot, a Northern blot to quantify mRNA transcription, a dot blot (DNA or RNA analysis) using an appropriately labeled probe, Alternatively, it can be measured directly in the sample by in situ hybridization. Those skilled in the art can readily envision how these methods can be modified if desired.

  PCR was originally developed as a means of amplifying DNA from impure samples. This technique is based on a temperature cycle in which the reaction solution is repeatedly heated and cooled to anneal the primers to the target sequence and extend the primers for the formation of overlapping daughter strands. RT-PCR uses an RNA template to produce a first cDNA strand by reverse transcriptase. This cDNA is then amplified according to standard PCR protocols. Repeated synthesis and denaturation cycles exponentially increase the copy number of the target DNA produced. However, as the reaction components become limiting, the rate of amplification decreases until a plateau is reached and there is little or no net increase in PCR product. The higher the starting copy number of the nucleic acid target, the faster this “end point” is reached. Primers can be designed using standard procedures in the art such as Taqman ™ technology.

  Real-time PCR is a quantitative assay endpoint PCR in that it uses a probe labeled with a fluorescent tag and is used to detect PCR products during accumulation rather than measuring product accumulation after a fixed number of cycles. Is different. This reaction is characterized in that amplification of the target sequence is first detected by a significant increase in fluorescence at a point in the cycle. The advantage of real-time PCR is that the amount of target sequence in the sample can be accurately determined. Suitable protocols are for example Meuer S. et al. (2000).

  The ribonuclease protection (RNase protection) assay is a very sensitive technique for quantifying specific RNAs in solution. Ribonuclease protection assays can be performed targeting total cellular RNA or poly (A) -selected mRNA. The sensitivity of the ribonuclease protection assay stems from the use of complementary in vitro transcription probes radiolabeled to high specific activity. The probe and target RNA are hybridized in solution, after which the mixture is diluted and treated with ribonuclease (RNase) to degrade any remaining single-stranded RNA. The hybridized portion of the probe is protected from digestion and can be visualized by electrophoresis of the mixture on a denaturing polyacrylamide gel followed by autoradiography. Since the protected fragments are analyzed by high resolution polyacrylamide gel electrophoresis, the ribonuclease protection assay can be used to accurately map the characteristics of the mRNA. When the probe is hybridized to the target RNA in molar excess, the resulting signal is directly proportional to the amount of complementary RNA in the sample.

  Gene expression can also be detected using a reporter system. Such reporter systems can include markers that are readily identifiable under the control of the expression system, such as, for example, the gene being monitored. Fluorescent markers that can be detected and selected by FACS are preferred. Particularly preferred are GFP and luciferase. Another type of preferred reporter is a cell surface marker, ie a protein that is expressed on the cell surface and is easily identifiable.

  In general, reporter constructs useful for detecting Notch signaling by reporter gene expression can be constructed according to the general teaching of Sambrook et al. (1989). Typically, a construct according to the invention comprises a promoter from the gene of interest and a coding sequence encoding the desired reporter construct, such as GFP or luciferase. Vectors encoding GFP and luciferase are known in the art and are commercially available.

  Sorting of cells based on detection of gene expression can be performed by any technique known in the art, as exemplified above. For example, cells can be sorted by flow cytometry or FACS. For general references, see Flow Cytometry and Cell Sorting: A Laboratory Manual (1992) A.A. See Radbruch, Springer Laboratory, New York.

  Flow cytometry is a powerful method for studying and purifying cells. Although flow cytometry has found wide application, particularly in immunology and cell biology, the ability of FACS can be applied in many other areas of biology. The acronym FACS stands for Fluorescence Activated Cell Sorting and is used interchangeably with “flow cytometry”. It is the principle of FACS that individual cells held in a fine fluid pass one or more laser beams, thereby scattering the light and the fluorescent dye emitting light at various frequencies. A photomultiplier tube (PMT) converts the light into an electrical signal that is interpreted by software to produce data about the cell. A subpopulation of cells with distinct characteristics can be identified and automatically sorted from suspension with very high purity (˜100%).

  FACS can be used to measure gene expression in cells transfected with recombinant DNA encoding a polypeptide. This can be achieved directly by labeling the protein product or indirectly using the reporter gene of the construct. Examples of reporter genes are β-galactosidase and green fluorescent protein (GFP). The activity of β-galactosidase can be detected by FACS using a fluorescent substrate such as fluorescein digalactoside (FDG). FDG is introduced into cells by hypotonic shock and is cleaved with an enzyme to produce a fluorescent product, which is captured in the cell. Thus, one enzyme can produce a large amount of fluorescent product. Cells expressing the GFP construct fluoresce without the addition of substrate. Variants of GFP that have different excitation frequencies but fluoresce in the same channel are available. In a two-laser FACS instrument, cells that are excited with different lasers can be distinguished, and therefore two transfections can be assayed simultaneously.

  Other means of sorting cells can also be used. For example, the invention includes the use of nucleic acid probes that are complementary to mRNA. Such probes can be used to identify cells that individually express the mRNA of a polypeptide so that it can later be sorted manually or using FACS sorting methods. Nucleic acid probes complementary to mRNA can be prepared according to the teachings described above using the general procedure described by Sambrook et al. (1989).

  In a preferred embodiment, the present invention involves the use of an antisense nucleic acid molecule complementary to mRNA conjugated to a fluorophore that can be used in FACS cell sorting.

  Methods have also been described for obtaining information on gene expression and identity using so-called gene chip arrays or high density DNA arrays (Chee). These high density arrays are particularly useful for diagnosis and prognosis. In vivo expression technology (IVET) can also be used (Camilli). IVET identifies genes that are up-regulated during treatment or disease, for example, when compared to laboratory culture.

  The advantage of using a protein assay is that Notch activation can be measured directly. Assay techniques that can be used to determine polypeptide levels are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis, antibody sandwich assays, antibody detection, FACS, and ELISA assays.

  A modulator of Notch signaling can also be an immune cell that has been treated to modulate the expression or interaction of Notch, a Notch ligand, or a Notch signaling pathway. Such cells are described, for example, by Lorantis Ltd., the original of which is hereby incorporated by reference. Can be easily prepared as described in WO 00/36089.

Pharmaceutical Composition Suitably the active agent is administered in combination with a pharmaceutically acceptable diluent, carrier or excipient (ie, as a pharmaceutical composition). The pharmaceutical composition can be for human or animal use in human and veterinary medicine.

  Carriers or diluents acceptable for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Ltd. (A. R. Gennaro, 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can include any suitable binder, lubricant, suspending agent, coating agent, solubilizer, as or in addition to a carrier, excipient or diluent. Preservatives, stabilizers, dyes, and flavoring agents can also be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

  For some applications, the active agent is in the form of a tablet containing an excipient such as starch or lactose, or alone or in combination with an excipient, in a capsule or egg, or a flavoring agent or It can be administered orally in the form of elixirs, solutions or suspensions containing colorants.

  Alternatively or in addition, the active agent can be administered in the form of inhalation, intranasal, aerosol, or suppository or vaginal suppository, or in the form of a lotion, solution, cream, ointment, or powder. It can also be applied locally. Another means of transdermal administration is through the use of skin patches. For example, it can be mixed into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. The active agent can also be mixed in a concentration of 1 to 10% by weight in an ointment consisting of a white wax or a white soft paraffin base, if necessary with the stabilizers and preservatives described above.

  Active agents such as polynucleotides and proteins / polypeptides can also be administered by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic planar amphiphile (CFA), and combinations thereof. Such delivery mechanism routes include, but are not limited to, mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes. The active agent can be delivered by conventional DNA delivery techniques such as DNA vaccination, or by injection or needle-free system, such as ballistic delivery of particles coated with DNA for delivery to other sites such as the epidermis or mucosal surface. Etc. can be administered.

  Typically, a physician will determine the actual dosage that is most appropriate for an individual patient, and that dosage will vary depending on the age, weight and response of the particular patient. The above dose is an example of an average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

  In general, a therapeutically effective oral or intravenous dose is likely to be in the range of 0.01 to 50 mg / kg, preferably 0.1 to 20 mg / kg of body weight of the subject to be treated. The conjugate can be administered by intravenous infusion and the dose is likely in the range of 0.001-10 mg / kg / hr.

  Conjugate tablets or capsules can be administered one or more at a time as needed. The conjugate can also be administered in a long acting formulation.

  The active agent can also be injected parenterally, for example, intracavity, intravenously, intramuscularly, intradermally or subcutaneously.

  For parenteral administration, the active agent can be used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or monosaccharides to make the solution isotonic with blood.

  For buccal or sublingual administration, the active agent can be administered in the form of tablets or lozenges that can be formulated in conventional manner.

  For oral, parenteral, buccal, and sublingual administration to a subject (such as a patient), the dosage levels of the active agents and their pharmaceutically acceptable salts and solvates are typically 10 to 500 mg (single or Divided dose). Thus, by way of example, a tablet or capsule may contain 5-100 mg of active agent as needed to administer one or more at a time. As noted above, the physician will determine the actual dosage that is most appropriate for the individual patient, and that dosage will vary depending on the age, weight and response of the particular patient. It should be noted that although the above doses are examples of the average case, there are of course individual cases where higher or lower dose ranges are advantageous and they are within the scope of the present invention.

  Skilled physicians can easily determine the optimal route of administration and dosage for any particular patient, depending, for example, on the patient's age, weight and condition, and so the route of administration and dosage described are guidelines. Only.

  As used herein, the term treatment or therapy should be considered to include diagnostic and prophylactic applications.

  The treatment of the present invention includes both human and veterinary applications.

  The active agent can also be administered by any suitable means including, but not limited to, conventional syringes, needle-free injection devices, or “microprojectile impact gene guns”. Alternatively, active agents such as polynucleotides can be introduced into cells that have been removed from an individual by various means. Such means include, for example, ex vivo transfection, electroporation, nucleoporation, microinjection, and microprojectile bombardment. After the factor is taken up by the cells, the cells can be reimplanted into the individual. Furthermore, it is contemplated that cells that are otherwise non-immunogenic with an integrated genetic construct can be transplanted into an individual even if the vaccinated cells were originally harvested from another individual. The

  According to some preferred embodiments of the present invention, the active agent can be administered to an individual using a needleless injection device. For example, the active agent can be administered to an individual via an intradermal, subcutaneous, and / or intramuscular route using a needleless injection device, or similarly, for example, mucosal tissue of the respiratory tract, gastrointestinal tract, or urogenital tract Can be delivered to. Needleless injection devices are well known and widely available. Needleless injection devices are particularly suitable for delivering genetic material to tissue. Needleless injection devices are particularly useful for delivering genetic material to skin and muscle cells. In some embodiments, for example, a needleless injection device can be used to spray a liquid containing DNA molecules onto the surface of an individual's skin. This liquid is sprayed at a sufficient rate so that the liquid will penetrate the surface of the skin upon impact with the skin and into the underlying skin and / or muscle tissue. Thus, genetic material is administered simultaneously or selectively intradermally, subcutaneously, and intramuscularly. In some embodiments, needleless injection devices can be used to deliver genetic material to tissues of other organs in order to introduce nucleic acid molecules into cells of the organ.

  Preferably, the pharmaceutical preparation according to the invention is sterile and pyrogen-free.

Drug Administration Typically, a physician will determine the actual dosage that is most appropriate for an individual subject, and that dosage will vary depending on the age, weight and response of the particular patient. The following doses are examples of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.

  In one embodiment, it is understood that the therapeutic agents used in the present invention can be administered directly to a patient in vivo. Alternatively or in addition, the agent can be administered to immune cells such as T cells and / or APCs in an ex vivo manner. For example, white blood cells such as T cells or APCs can be obtained from a patient or donor in a known manner, treated / incubated ex vivo in the manner of the present invention, and then administered to the patient.

  In general, a therapeutically effective daily dose of an active agent conjugate according to the invention will be in the range of, for example, 0.01 to 50 mg / kg body weight of the subject to be treated, preferably 0.1 to 20 mg / kg. it can.

  A skilled physician can easily determine the optimal route of administration and dosage for any particular patient, for example depending on the age, weight and condition of the patient. Preferably, the pharmaceutical composition is in unit dosage form. The present invention includes both human and veterinary applications.

  “Simultaneously” means that the modulator of the Notch signaling pathway and the pathogen antigen, antigenic determinant, or polynucleotide encoding the pathogen antigen or antigenic determinant are administered together substantially simultaneously, preferably in the same formulation. Means that.

  “Simultaneously” means that the modulator of the Notch signaling pathway and the pathogen antigen, antigenic determinant, or polynucleotide encoding the pathogen antigen or antigenic determinant are administered at close times, For example, the pathogen antigen, antigenic determinant, or polynucleotide encoding the pathogen antigen or antigenic determinant is administered within about 1 minute to about 1 day before or after the modulator of the Notch signaling pathway is administered. Any contemporaneous time is useful. However, in many cases, when not administered simultaneously, the modulator of the Notch signaling pathway and the pathogen antigen, antigenic determinant, or polynucleotide encoding the pathogen antigen or antigenic determinant are within about 1 minute to about 8 hours. Preferably within about 1 hour to less than about 4 hours. When administered at the same time, the modulator of the Notch signaling pathway and the pathogen antigen, antigenic determinant, or polynucleotide encoding the pathogen antigen or antigenic determinant are preferably administered at the same site in the animal. The term “same site” includes the exact location, but can be within about 0.5 to about 15 cm, preferably within about 0.5 to about 5 cm.

  As used herein, the term “individually” refers to a modulator of the Notch signaling pathway and a pathogen antigen, antigenic determinant, or a polynucleotide encoding the pathogen antigen or antigenic determinant, at intervals, such as about It is meant to be administered at intervals of 1 day to several weeks or months. The active agents may be administered in any order.

  Further, the modulator of the Notch signaling pathway can be administered more frequently than the pathogen antigen, antigenic determinant, or polynucleotide encoding the pathogen antigen or antigenic determinant, or vice versa.

  As used herein, the term “sequential” refers to a modulator of a Notch signaling pathway and a pathogen antigen, an antigenic determinant, or a polynucleotide encoding the pathogen antigen or antigenic determinant, in order, for example, minutes, It is meant to be administered at intervals of hours, days, or weeks. Where appropriate, the active agent is administered in a regular repetitive cycle.

Vaccine Compositions Vaccine compositions and preparations according to the present invention are susceptible to or are susceptible to disease by administering said vaccine by a mucosal route such as oral / buccal / intestinal / vaginal / rectal or nasal route. It can be used to protect or treat animals. Such administration can be in droplet, spray, or dry powder form. Nebulized or aerosolized vaccine formulations can also be used where appropriate.

  Enteric preparations such as gastric resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration may also be used. The invention can also be used to enhance the immunogenicity of an antigen applied to the skin, for example, by intradermal, transdermal, or transdermal delivery. Furthermore, the adjuvants of the invention can be delivered parenterally, for example, by intramuscular or subcutaneous administration.

  Various delivery devices can be used depending on the route of administration. For example, in the case of intranasal administration, a spray device such as a commercially available Accuspray (Becton Dickinson) can be used.

  A preferred spray device for use in the nasal cavity is a device whose operation does not depend on the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is ejected from the nozzle only when the threshold pressure is reached. With these devices, a spray having a constant droplet diameter can be easily obtained. Pressure threshold devices suitable for use in the present invention are known in the art and are described, for example, in WO 91/13281 and EP 3118863B. Such an apparatus is commercially available from Pfeiffer GmbH.

  For certain vaccine formulations, the formulation can include other vaccine components. For example, the adjuvant formulation of the present invention can further comprise a bile acid or a derivative of cholic acid. Suitably, the derivative of cholic acid is its salt, for example the sodium salt. Examples of bile acids include cholic acid itself, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, taurodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, and the above bile acids glyco, tauro, amidopropyl-1- , Propanesulfonic acid, and amidopropyl-2-hydroxy-1-propanesulfonic acid derivatives, or derivatives such as N, N-bis (3D gluconamidopropyl) deoxycholamide.

  Suitably, the adjuvant formulation of the invention may be in the form of an aqueous solution or suspension in non-vesicular form. Such formulations are convenient for manufacturing and sterilization (eg, final filtration of 450 or 220 nm pore membranes).

  Suitably, the route of administration to the host is a mucosal surface, such as skin, intramuscular, or nasal mucosa. When the mixture is administered via the nasal mucosa, the mixture can be administered as a spray, for example. The method of enhancing the immune response can be either priming or boosting the vaccine.

  As used herein, the term “adjuvant” includes an agent that has the ability to enhance an immune response to an antigen or antigenic determinant of the immune system of a vertebrate subject.

  The term “immune response” includes any response to an antigen or antigenic determinant by the subject's immune system. Immune responses include, for example, humoral immune responses (eg, production of antigen-specific antibodies), and cell-mediated immune responses (eg, lymphocyte proliferation).

  The term “cell-mediated immune response” includes the immune defense provided by lymphocytes, such as the protection provided by T cell lymphocytes when they approach their sacrificial cells.

  When measuring “lymphocyte proliferation”, the ability of lymphocytes to proliferate in response to a particular antigen can be measured. Lymphocyte proliferation includes B cell, T helper cell, or CTL cell proliferation.

  The compositions of the present invention can be used to formulate vaccines containing antigens from a wide variety of sources. For example, antigens can include human, bacterial, or viral nucleic acids, pathogen-derived antigens or antigen preparations, host-derived antigens including GnRH and IgE peptides, recombinantly produced proteins or peptides, and chimeric fusion proteins. .

  Preferably, the vaccine formulation of the present invention contains an antigen or antigen composition capable of eliciting an immune response against human pathogens. The one or more antigens can be, for example, peptides / proteins, polysaccharides, and lipids, and can be derived from pathogens such as viruses, bacteria, and parasites / fungi as follows.

Viral antigens Viral antigens or antigenic determinants can be derived, for example, from:
Cytomegalovirus (especially human, such as gB or derivatives thereof), Epstein-Barr virus (such as gp350), flavivirus (such as yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), hepatitis virus, such as B Hepatitis B viruses (eg, hepatitis B surface antigens such as PreS1, PreS2, and S antigens described in EP-A-414374, EP-A-0304578, and EP-A-198474), hepatitis A virus, Hepatitis C virus, Hepatitis E virus, etc. HIV-1 (such as tat, nef, gp120, or gp160), human herpes virus, eg gD or its derivatives, or early proteins such as ICP27 from HSV1 or HSV2 , Human nipple Virus (eg HPV6, 11, 16, 18), influenza virus (all live or inactivated virus, egg or MDCK cells or Vero cells or split influenza virus grown on whole flurosomes (Gluck, Vaccine, 1992, 10, 915-920) Or purified or recombinant proteins thereof such as NP, NA, HA, or M protein), measles virus, mumps virus, parainfluenza virus, rabies virus, RS virus (F and G protein), rotavirus (including attenuated virus), variola virus, varicella-zoster virus (eg gpI, II, and IE63), and HPV virus that causes cervical cancer (eg protein D carrier and Combination of early proteins E6 or E7, L2 of the combination, or E6 or E7 forming protein D-E6 or E7 fusions from HPV16 engaged (e.g. see WO96 / 26277)).

Bacterial antigens Bacterial antigens or antigenic determinants can be derived, for example, from:
B. Bacillus spp containing anthracis (eg botulinum toxin); Bordetella spp containing pertussis (eg, pertactin, pertussis toxin, fibrillary hemagglutinin, adenylate cyclase, pili); burgdorferi (eg OspA, OspC, DbpA, DbpB), B.I. garinii (eg OspA, OspC, DbpA, DbpB), B.I. afzelii (eg OspA, OspC, DbpA, DbpB), B.I. andersonii (eg, OspA, OspC, DbpA, DbpB), B.I. Borrelia spp including hermsii; C.I. jejuni (eg, toxins, attachment factors, and invasin) and C.I. Campylobacter spp containing E. coli; trachomatis (eg MOMP, heparin binding protein), C.I. pneumoniae (eg MOMP, heparin binding protein), C.I. Chlamydia spp containing psittaci; tetani (such as tetanus toxin), C.I. botulinum (eg botulinum toxin), C.I. C. difficile (eg, Clostridial toxin A or B) Clostridium spp; Corynebacterium spp containing diphtheriae (eg, diphtheria toxin); Ehrlicia spp containing factors of Equi and human Granulolytic Ehrlichosis; Rickettsia spp including rickettsii; faecalis, E .; Enterococcus spp containing faecium; E. coli (eg colonization factor, non-thermostable toxin or derivative thereof, heat stable toxin), enterohemorrhagic E. coli. E. coli, enteropathogenic E. coli. Escherichia spp containing E. coli (eg, Shiga toxin-like toxin); influenza B type (for example, PRP), atypical H.P. Haemophilus spp including H. influenzae, such as OMP26, high molecular weight attachment factor, P5, P6, protein D and lipoprotein D, fimbrin and fimbrin derived peptides (see, eg, US5843464); Helicobacter spp containing Pylori (eg urease, catalase, vacuolating toxin); Pseudomonas spp containing aeruginosa; Legionella spp containing pneumophila; Leptospira spp containing interrogans; Listeria spp including monocytogenes; M. also called Branhamella catarrhalis. Moraxella spp including catarrhalis (eg, high and low molecular weight attachment factors and invasin); Morexella Catarrhalis (including its outer membrane vesicles and OMP106 (see, eg, WO 97/41731)); tuberculosis (eg, ESAT6, antigen 85A, B, or C), M. et al. bovis, M.M. leprae, M.M. avium, M.M. paratuberculosis, M. et al. Mycobacterium spp containing smegmatis; gonorrhea and N.H. Neisseria spp including meningitidis (eg capsular polysaccharide and its conjugates, transferrin binding protein, lactoferrin binding protein, PilC, adhesion factor); Neisseria mengidis B (its outer membrane vesicles and NspA (see eg WO 96/29412)) S). typhi, S.M. paraphyfi, S .; choleraesuis, S .; Salmonella spp containing S. enteritidis; sonnei, S .; dysenteriae, S. et al. Shigella spp containing flexnerii; aureus, S .; Staphylococcus spp containing epidermidis; pneumoniae (eg capsular polysaccharide and its conjugates, PsaA, PspA, streptolysin, choline-binding protein), and protein antigen Pneumolysin (Biochem Biophys Acta, 1989, 67, 1007, Rubins et al., Microbiological Pathogenesis, 25, 337-334). And Streptococcus spp, including mutant detoxified derivatives thereof (see, eg, WO 90/06951, WO 99/03884); Pallidum (eg outer membrane protein), T. denticola, T.W. Treponema spp containing hyodysenteriae; Vibrio spp containing cholera (eg cholera toxin); enterocoliticica (eg, Yop protein), Y. et al. pestis, Y. et al. Yersinia spp containing pseudotuberculosis.

Parasite / fungal antigens Parasite / fungal antigens or antigenic determinants can be derived, for example, from:
B. Babesia spp containing microti; Candida spp containing C. albicans; Cryptococcus spp containing neoformans; Entamoebba spp containing histolytica; Giardia spp containing lamblia; Lessmania spp including major; Plasmodium faciparum (MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2, Sequestrin, PfEMMP1, Pf332, LSA1, LSA3, STARP, SALP, ESP / 45, Pfs230, and their analogs of Plasmodium spp); Pneumocystis spp containing S. carinii; Schistoma spp containing Mansoni; Trichomonas spp containing Vaginalis; Toxoplasma spp including Gondi (eg, SAG2, SAG3, Tg34); Trypanosoma spp containing cruzi.

  Approved / approved vaccines include, for example, anthrax vaccines such as Biothrax (BioPort Corp); tuberculosis (BCG) vaccines such as TICE BCG (Organon Teknika Corp) and Mycobax (Aventis Pasteur, Ltd); Tripediate (Aventis P Diphtheria, tetanus, cell-free pertussis (DTP) vaccines such as Infanrix (GlaxoSmithKline), and DAPTACEL (Aventis Pasteur, Ltd); Haemophilus b conjugate vaccines (eg, Lederle Lab DiverCiMITH Gates, meningococcal protein conjugates such as PedvaxHIB from Merck & Co, Inc, tetanus toxoid conjugates such as ActHIB, Aventis Pasteur, SA; hepatitis such as Havrix (GlaxoSmithKline) and VAQTA (Merck & Co, Inc x); Hepatitis A and Hepatitis B (recombinant) combined vaccines such as (GlaxoSmithKline); Recombinax HB (Merck & Co, Inc) and recombinant hepatitis B vaccines such as Engerix-B (GlaxoSmithKline); Fluvirin (EvansVaccineFlucineW) Laboratories, Inc), and Fluzone ( influenza virus vaccines such as Ventis Pasteur, Inc); Japanese encephalitis virus vaccines such as JE-Vax (Research Foundation for Microbiology of Osaka University); Attenuvax (Merck & Co, Inc) M vaccine; , Inc), measles, mumps virus vaccine; M-M-RII (Merck & Co, Inc), measles, mumps, rubella virus vaccine; Menomune-A / C / Y / W -Meningococcal polysaccharide vaccines such as -135 (Aventis Pasteur, Inc) (mixed A, C, Y, and W-135 groups); Mump Epidemic parotiditis virus vaccines such as svax (Merck & Co, Inc); For example, diphtheria CRM197 protein conjugates such as Lederle Lab Div, American Cyanamid Co's Prevnar; poliovirus vaccines such as Poliovax (Aventis Pasteur, Ltd); polio virus vaccines such as IPOL (Aventis Pasteur, SA); , SA) and RabAvert (C rabies vaccines such as hiron Behring GmbH &Co; rubella virus vaccines such as Meruvax II (Merck & Co, Inc); typhoid Vi polysaccharide vaccines such as TYPHIM Vi (Aventis Pasteur, SA); -Yellow fever vaccines such as Vax (Aventis Pasteur, Inc) are included.

  In accordance with this aspect of the invention, it is understood that antigens and antigenic determinants may be used in many different forms. For example, antigens and antigenic determinants may exist as isolated proteins or peptides (eg, so-called “subunit vaccines”), or as cell-related or virus-related antigens or antigenic determinants (eg, live or dead pathogen strains). it can. Live pathogens are preferably attenuated in a known manner. Alternatively, an antigen or antigenic determinant can be produced in situ in a subject using a polynucleotide encoding the antigen or antigenic determinant (so-called “DNA vaccination”, but discussed above) Thus, it is understood that the polynucleotides that can be used in this approach are not limited to DNA, but may include RNA and modified polynucleotides).

  As used herein, the term “genetic vaccine” refers to a pharmaceutical formulation comprising a polynucleotide (eg, DNA). Genetic vaccines include pharmaceutical formulations useful for eliciting prophylactic and / or therapeutic immune responses. The therapeutic vaccine is also referred to as “pharmactin”.

  As discussed in, for example, US6025341, direct injection of polynucleotides such as DNA is a promising method of delivering antigens for immunization (Barry et al., BioTechniques, 1994, 16, 616-619, Davis et al., Hum. Mol. Genet. 1993, 11, 1847-1851, Tang et al., Nature, 1992, 356, 152-154, Wang et al., J. Virol. 1993, 67, 3338-3344, and Wolf et al., Science. 1990, 247, 1465-1468). This approach has been successfully used to generate protective immunity against influenza virus in mice and chickens, against bovine herpesvirus 1 in mice and cows, and against rabies virus in mice (Cox et al. J. Virol. 1993, 67, 5664-5667, Fynan et al., DNA and Cell Biol. 1993, 12, 785-789, Ulmer et al., Science, 1993, 259, 1745-1749, and Xiang et al., Virol. 1994, 199, 132-140). In most cases, powerful but highly variable antibodies and cytotoxic T cell responses were associated with infection control. Indeed, the potential to generate long-term memory CTL without the use of liver vectors makes this approach particularly attractive compared to approaches involving inactivated vaccines, and the generation of CTL responses protects against acute infections. As well as potentially eradicating persistent viral infections (Wolff et al., Science, 1990, 247, 1465-1468, Wolff et al., Hum. Mol. Genet. 1992, 1, 363-369, Manthorpe Et al., Human Gene Therapy, 1993, 4, 419-431, Ulmer et al., Science, 1993, 259, 1745-1749, Yankuckas et al., DNA and Cell Biol. 1993, 12, 777-783, Montgomery et al., DNA nd Cell Biol. 1993, 12, 777-783, Fynan et al., DNA and Cell Biol. 1993, 12, 785-789, Wang et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 4156-4160, Wang DNA and Cell Biol. 1993, 12, 799-805, Xiang et al., Virol. 1994, 199, 132-140, and Davis et al., Hum. Mol. Genet. 1993, 11, 1847-1851), its HCV and HBV is a serious human disease worldwide.

  Suitable genetic vaccines for use in accordance with the present invention include, for example, about 1 nanogram to about 1000 micrograms of polynucleotide, such as DNA, suitably about 10 nanograms to about 800 micrograms, suitably about 0.1 to about 500. A microgram, suitably about 1 to about 350 micrograms, suitably about 25 to about 250 micrograms of polynucleotide such as DNA.

  The amount of protein in the vaccine dose is selected as the amount that induces an immune defense response in a typical recipient without significant adverse side effects. Such amount will depend on which particular immunogen is used and how it is presented. Typically, each dose is expected to contain 1-1000 μg, preferably 1-500 μg, preferably 1-100 μg, most preferably 1-50 μg of protein. After the initial vaccination, the subject can receive one or several booster immunizations at appropriate intervals.

  The vaccine of the present invention can also be administered orally. In such cases, pharmaceutically acceptable excipients also include alkaline buffers, or enteric capsules or microgranules. The vaccine of the present invention can also be administered by the vaginal route. In such cases, pharmaceutically acceptable excipients include emulsifiers, polymers such as CARBOPOL, vaginal creams and other known stabilizers of suppositories. The vaccines of the present invention can also be administered by the rectal route. In such cases, excipients include waxes and polymers known in the art for the formation of rectal suppositories.

  The formulations of the present invention can be used for both prophylactic and therapeutic purposes. Accordingly, the present invention provides a method of treating a mammal susceptible to or suffering from an infection. In other aspects of the invention, there are provided adjuvant combinations and vaccines as described herein for use in medicine. Vaccine preparations are generally described in New Trends and Developments in Vaccines, Voller et al., University Park Press, Baltimore, Maryland, U.S. Pat. S. A. 1978.

  It will be appreciated that the adjuvants of the present invention may further be combined with other adjuvants, such as cholera toxin and its B subunit, E.I. Coli heat labile enterotoxin LT, its B subunit LTB, and its detoxified forms such as mLT, immunologically active saponin fractions such as Quil A and its derivatives from the bark of the South American tree Quillaja Saponaria Molina (eg QS21, described in US Pat. No. 5,057,540), oligonucleotide adjuvant system CpG (described in WO96 / 02555), in particular 5′TCG TCG TTT TGT CGT TTT GTC GTT3 (SEQ ID NO: 1), and monophosphoryl lipid A and its non-toxic derivative 3-O -Deacylated monophosphoryl lipid A (3D-MPL, described in GB 2220211) is included.

  The present invention provides an increase in the magnitude and / or duration of the immune response. Preferably, the present invention provides an increased protective immune response.

  The present invention further contemplates generating selective Th1 or Th2 immunity. In general, T cells can act in different subpopulations that exhibit different effector functions. The T cell response can be inflammatory T helper type 1 (Th1), characterized by secretion of interferon gamma (IFN-gamma) and interleukin 2 (IL-2). Although Th1 cells are cytoprotective helper cells, they are of little use for antibody secretion. Other types of T cell responses are generally anti-inflammatory and produce IL-4, IL-5, and IL-10, but are mediated by Th2 cells that produce little or no IL-2 or IFN-gamma. Is done. Th2 cells are antibody-producing helper cells. Both CD4 + and CD8 + cells occur in these subpopulations. Th1 / Th2: CD4, Tc1 / Tc2: CD8.

  For each type of pathogen / infection, an “appropriate” (and different) type of T cell response (eg, Th1 vs. Th2, CD4 + vs. CD8 +) that counters infectious agents in the subject but does not cause excessive tissue damage. ) May exist. If an “inappropriate” type of T cell response is induced (Th1, not Th2, or vice versa), it can be harmful to the subject. In general, it is desirable that a Th1 response clears intracellular pathogens such as viruses or intracellular bacteria, and a Th2 response clears extracellular pathogens.

  It is understood that the present invention may be used for both so-called prophylactic vaccines and so-called therapeutic vaccines.

  For example, prophylactic vaccines can be used to provide non-infected subjects with protective immunity and provide protection against the establishment of future infections.

  Conversely, therapeutic vaccines can be used to increase the immune response to an infection, for example after an infection has been established (eg, acute or chronic infection). Suitably, therapeutic vaccines can be used to combat chronic infections, eg, bacterial infections (such as tuberculosis), parasitic infections such as malaria infections, or viral infections (such as HPV, HCV, HBV, or HIV infection). Can be.

  Examples of chronic infections with significant morbidity and early death cause human hepatitis viruses such as hepatitis A, B, C, D, and E, such as chronic hepatitis, cirrhosis, and liver cancer Hepatitis B virus (HBV) and hepatitis C virus (HCV) are included.

  Further examples of chronic infections caused by viral infectious agents include human retroviruses, human immunodeficiency viruses (HIV-1 and HIV-2) that cause acquired immunodeficiency syndrome (AIDS), T cell leukemia and myelopathy And those caused by human T lymphocyte-tropic viruses (HTLV-1 and HTLV-2). Human herpesviruses, including herpes simplex virus (HSV) types 1 and 2, Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6) Many other infections such as) are often not eradicated by host mechanisms, become chronic and can cause disease in that state. Chronic infection with human papillomavirus is associated with cervical cancer. Numerous other viruses and other infectious agents replicate in the cell and can become chronic when the host defense mechanisms cannot eliminate them. They include pathogenic protozoa (eg Pneumocystis carinii, Trypanosoma, Leishmania, Plasmodium (causes of malaria), and Toxoplasma gondii), bacteria (eg, Mycobacteria causing tuberculosis, Salmonella terrier, ), And fungi (eg, Candida and Aspergillus).

  A pathogen antigen is suitably an antigen naturally encoded by a pathogen for which an enhanced or increased immune response is desired.

  Numerous bacterial, protozoan, and viral nucleotide sequences, including different species, strains, and isolates, are known in the art (eg, Levy, Microbiological Reviews, 57: 183-289 (1993) (HIV)). Choo et al., Seminars in Liver Disease, 12: 279-288 (1992) (HCV)). Particularly suitable target antigens are those that induce T cell responses, particularly CTL responses, during infection. These can include, for example, HBV core antigen (HBcAg) E antigen, surface antigen (HBsAg). The polynucleotide sequences of HBsAg, including various surface antigen subtypes of pre-S1, pre-S2, and S regions, are well known in the art (eg, Okamoto et al., J. Gen. Virol. 67: 1383). ~ 1389 (1986), GenBank accession numbers D00329 and D00330). Polynucleotide sequences encoding the HIV glycoprotein gp160 and other antigenic HIV regions are known in the art (Lautenberger et al., Nature, 313: 277-284 (1985), Starrich et al., Cell, 45: 637-648 (1986), Wiley et al., Proc. Natl. Acad. Sci. USA, 83: 5038-5042 (1986), and Modrow et al., J. Virol. 61: 570-578 (1987)).

  For example, the human immunodeficiency virus type 1 genome (HXB2, HIV1 / HTLV-III / LAV reference genome) is provided in GenBank accession number K03455, and the sequences of various HIV antigen proteins have been reported.

  Numerous genomic sequences (including antigenic protein sequences) of HAV, HBV, and HCV strains are available from GenBank, for example AY057948 (hepatitis B virus isolated Tibet 127, whole genome), AY0579947 (hepatitis B virus isolated Tibet 705, Whole genome), NC_003977 (hepatitis B virus, whole genome), NC_004102 (hepatitis C virus, whole genome), AF139594 (hepatitis C virus strain HCV-N, whole genome), M16632 (hepatitis A virus strain (HM) -175 strain, attenuated)).

  In one embodiment, a modulator / inhibitor of Notch signaling increases a cytotoxic (CD8 +) T cell response to an antigen.

As described above, the present invention further provides a conjugate comprising a first and second sequence, the first sequence comprising a pathogen antigen, or a polynucleotide encoding such an antigen, The second sequence includes a polypeptide or polynucleotide for modulating Notch signaling. The conjugates of the invention can be protein / polypeptide or polynucleotide conjugates.

  Where the conjugate is a polynucleotide conjugate, suitably a polynucleotide such as a plasmid comprising a polynucleotide sequence encoding a pathogen antigen or antigenic determinant and a polynucleotide sequence encoding a modulator of the Notch signaling pathway It can take the form of a vector, preferably each sequence is operably linked to regulatory elements required for expression in eukaryotic cells. An outline of such a form of vector is shown in FIG.

  Suitably, the polynucleotide sequence encoding a modulator of the Notch signaling pathway is a nucleotide sequence encoding a Notch ligand, such as Delta1, Delta3, Delta4, Jagged1, or Jagged2, or the biological activity of such a sequence. It can be a fragment, derivative or homologue. Where human therapy is intended, sequences based on human sequences can suitably be used.

  Preferably, the polynucleotide sequence encoding the modulator of the Notch signaling pathway comprises a Notch ligand DSL domain and at least 1 to 20, suitably at least 2 to 15, suitably at least 2 to 10, such as at least 3 to 8 The nucleotide sequence encoding the EGF-like domain. Suitably, the DSL and EGF-like domain sequences are mammalian sequences or correspond to mammalian sequences. Suitably, the polynucleotide sequence encoding a modulator of the Notch signaling pathway may further comprise a transmembrane domain, and suitably a Notch ligand intracellular domain. Preferred sequences include human sequences such as human Delta1, Delta3, Delta4, Jagged1 or Jagged2 sequences.

  If desired, the polynucleotide sequence encoding the pathogen antigen or antigenic determinant can further include a nucleotide sequence encoding a signal sequence that directs transport of the antigen or antigenic determinant within the administered cell. For example, such signal sequences can direct antigens or antigenic determinants that are secreted or localized in the cytoplasm, cell membrane, endoplasmic reticulum, or lysosome.

  Regulators of DNA expression include promoters and polyadenylation signals. In addition, other factors such as the Kozak region can be included if desired. Initiation and termination signals are regulatory elements that are often considered part of the coding sequence.

  Examples of suitable promoters include, but are not limited to, human immunodeficiency viruses (HIV) such as simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, HIV long terminal repeat (LTR) promoter. ), Moloney virus, ALV, CMV early promoter such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoter, and human actin, human myosin, human hemoglobin, human muscle creatine And promoters derived from human genes such as human metallothionein. Tissue specific promoters specific for lymphocytes, dendritic cells, skin, brain cells, intraocular epithelial cells are particularly preferred, for example CD2, CD11c, keratin, Wnt-1 and rhodopsin promoters, respectively. Suitably, an epithelial cell promoter such as SPC may be used.

  Examples of suitable polyadenylation signals include, but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. For example, the SV40 polyadenylation signal used in plasmid pCEP4 (Invitrogen, San Diego Calif.), Referred to as the SV40 polyadenylation signal, can be used.

  In addition to the regulatory factors required for DNA expression, the conjugate can also include other factors. Such additional factors include enhancers that can be selected from, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers derived from CMV, RSV, EBV, and the like.

  When administered and taken up by a cell, the nucleotide conjugate can remain present in the cell, for example, as a functional extrachromosomal molecule and / or be incorporated into the chromosomal DNA of the cell. DNA can be introduced into cells where it remains genetic material isolated in the form of one or more plasmids. Alternatively, linear DNA that can be integrated into the chromosome can be introduced into the cell. When the DNA is introduced into the cell, a reagent that promotes integration of the DNA into the chromosome can be added. DNA sequences useful for facilitating integration can also be included in the DNA molecule. Alternatively, RNA can be administered to the cells. For example, the conjugate can be provided in the form of a minichromosome comprising a centromere, a telomere, and an origin of replication.

  If desired, the conjugate can include a mammalian origin of replication to maintain the construct extrachromosomally and produce multiple copies of the construct within the cell. For example, plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) Contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region that produces large copies of episomal replication without integration.

  To maximize protein production, regulatory sequences suitable for gene expression in the cell type to which the construct is administered can be selected. In addition, the codon that is most efficiently transcribed in the cell can be selected.

  Such conjugates can be used in vivo or ex vivo by a “gene vaccination” approach to provide cells or tissues with the expression of both inhibitors of Notch signaling and pathogen antigens or antigenic determinants. it can.

Accelerator In some embodiments, the polynucleotide can be delivered in conjunction with administration of the accelerator. Accelerators administered in conjunction with nucleic acid molecules can be administered as a mixture with nucleic acid molecules, or can be administered separately at the same time, prior to administration, or after administration of nucleic acid molecules. Examples of accelerators include benzoic acid esters, anilides, amidines, urethanes, and hydrochlorides thereof, such as local anesthetics.

  Examples of esters include benzoic acid esters such as piperocaine, meprilucaine, and isobucaine, paraaminobenzoic acid esters such as procaine, tetracaine, butetamine, propoxycaine, and methacrylocaine, including metabutamine and primacaine, Paraethoxybenzoic acid esters such as parethoxycaine are included. Examples of anilide include lidocaine, etidocaine, mepivacaine, bupivacaine, pilocaine, and prilocaine. Other examples of such compounds include dibucaine, benzocaine, dichronin, pramoxine, proparacaine, butacaine, benoxinate, carbocaine, methyl bupivacaine, butasin picrate, phenacine, diothane, lucaine, intracaine, Included are nupelcaine, metabutoxycaine, pyridocaine, biphenamine, and bicyclics of plant origin, such as cocaine, cinnamoylcocaine, truxillin, and cocaethylene, and all those compounds complexed with hydrochloride.

  The enhancer can be administered prior to, simultaneously with, or following the gene construct. Accelerators and gene constructs can be formulated in the same composition.

  Bupivacaine-HCl is chemically represented as 2-piperidinecarboxamide, 1-butyl-N- (2,6-dimethylphenyl) monohydrochloride monohydrate and is sold by Astra Pharmaceutical Products Inc. (Westboro, Mass), Sanofi Wintrop Pharmaceuticals (New York, NY), Eastman Kodak (Rochester, NY) and are widely available for drug use. Bupivacaine is formulated commercially with or without methylparaben and with or without epinephrine. Any such formulation can be used. It is commercially available at concentrations of 0.25%, 0.5%, and 0.75% for use as a drug and can be used in the present invention. If desired, other concentrations eliciting the desired effect can be prepared, in particular from 0.05% to 1.0%. Suitably, for example, about 250 μg to about 10 mg of bupivacaine can be administered.

Antigen presenting cells If desired, antigen presenting cells (APCs) can be “professional” antigen presenting cells or other cells that can be induced to present antigens to T cells. Alternatively, APC precursors that differentiate or are activated under culture conditions that produce APC can be used. The APC used in the ex vivo method of the invention is typically isolated from tumors or peripheral blood found in the patient's body. Preferably, the APC or precursor is human. However, if APC is used in a preliminary in vitro screening procedure to identify and test a suitable nucleic acid sequence, APC from any suitable source, such as a healthy patient, can be used.

APC can be activated by transfection of dendritic cells (DC), such as interconnected DCs or follicle DCs, Langerhans cells, PBMCs, macrophages, B lymphocytes, or epithelial cells, fibroblasts, or endothelial cells. Other cell types that are engineered to express MHC molecules (class I or II) on their surface are included. APC precursors include CD34 ⁺ cells, monocytes, fibroblasts, and endothelial cells. APCs or precursors can be modified depending on the culture conditions or, for example, selected cytokine combinations that promote antigen presentation and / or immune synergy (eg, IL-2, IL-12, IFN-γ, TNF- It can also be genetically modified by transfection of one or more genes encoding proteins that play a role in α, IL-18, etc.). Such proteins include MHC molecules (class I or II), CD80, CD86, or CD40. Most preferably, DC or a DC precursor is included as a source of APC.

Dendritic cells (DCs) can be isolated / prepared by several means, for example, dendritic cells are purified directly from peripheral blood or transferred to peripheral blood, eg, by treatment with GM-CSF, and then CD34 ⁺ It can arise directly from precursor cells or from bone marrow. Adhesion precursors from peripheral blood can be treated with a GM-CSF / IL-4 mixture (Inaba K et al. (1992) J. Exp. Med. 175: 1157-1167 (Inaba)) or non-derived from bone marrow. Adherent CD34 ⁺ cells can be treated with GM-CSF and TNF-α (Caux C et al. (1992) Nature 360: 258-261 (Caux)). DC uses purified peripheral blood mononuclear cells (PBMC) and treats adherent cells with GM-CSF and IL-4 for 2 hours Salusto and Lanzavecchia (Sallusto F and Lanzvecchia A (1994) J. Exp. Med. 179: Similarly to the method of 1109 to 1118), it can also be conventionally prepared from the peripheral blood of human volunteers. If necessary, they can remove CD19 ⁺ B cells, and CD3 ⁺ , CD2 ⁺ T cells using magnetic beads (Coffin RS et al. (1998) Gene Therapy 5: 718-722 (Coffin)). Culture conditions may include the activity of other cytokines such as GM-CSF or IL-4 to maintain, or dendritic cells or other antigen presenting cells.

  Accordingly, it is understood herein that the term “antigen-presenting cell or the like” is not limited to APC. Those skilled in the art will appreciate that any vehicle that can be presented to a T cell population can be used, but for convenience we use the term APC to refer to all of these. As noted above, preferred examples of suitable APCs include synthetic APCs such as dendritic cells, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B cells, or lipid membranes.

T cells can be used as needed from any suitable source, such as a healthy patient, and can be obtained from blood or other sources (such as lymph nodes, spleen, or bone marrow). T cells can optionally be enriched or purified by standard procedures. T cells can be used in combination with other immune cells obtained from the same or different individuals. Alternatively, whole blood, leukocyte-enriched blood or purified leukocytes can be used as a source of T cells and other cell types. The use of helper T cells (CD4 ⁺ ) is particularly preferred. Alternatively, other T cells such as CD8 ⁺ cells can be used. It is also convenient to use a cell line such as a T cell hybridoma.

  Accordingly, it is understood herein that the term “antigen-presenting cell or the like” is not limited to APC. Those skilled in the art will appreciate that any vehicle that can be presented to a T cell population can be used, but for convenience we use the term APC to refer to all of these. As noted above, preferred examples of suitable APCs include synthetic APCs such as dendritic cells, L cells, hybridomas, fibroblasts, lymphomas, macrophages, B cells, or lipid membranes.

Exposure of factors to APC and T cells T cells / APC / tumor cells can be cultured as described above. APC / T cells / tumor cells can be incubated / exposed to substances that can prevent or down-regulate Notch or Notch ligand expression. The resulting T cells / APC / tumor cells with Notch or Notch ligand expression down-regulated are ready for use. For example, it can be prepared for administration to a patient or can be incubated with T cells in vitro (ex vivo).

  For example, tumor material is isolated and transfected with a nucleic acid sequence encoding, for example, a Toll-like receptor or BMP receptor and / or a costimulatory molecule (suitable costimulators are described above) and / or -Treated with cytokines such as γ, TNF-α, IL-12, and then used in vitro to prime TRL and / or TIL cells.

When treated ex vivo, the modified cells of the invention are administered to the host, preferably by direct injection into the patient's lymph nodes. Typically, 10 ⁴ to 10 ⁸ treated cells, preferably 10 ⁵ to 10 ⁷ cells, more preferably about 10 ⁶ cells are administered to the patient. Preferably, the cells take the form of an enriched cell population.

  As used herein, the term “enrichment” as applied to a cell population of the invention refers to a more homogeneous cell population that has fewer other cells with which it is naturally associated. An enriched population of cells can be obtained by several methods known in the art. For example, an enriched population of T cells can be obtained using immunoaffinity chromatography using monoclonal antibodies specific for determinants found only in T cells.

  Enriched populations can also be obtained from mixed cell suspensions by positive selection (collecting only desired cells) or negative selection (removing unwanted cells). Techniques for obtaining specific cells of affinity material are well known in the art (Wigzel et al., J. Exp. Med. 128: 23, 1969, Mage et al., J. Immunol. Meth. 15:47, 1977). Wysocki et al., Proc. Natl. Acad. Sci. USA 75: 2844, 1978, Schrempf-Decker et al., J. Immunol Meth. 32: 285, 1980, Muller-Sieburg et al., Cell 44: 653. 1986).

  Monoclonal antibodies against antigens specific for mature differentiated cells are used in various negative selection methods to remove unwanted cells, eg, deplete T cells or malignant cells from allogeneic or autologous bone marrow grafts, respectively. (Gee et al., JNCI 80: 154, 1988). Purification of human hematopoietic cells by negative selection using monoclonal antibodies and immunomagnetic microspheres can be achieved using multiple monoclonal antibodies (Griffin et al., Blood, 63: 904, 1984).

  Procedures for separating cells include magnetic separation using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents combined with or combined with monoclonal antibodies, such as complement and cytotoxins, and solid matrices such as plates. “Panning” using other antibodies, or other convenient techniques can be included. Techniques that provide accurate separation include fluorescence activated cell separation devices, which have varying degrees of accuracy, such as multiple color channels, small and obtuse light scattering detection channels, impedance channels, and the like.

  In one embodiment, it is understood that the therapeutic agents used in the present invention can be administered directly to a patient in vivo. Alternatively or in addition, the agent can be administered to cells such as T cells and / or APCs in an ex vivo manner. For example, white blood cells such as T cells or APCs can be obtained from a patient or donor in a known manner, treated / incubated ex vivo in the manner of the present invention, and then administered to the patient. It will be further appreciated that combinations of routes of administration may be used if desired. For example, one suitable component (such as a modulator of Notch signaling) can be administered ex vivo and the other component can be administered in vivo, or vice versa.

Introduction of nucleic acid sequences into APCs and T cells The T cells and APCs described above are optionally cultured in a suitable medium such as DMEM or other defined medium in the presence of fetal calf serum.

  A polypeptide substance is introduced into a T cell and / or APC by introducing a nucleic acid construct / viral vector encoding the polypeptide into the cell under conditions that allow expression of the polypeptide in the T cell and / or APC. Can be administered. Similarly, a nucleic acid construct encoding an antisense construct can be introduced into T cells and / or APCs by transfection, viral infection, or viral transduction.

  In a preferred embodiment, the nucleotide sequence encoding a modulator of Notch signaling is operably linked to a control sequence, including promoter / enhancer and other expression control signals. The term “operably linked” means that the components described are in a relationship in which they can function in the intended manner. A regulatory sequence “operably linked” to a coding sequence is preferably ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

  The promoter is typically selected from promoters that function in mammalian cells, although eukaryotic promoters and promoters that function in other eukaryotic cells can also be used. The promoter is typically derived from a viral or eukaryotic gene promoter sequence. For example, it can be obtained from the genome of the cell in which expression occurs. For eukaryotic promoters, it must be a promoter that functions in an ubiquitous manner (a-actin, b-actin, tubulin promoter, etc.) or tissue-specific manner (eg, pyruvate kinase gene promoter). Can do. Tissue specific promoters specific for lymphocytes, dendritic cells, skin, brain cells, and intraocular epithelial cells are particularly preferred, such as CD2, CD11c, keratin 14, Wnt-1, and rhodopsin promoters, respectively. Preferably, the epithelial cell promoter SPC is used. It may be a promoter that responds to a specific stimulus, such as a promoter that binds to a steroid hormone receptor. Viral promoters can also be used, such as the Moloney murine leukemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter, or the human cytomegalovirus (CMV) IE promoter.

  It may also be advantageous that the promoter is inducible so that the expression level of the heterologous gene can be regulated during the lifetime of the cell. Inducible means that the level of expression obtained using a promoter can be regulated.

  Any of the promoters described above can be modified by adding additional regulatory sequences, such as enhancer sequences. Chimeric promoters comprising sequence elements of two or more different promoters can also be used.

  Alternatively (or in addition) the regulatory sequence can be cell specific so that the gene of interest is expressed only in the cells used in the present invention. Such cells include, for example, APC and T cells.

  Resulting T cells and / or APCs containing nucleic acid constructs that can up-regulate Notch ligand expression are ready for use. If necessary, small aliquots of cells can be tested for upregulation of Notch ligand expression as described above. The cells can be prepared for administration to a patient or can be incubated with T cells in vitro (ex vivo).

  Any of the assays described above (see “Assays”) can be adapted to monitor or detect responsiveness in immune cells for use in clinical applications. Such assays include, for example, detection of Notch ligand activity in host cells, or monitoring of Notch cleavage in donor cells. Additional methods for monitoring immune cell activity are described below.

  Immune cell activity can be monitored by any suitable method known to those skilled in the art. For example, cytotoxic activity can be monitored. Natural killer (NK) cells demonstrate enhanced cytotoxic activity after activation. Thus, reduction or stabilization of cytotoxicity suggests a reduction in responsiveness.

  After activation, leukocytes express a variety of new cell surface antigens. NK cells express, for example, transferrin receptor, HLA-DR, and CD25 IL-2 receptor after activation. Therefore, a reduction in responsiveness can be assessed by monitoring the expression of these antigens.

  Hara et al., Human T-cell Activation: III, Rapid Induction of a Phosphorylated 28 kD / 32 kD Displaced Linked Early Activation Anti (3-A-1) Exp. Med. 164: 1988 (1986) and Cosulich et al., Functional Charactorization of an Antigen (MLR3) Involved in an Early Step of T-Cell Activation, PNAS, 84: 4205 (1987) activated in T cells. A surface antigen is described. These antigens, EA-1 and MLR3 are glycoproteins having major components of 28 kD and 32 kD, respectively. EA-1 and MLR3 are not HLA class II antigens, and MLR3 Mab blocks IL-1 binding. These antigens appear on activated T cells within 18 hours and can therefore be used to monitor immune cell responsiveness.

  In addition, leukocyte responsiveness can be monitored as described in EP0325489, which is hereby incorporated by reference. Briefly, this is done using a monoclonal antibody that interacts with a cellular antigen recognized by the monoclonal antibody produced by the hybridoma designated ATCC No. HB-9627 (“Anti-Leu23”).

  Anti-Leu23 recognizes cell surface antigens of activated and antigen-stimulated leukocytes. In activated NK cells, the antigen Leu23 is expressed within 4 hours after activation, and continues to be expressed for 72 hours after activation if it is longer. Leu23 is a disulfide-linked homodimer consisting of a 24 kD subunit with at least two N-linked carbohydrates.

  Since the appearance of Leu23 in NK cells correlates with the occurrence of cytotoxicity, and the appearance of Leu23 in certain T cells correlates with stimulation of the T cell antigen receptor complex, Anti-Leu23 is a leukocyte response. Useful for monitoring sex.

  Further details of techniques for monitoring immune cell responsiveness can be found in “The Natural Killer Cell” Lewis C., which is incorporated herein by reference. E. And J.A. O'D. McGee, 1992, Oxford University Press, Trichieri G. “Biology of Natural Killer Cells” Adv. Immunol. 1989, 47, pp. 187-376, Chapter 7 of “Handbook of the Immunity Response Genes” “Cytokines of the Immunity Response”, Mak T. et al. W. And J.A. J. et al. L. Can be found in Simard, 1998.

Preparation of primed APCs and lymphocytes According to one aspect of the present invention, immune cells can be used to present antigens or allergens and / or expression of Notch, Notch ligand, or Notch signaling pathway or It can be processed to modulate the interaction. Thus, for example, antigen presenting cells (APC) can be cultured in a suitable medium such as DMEM or other defined medium, optionally in the presence of serum such as fetal calf serum. The optimal cytokine concentration can be determined by titration. Typically, one or more substances capable of up-regulating or down-regulating the Notch signaling pathway are then added to the medium along with the antigen of interest. The antigen can be added before, after, or substantially simultaneously with the substance. The cells are typically incubated at 37 ° C. with the substance and antigen for at least 1 hour, preferably at least 3 hours. If necessary, small aliquots of cells can be tested for regulated target gene expression as described above. Alternatively, cellular activity can be measured by inhibition of T cell activation by monitoring surface markers, cytokine secretion or proliferation, as described in WO 98/20142. For example, APC transfected with a nucleic acid construct that directs the expression of Serrate can be used as a control.

  As discussed above, administering a polypeptide substance to an APC by introducing into the cell a nucleic acid construct / viral vector encoding the polypeptide under conditions that allow expression of the polypeptide in APC. it can. Similarly, a nucleic acid construct encoding an antigen can be introduced into an APC by transfection, viral infection, or viral transduction. The resulting APC that shows an increased Notch signaling level is ready to use.

Tolerization Assays Any of the assays described above (see “Assays”) can be adapted to monitor or detect the degree of responsiveness and tolerance in immune cells for use in clinical applications. Such assays include, for example, detecting reduced Notch signaling activity in host cells, or monitoring Notch cleavage in donor cells. Additional methods for monitoring immune cell activity are described below.

  Immune cell activity can be monitored by any suitable method known to those skilled in the art. For example, cytotoxic activity can be monitored. Natural killer (NK) cells demonstrate enhanced cytotoxic activity after activation. Thus, reduction or stabilization of cytotoxicity suggests a reduction in responsiveness.

  After activation, leukocytes express a variety of new cell surface antigens. NK cells express, for example, transferrin receptor, HLA-DR, and CD25 IL-2 receptor after activation. Therefore, a reduction in responsiveness can be assessed by monitoring the expression of these antigens.

  Hara et al., Human T-cell Activation: III, Rapid Induction of a Phosphorylated 28 kD / 32 kD Displaced Linked Early Activation Anti (3-A-1) Exp. Med. 164: 1988 (1986) and Cosulich et al., Functional Charactorization of an Antigen (MLR3) Involved in an Early Step of T-Cell Activation, PNAS, 84: 4205 (1987) activated in T cells. A surface antigen is described. These antigens EA-1 and MLR3 are glycoproteins having major components of 28 kD and 32 kD, respectively. EA-1 and MLR3 are not HLA class II antigens, and MLR3 Mab blocks IL-1 binding. These antigens appear on activated T cells within 18 hours and can therefore be used to monitor immune cell responsiveness.

  In addition, leukocyte responsiveness can be monitored as described in EP0325489, which is hereby incorporated by reference. Briefly, this is done using a monoclonal antibody that interacts with a cellular antigen recognized by a monoclonal antibody produced by a hybridoma designated ATCC number HB-9627 (“Anti-Leu23”).

  Anti-Leu23 recognizes cell surface antigens of activated and antigen-stimulated leukocytes. In activated NK cells, the antigen Leu23 is expressed within 4 hours after activation, and continues to be expressed for 72 hours after activation if it is longer. Leu23 is a disulfide-linked homodimer consisting of a 24 kD subunit with at least two N-linked carbohydrates.

  Since the appearance of Leu23 in NK cells correlates with the occurrence of cytotoxicity, and the appearance of Leu23 in certain T cells correlates with stimulation of the T cell antigen receptor complex, Anti-Leu23 is a leukocyte response. Useful for monitoring sex.

  Further details of techniques for monitoring immune cell responsiveness can be found in “The Natural Killer Cell” Lewis C., which is incorporated herein by reference. E. And J.A. O'D. McGee, 1992, Oxford University Press, Trichieri G. “Biology of Natural Killer Cells” Adv. Immunol. 1989, 47, pp. 187-376, Chapter 7 of “Handbook of the Immunity Response Genes” “Cytokines of the Immunity Response”, Mak T .; W. And J.A. J. et al. L. Can be found in Simard, 1998.

  Various preferred features and embodiments of the present invention are described in further detail by non-limiting examples.

Preparation of Notch signaling inhibitor (hDelta1-IgG4Fc fusion protein)
See fusion protein comprising the extracellular domain of human Delta1 (“hDelta1-IgG4Fc”) fused to the Fc domain of human IgG4, nucleotide sequence encoding the extracellular domain of human Delta1 (see, eg, Genbank accession number AF003522) ) Was inserted into the expression vector pCONγ (Lonza Biology, Slow, UK) and prepared by expressing the resulting construct in CHO cells.

i) A 1622 bp extracellular (EC) fragment of cloned human Delta-like ligand 1 (see hECDLL-1, GenBank accession number AF003522), according to the manufacturer's instructions, Qiagen QIAquick ™ Gel Extraction Kit (Cat. No. 28706) Was used to purify the gel. The fragment was then ligated into a HindIII-BsiWI cut pCR Blunt cloning vector (Invitrogen, UK) to remove the HindIII, BsiWI, and Apal sites.

This ligation was transformed into DH5α cells, streaked on LB + Kanamycin (30 μg / ml) plates and incubated overnight at 37 ° C. Colonies were picked from the plates in 3 ml LB + Kanamycin (30 μg / ml) and grown overnight at 37 ° C. Plasmid DNA was purified from the culture using the Qiagen Qiaquick Spin Miniprep kit (Cat. No. 27106) according to the manufacturer's instructions and then diagnostically digested with HindIII. Clones were selected and streaked onto LB + Kanamycin (30 μg / ml) plates using a modified glycerol stock of pCRBlunt-hECDLL-1 and incubated overnight at 37 ° C. Colonies from this plate were taken up in 60 ml LB + kanamycin (30 μg / ml) and incubated overnight at 37 ° C. Cultures were maxiprepped using the Clontech Nucleobond Maxi Kit (Cat. No. K3003-2) according to the manufacturer's instructions and the final DNA pellet was resuspended in 300 μl dH ₂ O and stored at −20 ° C.

  5 μg of modified pCRBlunt-hECDLL-1 vector was linearized with HindIII and partially digested with ApaI. The 1622 bp hECDLL-1 fragment was then gel purified using the Clontech Nucleospin® Extraction Kit (K3051-1) according to the manufacturer's instructions. After passing the DNA through another Clontech Nucleospin® column and isolation by PCR protocol, the concentration of the sample was examined by agarose gel analysis which can be used for ligation.

  The plasmid pconγ (Lonza Biology, UK) was cut with HindIII-ApaI and the following oligo was ligated (SEQ ID NO: 2).

This ligation was transformed into DH5α cells and LB + Amp (100 μg / ml) plates were streaked with 200 μl of transformants and incubated overnight at 37 ° C. The next day, 12 clones were taken up in 2 × YT + ampicillin (100 μg / ml) and grown at 37 ° C. all day. Plasmid DNA was purified from the culture using the Qiagen Qiaquick Spin Miniprep kit (Catalog No. 27106) and diagnostically digested with NotI. A clone (referred to as “pDev41”) was selected and an LB + Amp (100 μg / ml) plate was streaked with a glycerol stock of pDev41 and incubated at 37 ° C. overnight. The next day, clones from this plate were taken up in 60 ml LB + Amp (100 μg / ml) and incubated overnight at 37 ° C. with shaking. This clone was maxiprepped using the Clontech Nucleobond Maxi Kit (catalog number K3003-2) according to the manufacturer's instructions and stored at -20 ° C. PDev41 clone 5 maxiprep was then digested with ApaI-EcoRI to generate an IgG4Fc fragment (1624 bp). The digest was purified on a 1% agarose gel, the major band was excised and purified using the Clontech Nucleospin Extraction Kit (K3051-1).

  The polynucleotide is then SV40 polyadenylation signal (glutamine free) that has an hCMV promoter that directs transcription of the desired gene downstream of the strong hCMV promoter enhancer region (hCMV-MIE) and under the control of the late SV40 promoter. Upstream of pEE14. Encoding GS gene required for selection in medium; contains GS minigene-GS cDNA, which contains the polylinker adenylation signal and the last intron of the wild type hamster GS gene. 4 (Lonza Biologicals, UK) cloned into the polylinker region. pEE14.4 maxiprep 5 μg was digested with HindIII-EcoRI and the product gel extracted and treated with alkaline phosphatase.

ii) Generation of Expression Constructs Three fragment ligation was performed using HindIII-EcoRI cleaved pEE14.4, modified pCR Blunt ECDLL-1 (HindIII-ApaI), and an IgG4Fc fragment (ApaI-EcoRI) cleaved from pDev41. . This was transformed into DH5α cells and LB + Amp (100 μg / ml) plates were streaked with 200 μl of transformation and incubated at 37 ° C. overnight. The next day, 12 clones were taken up in 2 × YT + Amp (100 μg / ml) and grown at 37 ° C. throughout the day. Plasmid DNA is purified from the prep using the Qiagen Qiaquick Spin Miniprep kit (Cat. No. 27106), diagnostically digested (EcoRI and HindIII), and a clone (clone 8, designated “pDev44”) for maxiprep. Selected. The glycerol stock of pDev44 clone 8 was streaked onto LB + Amp (100 μg / ml) plates and incubated overnight at 37 ° C. The next day, the colonies were taken up in 60 ml LB + Amp (100 μg / ml) culture and incubated at 37 ° C. overnight. This plasmid DNA was isolated using the Clontech Nucleobond Maxiprep Kit (catalog number K3003-2).

iii) Addition of optimal KOZAK sequence The Kozak sequence was inserted into the expression construct as follows.
The oligonucleotide was kinased and annealed to yield the following sequence:

pDev44 was digested with HindIII-BstBI, gel purified and treated with alkaline phosphatase. The digest was ligated with oligos and transformed into DH5α cells by heat shock. 200 μl of each transformant was streaked onto an LB + Amp plate (100 μg / ml) and incubated overnight at 37 ° C. Minipreps were grown in 3 ml, 2 × YT + ampicillin (100 μg / ml). Plasmid DNA was purified from the miniprep using the Qiagen Qiaquick spin miniprep kit (Cat. No. 27106) and diagnostically digested with NcoI. A clone (pDev46) was selected and the sequence was confirmed. The glycerol stock was streaked, the culture was grown, and the plasmid was maxiprepped.

iv) Transfection About 100 μg of pDev46 clone 1 DNA was linearized with the restriction enzyme PvuI. The resulting DNA sample was washed using phenol / chloroform / IAA extraction, then washed with ethanol and precipitated. The pellet was resuspended in sterile water and linearization and quantification were examined by agarose gel electrophoresis and UV spectrometry.

40 μg of linearized DNA (pDev46 clone 1) and 1 × 10 ⁷ CHO-K1 cells were mixed with serum-free DMEM in a 4 mm cuvette at room temperature. Cells were then electroporated at 280 v, 950 μF, washed into non-selective DMEM, diluted into 96 well plates and incubated. After 24 hours, the medium was removed and replaced with selective medium (25 μM, L-MSX). After 6 weeks, the media was removed and analyzed by IgG4 sandwich ELISA. The selection medium was changed. Positive clones were identified and passaged to 25 μm selective medium L-MSX.

v) Expressed cells were grown in selective DMEM (25 μm, L-MSX) to semi-confluent. Thereafter, the medium was replaced with serum-free medium (UltraCHO) for 3-5 days. The protein (hDelta1-IgG4Fc fusion protein) was purified from the resulting medium by HPLC.

  The amino acid sequence of the obtained expression fusion protein was as follows (SEQ ID NO: 5).

In the sequence, the first underlined sequence is the signal peptide (cleaved from the mature protein), and the second underlined sequence is the IgG4Fc sequence. This protein usually exists as a dimer linked by cysteine disulfide bonds (see, eg, the schematic diagram in FIG. 10). The domain structure of this expressed fusion protein is shown in more detail in FIG.

Notch signaling inhibitors include or include Flushshield ™ full vaccine (5 micrograms, Roche USA) that enhances immune response to flu antigen , 100 micrograms hDelta1-IgG4Fc (Example 1 above) Not emulsified in Freund's incomplete adjuvant. Six to eight week old BALB / c mice (8 per group) were immunized subcutaneously at the base of the tail, 14 days later they were challenged in the right ear with 1.8 micrograms of Fullfield full vaccine in saline. . Ear response (ear thickness measured with calipers) was measured after 1, 2 and 6 days.

  The results expressed as an increase in ear swelling (right ear-left ear) are shown in FIG.

Notch signaling inhibitor enhances immune response to KLH 6-8 week old BALB / c mice (8 per group) subcutaneously at the base of the tail, 50 ng or 0.5 ng per mouse as described in Example 1 above. Immunization with Pierce scallop hemocyanin (KLH) emulsified in incomplete Freund's adjuvant (IFA) with or without hDelta1-IgG4Fc protein (100 micrograms). Some mice also received additional hDelta1-IgG4Fc (400 micrograms) in the adjacent subcutaneous site one day later. 14 days after the first KLH priming, the mice were challenged in the right ear with 20 micrograms of KLH and the ear immune response was measured with calipers as an increase in ear thickness due to an inflammatory response induced 24 hours later. did.

  The results are shown in FIG.

Notch signaling inhibitor enhances immune response to full vaccine 6-8 weeks old BALB / c mice (8 per group) subcutaneously at the base of the tail, 5 μg per mouse, hDelta1 of Example 1 above Immunization was performed with Flushshield ™ full vaccine emulsified in Freund's incomplete adjuvant (IFA) containing IgG4Fc protein (100 micrograms) or isotype control hIgG4 (Sigma, UK) 100 μg / IFA control. 14 days after the first Flushshield ™ full vaccine priming, the mice were challenged in the right ear with Flushshield ™ and the ear immune response as an increase in ear thickness due to an inflammatory response induced 24 hours later. Was measured with a caliper.

  The results are shown in FIG.

Regulation of cytokine production induced by Delta1 beads is inhibited by addition of soluble hDelta1-IgG4Fc
i) Preparation of beads coated with hDelta1-IgG4Fc fusion protein Anti-human IgG4 biotinylation by spinning M450 Streptavidin Dynabead ™ magnetic beads (Dynal, USA) in the presence of antibodies for 30 minutes at room temperature Coated with monoclonal antibody (BD Bioscience, 555879). The beads were washed 3 times with PBS (1 ml). They were further incubated with hDelta1-hIgG4 (see Example 1 above) for 2 hours at room temperature and washed 3 times with PBS (1 ml).

ii) Investigation of Notch signaling by ELISA Human peripheral blood mononuclear cells (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia). Briefly, 28 ml of blood was placed on 21 ml of Ficoll-Paque separation medium and centrifuged at 400 g for 40 minutes at 18-20 ° C. PBMCs were recovered from the interface, washed 3 times and used for CD4 + T cell purification.

CD4 + T cells were incubated in triplicate using 96 well plates (flat bottom) at 10 ⁵ CD4 / well / 200 μl in RPMI medium containing 10% FCS, glutamine, penicillin, streptomycin, and β ₂ -mercaptoethanol.

  Cytokine production is performed at a 1: 1 (bead: cell) ratio of cells in the presence of beads coated with hDelta1-IgG4Fc fusion protein (Example 1 above) at a 5: 1 (bead: cell) ratio. Induced by stimulation with anti-CD3 / CD28 T cell expansion beads. In some wells, an increased amount of soluble hDelta1-IgG4Fc fusion protein was further added.

After 3 days incubation at 37 ° C./5% CO ₂ / humidified atmosphere, the supernatant was removed and cytokine production was determined for Pharmingen kit OptEIA Set human IL10 according to the manufacturer's instructions for IL-10 and IL-5, respectively (catalog no. 555157), OptEIA Set human IL-5 (Cat. No. 555202) and evaluated by ELISA.

  The results showing the effect of increasing the concentration of added soluble hDelta1-IgG4Fc are shown in FIG.

  As can be seen from these results, bead-immobilized human Delta1 enhances IL-10 production by activating human CD4 + T cells. This effect was inhibited when hDelta1-IgG4Fc was added to the medium.

Modulation of cytokine production induced by Delta1 beads is inhibited by addition of soluble Notch1EC domain / Fc fusion protein Human peripheral blood mononuclear cells (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia) . Briefly, 28 ml of blood was placed on 21 ml of Ficoll-Paque separation medium and centrifuged at 400 g for 40 minutes at 18-20 ° C. PBMCs were recovered from the interface, washed 3 times and used for CD4 + T cell purification.

CD4 + T cells were incubated in triplicate using 96 well plates (flat bottom) at 10 ⁵ CD4 / well / 200 μl in RPMI medium containing 10% FCS, glutamine, penicillin, streptomycin, and β ₂ -mercaptoethanol.

  Cytokine production is performed at a 1: 1 (bead: cell) ratio of cells in the presence of beads coated with hDelta1-IgG4Fc fusion protein (Example 1 above) at a 5: 1 (bead: cell) ratio. Induced by stimulation with anti-CD3 / CD28 T cell expansion beads. In some wells, an increased amount of soluble rat Notch1 extracellular domain-hIgG1 fusion protein (R & D Systems, catalog number 1057-TK) was further added.

After 3 days incubation at 37 ° C./5% CO ₂ / humidified atmosphere, the supernatant was removed and cytokine production was determined according to the manufacturer's instructions for Pharmingen kit OptEIA Set human IL10 (catalog no. 555157), OptEIA Set human IL-5 (catalog number 555202) and evaluated by ELISA.

  The results showing the effect of increasing the concentration of the added soluble rat Notch1EC-hIgG1Fc fusion protein are shown in FIG.

  As can be seen from these results, bead-immobilized human Delta1-Fc enhances IL-10 production by activating human CD4 + T cells. This effect was inhibited when soluble rat Notch1-hIgG1Fc was added to the medium.

Preparation of Notch signaling inhibitor: truncated human Jagged1 fusion protein (hJagged1EGF1 & 2-IgG4Fc)
A fusion protein capable of acting as an inhibitor of Notch signaling containing the human Jagged1 sequence (leader sequence, amino terminus, DSL, EGF1 + 2) up to the end of EGF2 fused to the Fc domain of human IgG4 (“hJagged1 (EGF1 + 2) -IgG4Fc “), A nucleotide sequence encoding human Jagged1 from the ATG to the end of the second EGF repeat (EGF2) was inserted into the expression vector pCONγ (Lonza Biologicals, Slow, UK) and added with an IgG4Fc tag. This complete fusion protein was then shuttled to the glutamine synthetase (GS) selection system vector pEE14.4 (Lonza Biologics). The resulting construct was transfected into CHO-K1 cells (Lonza Biologics) and expressed.

1. Cloning i) Preparation of DNA-pDEV47 and pDEV20 Human Jagged1 was cloned into pcDNA3.1 (Invitrogen) to obtain plasmid pLOR47. This pLOR47 Jagged 1 sequence was aligned to full-length human Jagged 1 (GenBank U61276) and was found to have only a few apparent silent changes.

  The plasmid pLOR47 was then modified to remove one of the two DraIII sites (at the same time maintaining and replacing the complete extracellular hJagged1 amino acid sequence) and a BsiWI site was added to simplify subsequent cloning. The resulting plasmid was named pDEV20.

  Plasmid pLOR47 was cut with DraIII. This removes the 1.7 kb fragment containing the 3 'end of the intracellular, transmembrane and extracellular regions of hJagged1 and part of the vector sequence, and almost all of the extracellular region (EC) of hJagged1. A large 7.3 kbp fragment of the major vector backbone was left behind. Cleaved DNA was run on an agarose gel, large fragments were excised and gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to manufacturer's instructions. A pair of oligonucleotides were arranged so that when ligated together, a double-stranded piece of DNA having a sticky end compatible with DraIII at the 5 'end and reacting with the original restriction site was obtained. This sequence was followed by a BsiWI site followed by another sticky end compatible with DraIII at the 3 'end that did not react with the restriction site.

This oligo pair was then ligated to DraII cut pLOR47, thereby maintaining the 5 ′ DraIII site, inserting BsiWI and removing the 3 ′ DraIII site. The resulting plasmid was named pDEV20.

ii) Preparation of hJagged1 IgG4 Fc fusion DNA Three-fragment ligation was required to reconstruct the complete hJagged1EC sequence by the addition of a modified 5 ′ Kozak sequence and 3 ′ end repair and 5 ′ end repair.

Fragment 1: EChJagged sequence pDev20 was cut with RsrII-DraIII to generate 3 fragments. 1270 + 2459 + 3621 bp. These fragments were run on an agarose gel, a 2459 bp band was excised, and the DNA was gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to the manufacturer's instructions. This contained the hJagged1 sequence, deleted the 3 ′ sequence (up to the RsrII site) and deleted a portion of the 5 ′ sequence at the end of the EC region.

Fragment 2: Modified Kozak sequence pUC19 (Invitrogen) was modified to insert a new restriction enzyme site and introduce a modified Kozak with a 5'hJagged1 sequence. The new plasmid was named pLOR49.
pLOR49 was generated by cleaving the pUC19 vector HindIII EcoRI and ligating it into 4 oligonucleotides (2 oligo pairs).
One pair has a HindIII sticky end followed by an optimal Kozac and 5′hJagged1 sequence followed by an RsrII sticky end.

The other pair has an attached RsrII end followed by a DraIII, KpnI, BsiWI site followed by an attached EcoRI site

Therefore, pLOR49 is a pUC19 backbone with HindIII, followed by optimal Kozac, and 5′hJagged1 sequences, and specific RsrII, DraIII, KpnI, BsiWI sites are introduced prior to reaction with the EcorI site.

  The plasmid pLOR49 was then cut with RsrII-BsiWI to obtain a 2.7 kbp vector backbone fragment that was run on an agarose gel, the band was excised, and the Qiagen QIAquick ™ Gel Extraction Kit ( The DNA was gel purified using catalog number 28706).

Fragment 3: Generation of 3'hJagged1EC with BsiWI site PCR fragment Using pLOR47 as a template for PCR, hJagged1EC was amplified and a 3'BsiWI site was added.
3 ′ site to the end of hJagged1EC having 5 ′ primer 3 ′ binding BsiWI site of RsrII of hJagged1 The resulting fragment was cleaved with DraIII and BsiWI to obtain a fragment of about 600 bp. This was run on an agarose gel, the band was excised, and the DNA was gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to the manufacturer's instructions.

The above three fragments 1) 2459 bp hJagged1 fragment of pDev20-cut RsrII-DraIII 2) 2.7 kbp optimized Kozak and 5′hJagged1 of RsrII-BsiWILOr49 cut
3) 600 bp of 3 ′ EChJagged 1 PCR fragment cleaved DraIII-BsiWI were ligated together to obtain plasmid pDEV21.

iii) Further ligation (pDEV10)
To eliminate foreign sequences, an additional three fragment ligation was performed and inserted into the vector pCONγ4 (Lonza Biology, Slowh, UK).

  Fragment 1: Plasmid pDEV21-4 was digested with HindIII-BglII to obtain a fragment of 4958 bp + 899 bp. They were run on an agarose gel, a small 889 bp fragment band was excised, and the DNA was gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to the manufacturer's instructions.

  Fragment 2: pCONγ4 (Lonza Biologicals) was cut with HindIII-ApaI to obtain a 6602 bp vector fragment lacking the first 5 amino acids of IgG4FC. The band of this fragment was excised and the DNA was gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to the manufacturer's instructions.

  Fragment 3: The linker oligonucleotide pair was sequenced to obtain a tight junction between the end of hJagged1EGF2 and the 3 'start of IgG4FC without the introduction of excess amino acids.

The above three fragments 1) 899 bp hJagged1 fragment HindIII-BglIII cleaved pDEV21-4
2) 6602 bp HindIIIApaI cleaved pConγ vector backbone 3) Oligo linker BglII-ApaI were ligated together to give plasmid pDEV10.

  Ligated DNA was transformed into competent DH5alpha (Invitrogen), plated on LBamp plates and incubated overnight at 37 ° C. A good ratio of control to vector and insert was evident, so only 8 colonies were taken up in 10 ml LBamp broth and incubated overnight at 37 ° C. Glycerol cultures were made and the bacterial pellets were frozen at -20 ° C. Later, plasmid DNA was extracted using the Qiagen miniprep spin kit and diagnostically digested with ScaI. Since clones 2, 4, and 5 were considered to be correct clones, clone 2 was streaked on LBamp plates and 1/100 was inoculated into 120 ml of LB + amp culture. Plates and cultures were incubated overnight at 37 ° C. Glycerol broths were made from those broths and the pellets were frozen for later maxi prep. Plasmid DNA was extracted with Clontech Maxiprep, subjected to diagnostic digestion with ScaI, and diluted for UV spectrophotometric quantification and quality inspection.

iv) pDEVII cloning The coding sequence of the hJagged1EGF1 + 2IgG4FC fusion was transferred from pCONγ4 (Lonza Biologics) downstream of the hCMV promoter region (hCMV-MIE) and upstream of the SV40 polyadenylation signal to pEE14.4 (Lonza Biologics). Stable cell lines selected using the system (Lonza Biology) were obtained. Plasmid pEE14.4 contains the GS minigene (GS cDNA containing the polylinker adenylation signal of the wild-type hamster GS gene and the final intron under the control of the late SV40 promoter), which is selected in medium without glutamine. Encodes the GS gene required for.

v) Insertion pDEV10 clone 2 was cut with HindIII-EcoRI to generate two fragments of 5026 bp + 2497 bp. 2497 bp contains the coding sequence of the hJagged1EGF1 + 2IgG4FC fusion, which was excised from the agarose gel and the DNA was gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to the manufacturer's instructions.

vi) Vector pEE14.4 (Lonza Biologics) was cut with HindIII-EcoRI to remove the IgG4FC sequence, resulting in two fragments of 5026 bp + 1593 bp. A large 5026 bp fragment was excised from the agarose gel and the DNA was gel purified using a Qiagen QIAquick ™ Gel Extraction Kit (Cat # 28706) according to the manufacturer's instructions. This pEE14.4 vector backbone and the hJagged1EGF1 + 2IgG4FC fusion insert were ligated to yield the final transfection plasmid pDEV11.

  This ligation was transformed into DH5α cells, streaked onto LB + ampicillin (100 μg / ml) plates and incubated at 37 ° C. overnight. Colonies were taken from the plates in 7 ml LB + ampicillin (100 μg / ml) and cultured overnight at 37 ° C. with shaking. Glycerol cultures were made and plasmid DNA was purified from the cultures using the Qiagen Qiaquick Spin Miniprep kit (Cat # 27106) according to the manufacturer's instructions. The DNA was then diagnostically digested with SapI.

vii) Maxiprep for transfection A correct clone (clone 1) was selected, 100 μg of its glycerol stock was inoculated into 100 ml of LB + ampicillin (100 μg / ml) and further streaked on an LB + ampicillin (100 μg / ml) plate. . Both plates and culture were incubated overnight at 37 ° C. The plate showed pure growth and therefore the culture was maxiprepped using the Clontech Nucleobond Maxi Kit (Cat. No. K3003-2) according to the manufacturer's instructions. The final DNA pellet was resuspended in 500 μl dH ₂ O. Thereafter, a sample of pLOR11 clone 1 DNA was diluted and the concentration and quality of the DNA was evaluated by UV spectrophotometry. The sample was further digested diagnostically with SapI to obtain the correct size band.

viii) Linearization of DNA About 100 μg of pDev11 clone 1 DNA was linearized with the restriction enzyme PvuI. The resulting DNA sample was washed using phenol / chloroform / IAA extraction and then washed with ethanol and deposited in a laminar flow hood. The pellet was resuspended in sterile water. Linearization was examined by agarose gel electrophoresis and quantification and quality were assessed at 260 and 280 nm by UV spectrophotometry.

2. Transfected 40 μg of linearized DNA (pDev11 clone 1) and 1 × 10 ⁷ CHO-K1 cells (Lonza) were mixed with 500 μl of serum-free DMEM in a 4 mm cuvette at room temperature. Cells were then electroporated at 280 v, 950 μF and washed away in non-selective DMEM (DMEM / glutamine / 10% FCS). From this diluted 6 × 96 well plate, seeded at 50 μl per well. A quarter dilution of the original stock was made and inoculated from this 8 × 96 well plate at 50 μl per well. An additional 1/10 dilution was made from the second stock and seeded from this 12 × 96 well plate at 50 μl per well. Plates were incubated overnight at 37 ° C., 5% CO2. After 24 hours, the medium was removed and replaced with 200 μl selective medium (25 μM, L-MSX). Four to six weeks after transfection, media was removed from the plates for analysis by IgG4 sandwich ELISA. The selection medium was changed. Positive clones were identified, passaged into 25 μm selective medium L-MSX and allowed to grow.

3 expressing cells were grown in selective DMEM (25 μm, L-MSX) to semi-confluent. Thereafter, the medium was replaced with serum-free medium (UltraCHO, Bio Whitaker) for 3-5 days. The protein (hJagged1EGF1 + 2-IgG4Fc fusion protein) was purified from the resulting medium by FPLC.

Amino acid sequence of expressed fusion protein (hJagged1EGF1 + 2IgG4FC)

(SEQ ID NO: 10) Bold = hJagged1 extracellular domain leader sequence, amino terminal region, DSL, EGF1 + 2, underlined = IgG4 Fc sequence This protein appears to exist as a dimer linked by cysteine disulfide bonds with signal peptide cleavage It has been.

Regulation of cytokine production induced by Delta1 beads is inhibited by addition of soluble Jagged1 (2EGF truncated) / Fc fusion protein using human peripheral blood mononuclear cells (PBMC) using Ficoll-Paque isolation medium (Pharmacia) Purified from blood. Briefly, 28 ml of blood was placed on 21 ml of Ficoll-Paque separation medium and centrifuged at 400 g for 40 minutes at 18-20 ° C. PBMCs were recovered from the interface, washed 3 times and used for CD4 + T cell purification.

CD4 + T cells were incubated in triplicate using 96 well plates (flat bottom) at 10 ⁵ CD4 / well / 200 μl in RPMI medium containing 10% FCS, glutamine, penicillin, streptomycin, and β ₂ -mercaptoethanol.

  Cytokine production is performed at a 1: 1 (bead: cell) ratio of cells in the presence of beads coated with hDelta1-IgG4Fc fusion protein (Example 1 above) at a 5: 1 (bead: cell) ratio. Induced by stimulation with anti-CD3 / CD28 T cell expansion beads. In some wells, an increased amount of soluble Jagged-1 (2EGF) -hIgG1 fusion protein (hJagged1EGF1 & 2-IgG4Fc, prepared as above) was further added.

After 3 days incubation at 37 ° C./5% CO ₂ / humidified atmosphere, the supernatant was removed and cytokine production was determined according to the manufacturer's instructions for Pharmingen kit OptEIA Set human IL10 (catalog no. 555157), OptEIA Set human IL-5 (catalog number 555202) and evaluated by ELISA.

  The results showing the effect of increasing the concentration of added soluble hJagged1EGF1 & 2-IgG4Fc are shown in FIG.

  As can be seen from these results, bead-immobilized human Delta1-Fc enhances IL-10 production by activating human CD4 + T cells. This effect was inhibited when soluble hJagged1EGF1 & 2-IgG4Fc fusion protein (hj1E2Fc) was added to the medium.

ELISA assay for detecting Notch signaling modulator activity in mouse CD4 + cells
(I) CD4 + cell purification Spleens were removed from 8-10 week old female Balb / c mice, and 2 mM L in 20 ml R10F medium (R10F-RPMI 1640 medium (Gibco catalog number 22409) from a 0.2 μM cell strainer. -Glutamine, 50 μg / ml penicillin, 50 μg / ml streptomycin, 5 × 10 ⁻⁵ M with 10% fetal bovine serum β-mercaptoethanol added). This cell suspension was centrifuged (1150 rpm, 5 minutes), and the medium was removed.

Cells were incubated for 4 minutes with 5 ml ACK lysis buffer (0.15 M NH ₄ Cl, 1.0 M KHCO ₃ , 0.1 mM Na ₂ EDTA in double distilled water) per spleen (to lyse red blood cells). The cells were then washed once with R10F medium and counted. CD4 + cells were prepared using Magnetic Associated Cell Sorter (MACS) column (Miltenyi Biotec, Bisley, UK, catalog number 130-042) using CD4 (L3T4) beads (Miltenyi Biotec catalog number 130-049-201) according to the manufacturer's instructions. -401) purified from suspension by positive selection.

(Ii) Antibody coated The following protocol was used to coat 96 well flat bottom plates with antibody.

  Plates were coated with DPBS containing 1 μg / ml anti-hamster IgG antibody (Pharmingen catalog number 554007) and 1 μg / ml anti-IgG4 antibody. 100 μl of coating mixture was added per well. Plates were incubated overnight at 4 ° C. and then washed with DPBS. Each well was then given 100 μl DPBS containing anti-CD3 antibody (1 μg / ml) or 100 μl DPBS containing anti-CD3 antibody (1 μg / ml) and hDelta1-IgG4Fc fusion protein (10 μg / ml). Plates were incubated at 37 ° C. for 2-3 hours, then washed again with DPBS, after which cells (prepared as described above) were added.

iii) Investigation of Notch signaling inhibition Mouse CD4 + T cells (prepared as described above) were prepared from plate-bound hDelta1-IgG4Fc fusion protein (prepared as described above) and soluble anti-CD28 (Pharmingne catalog number 553294, clone number 37) at a final concentration of 2 μg / ml. Incubate at 2 × 10 ⁵ / well in anti-CD3 coated plates with or without .51). Soluble hDelta1-IgG4Fc fusion protein was first added to the cultures at the indicated concentrations, and on day 3 supernatant IL-10 was measured by ELISA using an antibody pair from R & D Systems (Abingdon, UK). . The results (shown in FIG. 19) show that the increase in IL-10 release induced by the plate-bound hDelta1-IgG4Fc fusion protein is substantially reversed at all soluble hDelta1-IgG4Fc fusion protein concentrations tested. Yes.

CHO-N2 (N27) Luciferase Reporter Assay A) Construction of Luciferase Reporter Plasmid 10xCBF1-Luc (pLOR91) An adenovirus major late promoter TATA box motif with BglII and HindIII sticky ends was generated as follows.

This was cloned into plasmid pGL3-Basic (Promega) between the BgiII and HindIII sites, resulting in plasmid pGL3-AdTATA.

  A TP1 promoter sequence with BamH1 and BglII sticky ends (TP1 corresponds to 2CBF1 repeat) was generated as follows.

This sequence was pentamerized by repeated insertion into the BglII site, and the resulting TP1 pentamer (corresponding to 10CBF1 repeat) was inserted into pGL3-AdTATA at the BglII site, resulting in plasmid pLOR91.

B) Production of a stable CHO cell reporter cell line expressing full-length Notch2 and 10xCBF1-Luc reporter cassette A cDNA clone spanning the complete coding sequence of the human Notch2 gene (see, eg, GenBank accession number AF315356) was constructed as follows. . A 3 ′ cDNA fragment encoding the entire intracellular domain and a portion of the extracellular domain was isolated from a human placenta cDNA library (OriGene Technologies Ltd., USA) using a PCR-based screening method. The remaining 5 'coding sequence was isolated using the RACE (rapid amplification of cDNA ends) method and ligated to the existing 3' fragment using a unique restriction site common to both fragments (Cla I). The resulting full length cDNA was then cloned into the mammalian expression vector pcDNA3.1-V5-HisA (Invitrogen) without a stop codon, resulting in plasmid pLOR92. When expressed in mammalian cells, pLOR92 expresses a full-length human Notch2 protein with V5 and His tags at the 3 ′ end of the intracellular domain.

  Wild-type CHO-KI cells (see, eg, ATCC number CCL61) were transfected with pLOR92 (pcDNA3.1-FLNotch2-V5-His) using Lipfectamine 2000 ™ (Invitrogen) and full-length human Notch2 (N2) Resulted in stable CHO cell clones expressing. Transfectant clones were isolated from 10% heat-inactivated fetal calf serum ((HI) FCS), glutamine, penicillin / streptomycin (P / S), 1 mg / ml G418 (Geneticin) in 96-well plates using a limiting dilution method. (Trademark, Invitrogen) containing Dulbecco's Modified Eagle Medium (DMEM). Individual colonies were grown in DMEM containing 10% (HI) FCS, glutamine, P / S, 0.5 mg / ml G418. Clones were tested for N2 expression by Western blot of cell lysates using an anti-V5 monoclonal antibody (Invitrogen). Positive clones were then transiently transfected with the reporter vector pLOR91 (10xCBF1-Luc) and a stable CHO cell clone (CHO-Delta) expressing full length human Delta-like ligand 1 (DLL1, see eg GenBank accession number AF196571). ) And co-culture. CHO-Delta cells were prepared in the same manner as the CHONotch2 clone, using human DLL1 instead of Notch2. Strong positive clones were selected by Western blot of cell lysates using anti-V5 mAb.

  When transient transfection with pLOR91 (10xCBF1-Luc) and co-culture with stable CHO cell clones expressing full-length human DLL1 (CHO-Delta1), one CHO-N2 stable clone N27 was It was found to give a level of induction. A hygromycin gene cassette (available from pcDNA3.1 / hygro, Invitrogen) is inserted into pLOR91 (10xCBF1-Luc) using BamH1 and Sal1, and this vector (10xCBF1-Luc-hygro) is inserted into Lipfectamine 2000 (Invitrogen). Was used to transfect a CHO-N2 stable clone (N27). Transfectant clones were isolated using 10% (HI) FCS, glutamine, P / S, 0.4 mg / ml hygromycin B (Invitrogen), 0.5 mg / ml G418 (Invitrogen) in 96-well plates using limited dilution. ) Containing DMEM. Individual colonies were grown in DMEM containing 10% (HI) FCS, glutamine, P / S, 0.2 mg / ml hygromycin B, 0.5 mg / ml G418 (Invitrogen).

Clones were tested by co-culture with CHODelta (expressing full-length human Delta1 (DLL1)). Three stable reporter cell lines, N27 # 11, N27 # 17, and N27 # 36 were produced. N27 # 11 was selected for further use because of the low background signal in the absence of Notch signaling and thus high induction factor when signaling was initiated. Assays were performed in 96-well plates using 2 × 10 ⁴ N27 # 11 cells / well in 100 μl / well of DMEM containing 10% (HI) FCS, glutamine, P / S.

CHO-Delta cells (described above) were maintained in DMEM containing 10% (HI) FCS, glutamine, P / S, 0.5 mg / ml G418. Immediately before use, cells were removed from T80 flasks using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM containing 10% (HI) FCS, glutamine, P / S. 10 μl of cells were counted and the cell density was adjusted to 5.0 × 10 ⁵ cells / ml using fresh DMEM containing 10% (HI) FCS, glutamine, P / S.

To perform the CHO-Delta antagonist assay, 0.07% EDTA solution (Sigma) was used to remove N27 # 11 cells (T80 flask), spun down, 10% (HI) FCS, glutamine, P / Resuspended in 10 ml of DMEM containing S. 10 μl of cells were counted and the cell density was adjusted to 2.0 × 10 ⁵ cells / ml using fresh DMEM containing 10% (HI) FCS, glutamine, P / S. Reporter cells were plated at 100 μl per well (ie 2.0 × 10 ⁴ cells / well) in a 96 well plate, placed in an incubator and allowed to stand for at least 30 minutes.

HDelta1-IgG4Fc (Notch signaling soluble ligand inhibitor), prepared as described above, is diluted in complete DMEM to the final concentration required for assay x 5 and 50 μl of diluted ligand is added to 100 μl of N27 # 11 cells in a 96-well plate did. Then, to initiate signaling, 100 μl of CHO-Delta cells at 5 × 10 ⁵ cells / ml were added, bringing the final volume in each well to 250 μl. The plates were then placed in an incubator overnight at 37 ° C.

  The next day, 150 μl of supernatant was taken from all wells, 100 μl of SteadyGlo ™ Luciferase Assay Reagent (Promega) was added and the resulting mixture was allowed to stand at room temperature for 5 minutes. The mixture was pipetted up and down twice to ensure cell lysis and the contents of each well were transferred to a white 96 well plate (Nunc). Luminescence was then measured with a TopCount ™ counter (Packard). The same assay was performed using IgG4 as a control.

  The results are shown in FIG.

Soluble hJagged1 [2EGF] -IgG4Fc antagonizes Notch activation in CHO-N2 cells
CHO-Delta Cell Notch Signaling Antagonist Assay The procedure of Example 8 was repeated using hJagged1 & 2-IgG4Fc instead of hDelta1-IgG4Fc. For comparison, corresponding experiments were performed using hDelta1-IgG4Fc.

  The results are shown in FIG. It can be seen that the truncated Jagged protein with only 2EGF repeats (hjagged1EGF1 & 2-IgG4Fc) provided substantially the same Notch signaling inhibition as the corresponding protein containing the full-length human Delta1 extracellular domain (hDelta1-IgG4Fc).

mDLL1-Fc Coated Dynabeads Notch Signaling Antagonist Assay A fusion protein was prepared in the same manner as hDelta1-IgG4Fc described above using mouse Delta1 instead of human Delta1 (“mDelta1-IgG4Fc”).

The Fc-tagged Notch signaling modulator was combined with biotinylated α-IgG-4 (Pharmingen (Cat # 555879) clone JDC14, 0.5 mg / ml) as follows: Dynabeads (catalog number 115.21) “beads”, 4.0 × 10 ⁸ beads / ml).

An amount of Dynabeads beads corresponding to the total number required was removed from the bead stock at 4.0 × 10 ⁸ beads / ml. This was washed twice with 1 ml of PBS, resuspended in a final volume of 100 μl of PBS containing biotinylated anti-IgG4 antibody (Pharmingen (Cat # 555879) clone JDC14, 0.5 mg / ml) in a sterile Eppendorf tube and incubated at room temperature for 30 Place on shaker for minutes. The amount of biotinylated anti-IgG4 antibody required to coat the beads was calculated from the fact that 1 × 10 ⁷ streptavidin Dynabeads bind to up to 2 μg of antibody.

The beads were coated with antibody, washed 3 times with 1 ml PBS, and finally resuspended in mDelta1-IgG4Fc protein diluted in PBS. The beads are coated in a 2 μg / ml protein solution (usually 5 μg mDelta1-IgG4Fc protein is added per 10 ⁷ coated beads) on a rotary shaker to keep the beads in suspension at room temperature The ligand was bound to the beads in a 1 ml volume for 2 hours (or overnight at 4 ° C.). After coating the beads with mDelta1-IgG4Fc, the beads were washed 3 times with 1 ml PBS, and finally 100 μl of this was added to the wells of 2 × 10 ⁴ reporter cells so that a ratio of 100 beads: cells was obtained. Resuspended in complete DMEM with 2 × 10 ⁷ beads per ml.

To perform the bead antagonist assay, remove N27 # 11 cells (T80 flask) using 0.02% EDTA solution (Sigma), centrifuge, and add 10% (HI) FCS, glutamine, P / S. Resuspended in 10 ml of DMEM. 10 μl of cells were counted and the cell density was adjusted to 2.0 × 10 ⁵ cells / ml using fresh DMEM containing 10% (HI) FCS, glutamine, P / S. Reporter cells were plated at 100 μl per well (ie 2.0 × 10 ⁴ cells / well) in a 96 well plate, placed in an incubator and allowed to stand for at least 30 minutes.

Purified mDelta1-IgG4Fc was diluted in complete DMEM to the final concentration required for the assay × 5 and 50 μl of diluted ligand was added to 100 μl of N27 # 11 cells in a 96 well plate. Then, to initiate signaling, 100 μl of mDelta1-IgG4Fc Dynabeads at 2 × 10 ⁷ beads / ml was added, bringing the final volume of each well to 250 μl. The plates were then placed in an incubator overnight at 37 ° C.

  The next day, 150 μl of supernatant was taken from all wells, 100 μl of SteadyGlo ™ Luciferase Assay Reagent (Promega) was added and the resulting mixture was allowed to stand at room temperature for 5 minutes. The mixture was pipetted up and down twice to ensure cell lysis and the contents of each well were transferred to a 96-well plate (with V-type wells) and spun at 1000 rpm in a plate holder for 5 minutes at room temperature. Thereafter, the bead pellet was left and the clear supernatant was transferred to a white 96-well plate (Nunc). Luminescence was then measured with a TopCount ™ counter (Packard). The results are shown in FIG.

Soluble hJagged1EGF1 & 2-IgG4Fc antagonizes Notch activation in CHO-N2 cells
Delta Bead Notch Signaling Antagonist Assay The procedure of Example 8B was repeated using hJagged1EGF1 & 2-IgG4Fc instead of mDelta1-IgG4Fc. For comparison, corresponding experiments were performed using hDelta1-IgG4Fc and IgG4Fc as a control.

  The results are shown in FIG. It can be seen that the truncated Jagged protein with only 2EGF repeats (hjagged1EGF1 & 2-IgG4Fc) provided substantially the same Notch signaling inhibition as the corresponding protein containing the full-length human Delta1 extracellular domain (hDelta1-IgG4Fc). In either case, significant inhibition occurred compared to the control.

Reporter assay using Jurkat cell line Since Jurkat cells cannot be cloned by a simple limiting dilution method, a methylcellulose-containing medium (ClonaCell ™ TCS) was used with these cells.

  Jurkat E6.1 cells (lymphoblast cell line, ATCC TIB-152) using ClonCell ™ transfected cell selection (TCS) medium (StemCell Technologies, Vancouver, Canada, and Meylan, France) according to manufacturer guidelines, Cloned.

  Plasmid pLOR92 (prepared as described above) was electroporated into Jurkat E6.1 cells using a Biorad Gene Pulser II electroporator as follows.

Actively dividing cells are spun down and resuspended at 2.0 × 10 ⁷ cells / ml in ice-cold RPMI medium (complete RPMI) containing 10% heat-inactivated FCS, glutamine, penicillin / streptomycin. It became cloudy. After 10 minutes on ice, 0.5 ml of cells (ie 1 × 10 ⁷ cells) was placed in a pre-cooled 4 mm electroporation cuvette containing 20 μg of plasmid DNA (Endo-free Maxiprep DNA dissolved in sterile water). . The cells were electroporated at 300 v, 950 μF and then rapidly removed in 0.5 ml of warm complete RPMI medium in an Eppendorf tube. The cells were spun at 3000 rpm for 1 minute in a microcentrifuge and placed at 37 ° C. for 15 minutes to recover from electroporation. Thereafter, the supernatant was removed, and the cells were plated into wells of a complete RPMI 4 ml 6-well dish and left at 37 ° C. for 48 hours to express antibiotic resistance markers.

  After 48 hours, cells were resuspended in 10 ml of fresh and complete RPMI. This was then divided into 10 × 15 ml Falcon tubes, 8 ml of pre-warmed Cloncell-TCS medium was added, followed by 10 × final concentration of 1 ml of antibiotic used for selection. For G418 selection, the final concentration of G418 was 1 mg / ml and a 10 mg / ml solution in RPMI was prepared and 1 ml was added to each tube. The tube was mixed well by inversion, allowed to stand at room temperature for 15 minutes, and then plated on a 10 cm tissue culture dish. It was then placed in a CO2 incubator for 14 days and visible colonies were examined.

  Visible colonies were picked from the plates by visual inspection and these colonies were grown from 96-well plates into 24-well plates, T25 flasks.

  Clones were selected and transiently transfected with pLOR91 reporter construct using Lipofectamine 2000 reagent and then plated into 96 well plates containing plate-bound immobilized hDLL1-Fc (purified Notch ligand in sterile PBS on each plate) The plate was coated by adding 10 μg protein, sealing the plate lid with parafilm and incubating overnight at 4 ° C. or 2 hours at 37 ° C. and washing the plate with 200 μl PBS before use).

  Thereafter, a luciferase assay was performed as generally described above. The results are shown in FIG.

Antagonism of A20-Delta and A20-JaggedNotch signaling by soluble hDLL-1Fc
Remove the IVS, IRES, Neo, pA elements from the A20-Delta and A20-Jagged cell plasmid pIRESneo2 (Clontech, USA) and insert it into the pUC cloning vector downstream of the chicken beta actin promoter (see, eg, GenBank accession number E02199) did. Mouse Delta-1 cDNA (eg GenBank accession number NM — 007865) was inserted between the actin promoter and the IVS element, and a sequence with multiple stop codons in all three reading frames was inserted between the Delta and IVS elements.

  The resulting construct was transfected into A20 cells using electroporation and G418 to obtain A20 cells expressing mouse Delta1 on the surface (A20-Delta).

  Corresponding cells (A20-Jagged) were prepared using human Jagged1 cDNA (see, eg, GenBank accession number U61276).

The procedure of the example was repeated using A20-Delta or A20-Jagged cells (1 × 10 ⁵ / well) instead of CHO-Delta cells. IgG4 was used as a control. The results are shown in FIG. These results indicate that hDelta1-IgG4Fc was able to inhibit Jagged1 as well as Delta Notch signaling.

  A human Jagged1 was used instead of human Delta1, and a fusion protein was prepared in the same manner as the above-described hDelta1-IgG4Fc (hJagged1-IgG4Fc).

  The procedure of Example 8 was repeated using hJagged1-IgG4Fc instead of hDelta1-IgG4Fc, and corresponding repeated experiments were performed using hDelta1-IgG4Fc for comparison. The results are shown in FIG.

Notch signaling inhibitors reduce the induction of tolerance to KLH in BALB / c mice (8 per group) by intranasal route, i) PBS, ii) KLH (10 mg) alone, or iii) KLH (10 mg) + hDelta1 -Treated with IgG4Fc (100 mg). After 14 days, the mice were given KLH 50 mg / IFA subcutaneously. 28 days after the first KLH priming, the mice were challenged subcutaneously with KLH 50 mg / IFA and the ear immune response measured with calipers as an increase in ear thickness due to an inflammatory response induced 48 hours later. did.

  The results are shown in FIG.

Regulation of cytokine production by γ-secretase inhibitor in human CD4 + T cells Human peripheral blood mononuclear cells (PBMC) were purified from blood using Ficoll-Paque separation medium (Pharmacia). Briefly, 28 ml of blood was placed on 21 ml of Ficoll-Paque separation medium and centrifuged at 400 g for 40 minutes at 18-20 ° C. PBMCs were recovered from the interface, washed 3 times and used for CD4 + T cell purification.

Human CD4 + T cells were isolated by positive selection using Miltenyi Biotech anti-CD4 microbeads according to the manufacturer's instructions. The CD4 + T cells were incubated in triplicate using 96 well plates (flat bottom) at 10 ⁵ CD4 / well / 200 ml in RPMI medium containing 10% FCS, glutamine, penicillin, streptomycin, and b ₂ -mercaptoethanol.

  Cytokine production was induced by stimulating cells at a 1: 1 (bead: cell) ratio with Dynal's anti-CD3 / CD28 T cell expansion beads. Dynal beads coated with hDelta1-IgG4Fc fusion protein or control beads are added to several wells at a ratio of 5: 1 (beads: cells) and the γ-secretase inhibitor MW167 (Calbiochem γ-secretase inhibitor II, Catalog number 565755) was added at various final concentrations of 0, 0.4 mM, 2 mM, and 10 mM (in DMSO).

After 3 days incubation at 37 ° C./5% CO ₂ / humidified atmosphere, the supernatant was removed and cytokine production was determined for Pharmingen kit OptEIA Set human IL10 according to the manufacturer's instructions for IL-10 and IL-5, respectively (catalog no. 555157), OptEIA Set human IL-5 (Cat. No. 555202) and evaluated by ELISA.

  The results are shown in FIG. 22, and it can be seen that the γ-secretase inhibitor substantially reversed the Delta-mediated increase in IL-10 expression and further reversed the Delta-mediated decrease in IL-5 expression. .

Effect of γ-secretase inhibitors on Delta-mediated activation of Notch signaling in Jurkat-N2 cells Since Jurkat cells cannot be cloned by a simple limiting dilution method, a medium containing methylcellulose (ClonaCell ™ TCS) ) Was used.

  Jurkat E6.1 cells (lymphoblast cell line, ATCC TIB-152) using ClonCell ™ transfected cell selection (TCS) medium (StemCell Technologies, Vancouver, Canada, and Meylan, France) according to manufacturer guidelines, Cloned.

Plasmid pLOR92 (prepared as described above) was electroporated into Jurkat E6.1 cells using a Biorad Gene Pulser II electroporator as follows.
Actively dividing cells are spun down and resuspended at 2.0 × 10 ⁷ cells / ml in ice-cold RPMI medium (complete RPMI) containing 10% heat-inactivated FCS, glutamine, penicillin / streptomycin. It became cloudy. After 10 minutes on ice, 0.5 ml of cells (ie 1 × 10 ⁷ cells) was placed in a pre-cooled 4 mm electroporation cuvette containing 20 μg of plasmid DNA (Endo-free Maxiprep DNA dissolved in sterile water). . The cells were electroporated at 300 v, 950 μF and then quickly removed into 0.5 ml of warm, complete RPMI medium in an Eppendorf tube. The cells were spun at 3000 rpm for 1 minute in a microcentrifuge and placed at 37 ° C. for 15 minutes to recover from electroporation. Thereafter, the supernatant was removed, and the cells were plated into wells of a complete RPMI 4 ml 6-well dish and left at 37 ° C. for 48 hours to express antibiotic resistance markers.

  After 48 hours, the cells were spun down and resuspended in 10 ml fresh complete RPMI. This was then divided into 10 × 15 ml Falcon tubes, 8 ml of pre-warmed Cloncell-TCS medium was added, followed by 10 × final concentration of 1 ml of antibiotic used for selection. For G418 selection, the final concentration of G418 was 1 mg / ml and a 10 mg / ml solution in RPMI was prepared and 1 ml was added to each tube. The tube was mixed well by inversion, allowed to stand at room temperature for 15 minutes, and then plated on a 10 cm tissue culture dish. It was then placed in a CO2 incubator for 14 days and visible colonies were examined.

  Visible colonies were picked from the plates by gross examination and these colonies were grown from 96-well plates to 24-well plates, T25 flasks in complete RPMI containing 1 mg / ml G418.

  The resulting clones were transiently transfected with pLOR91, each using Lipofectamine 2000 reagent (according to the manufacturer's protocol) and then plated into 96 well plates containing plate-bound immobilized hDelta1-IgG4Fc (prepared as follows). Cultured. A good performing clone (# 24) was selected and used for further studies.

  10 μg of purified hDelta1-IgG4Fc fusion protein was added to sterile PBS in a sterile Eppendorf tube to obtain a final volume of 1 ml and 100 μl was added to the wells of a 96-well tissue culture plate. The lid of the plate was sealed with parafilm and the plate was left overnight at 4 ° C. or 2 hours at 37 ° C. The protein was then removed and the plate was washed twice with 200 μl PBS.

The assay was performed in 96-well plates coated with 2 × 10 ⁵ Jurkat cells per well in 100 μl per well of 10% (HI) FCS, glutamine, P / S containing DMEM. MW167 was diluted from a 10 mM stock solution in DMSO to complete RPMI to a final concentration of 20 μM. Control wells were prepared with the same dilution of DMSO alone. The plate was left overnight in a CO ₂ incubator.

  The supernatant was removed from all wells, leaving 100 μl of cells and medium, 100 μl of SteadyGlo ™ luciferase assay reagent (Promega) was added, and the cells were left at room temperature for 5 minutes. The mixture was pipetted up and down twice to ensure cell lysis and the contents of each well were transferred to a white 96-well OptiPlate ™ (Packard). Luminescence was measured with a TopCount ™ counter (Packard).

  The results of the sample assay using the Jurkat cells described above with the plate-immobilized hDelta1-IgG4Fc fusion protein are shown in FIG. 29 (expressed as the activation factor of reporter activity compared to cells cultured in the absence of Delta).

Preparation of Notch inhibitor construct with human Jagged1DSL domain and EGF repeat 1-2 ("hJagged1 [2EGF] -IgG4Fc")
A human Jagged 1 (JAG-1) deletion encoding the DSL domain and only the first two natural EGF repeats (ie, EGF repeats 3 through 16 were deleted) using the following primer pair, the JAG-1 clone (The sequence of human JAG-1 was generated by PCR from FIG. 4 and see, for example, Genbank accession number U73936).

These primers have leader peptide regions R to G aa. This produces an array that changes 2.

PCR conditions are as follows.
18 cycles at 95 ° C / 2 minutes (1 cycle at 95 ° C / 30 seconds, 60 ° C / 30 seconds, 72 ° C / 11/2 minutes) and 1 cycle at 72 ° C / 10 minutes. Isolated from a 1% agarose gel in (Tris / Boric acid / EDTA).

  pConγ (Lonza Biology, UK) was cut with HindIII and ApaI and the following adapter oligonucleotide sequences were ligated to introduce an XhoI site and subsequently cloned into DH5α cells.

pConγX was cut with HindIII and XhoI, then treated with shrimp alkaline phosphatase (Roche) and gel purified. The purified JAG-1 PCR product was cut with HindIII and XhoI, ligated into restriction pCONγX, and subsequently cloned into DH5α cells (InVitrogen). Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The resulting construct (pCONγhJ1E2) encoded the following JAG-1 amino acid sequence (SEQ ID NO: 16) fused to the IgG Fc domain encoded by the pCONγ vector.

(In the sequence, one underlined bold part sequence is a DSL domain, two underlined bold part sequences are EGF repeats 1 and 2, respectively, and italic is a linker / hinge. )
J1E2. DNA encoding the Fc4 sequence was excised with EcoRI and HindIII and ligated into EcoRI and HindIII restricted pEE14.4. The resulting plasmid pEE14. J1E2. Fc4 was cloned into DH5α (Invitrogen). Plasmid DNA was produced using the Qiagen Endofree Maxiprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the product was confirmed by sequencing.

  A series of shortened forms based on human Delta1 containing various numbers of EGF repeats were prepared as follows.

A) Delta1 DSL domain and EGF repeat 1-2
A human Delta1 (DLL-1) deletion encoding the DSL domain and only the first two natural EGF repeats (ie, deleting EGF repeats 3 through 8) was transferred to the DLL- One extracellular (EC) domain / V5His clone (the sequence of the human DLL-1 EC domain was generated by PCR from the figure and see, eg, Genbank accession number AF003522).

PCR conditions are as follows.
18 cycles at 95 ° C / 3 minutes (1 cycle at 95 ° C / 1 minute, 60 ° C / 1 minute, 72 ° C / 2 minutes) and 1 cycle at 72 ° C / 2 minutes. Isolated from a 1% agarose gel in SafeTAE (Tris / acetic acid / EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and used as a template for PCR with the following primers.

PCR conditions are as follows.
1 cycle at 94 ° C / 3 minutes (94 ° C / 1 minute, 68 ° C / 1 minute, 72 ° C / 2 minutes) and 1 cycle at 72 ° C / 10 minutes This fragment was ligated to pCRbluntITOPO (Invitrogen) Cloned into TOP10 cells (Invitrogen). Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The IgFc fusion vector pCONγ (Lonza Biology, UK) was cut with ApaI and HindIII, then treated with shrimp alkaline phosphatase (Roche) and gel purified.

  The DLL-1 deletion cloned into pCRbluntII was cut with HindIII (and EcoRV to facilitate subsequent selection of the desired DNA product) followed by ApaI partial restriction. This sequence was then gel purified and ligated into the pCONγ vector, which was cloned into TOP10 cells.

  Plasmid DNA was produced using a Qiagen Miniprep kit (QIAprep ™) according to the manufacturer's instructions.

  The resulting construct (pCONγhDLL1EGF1-2) encoded the following DLL-1 amino acid sequence (SEQ ID NO: 21) fused to the IgGFc domain encoded by the pCONγ vector.

(In the sequence, one underlined bold part sequence is a DSL domain, and two underlined bold part sequences are EGF repeats 1 and 2, respectively)
B) Delta1 DSL domain and EGF repeat 1-3
A human Delta1 (DLL-1) deletion encoding the DSL domain and only the first three natural EGF repeats (ie, EGF repeats 4 to 8 were deleted) using a primer pair: Produced by PCR from 1DSL and EGF repeat 1-4 clones.

PCR conditions are as follows.
One cycle at 94 ° C./3 minutes (94 ° C./1 minute, 68 ° C./1 minute, 72 ° C./2.5 minutes) and one cycle at 72 ° C./10 minutes. Isolated from a 1% agarose gel in V-SafeTAE (Tris / acetic acid / EDTA) buffer (MWG-Biotech, Ebersberg, Germany), ligated into pCRbluntITOPO and cloned into TOP10 cells (Invitrogen). Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The IgFc fusion vector pCONγ (Lonza Biology, UK) was cut with ApaI and HindIII, then treated with shrimp alkaline phosphatase (Roche) and gel purified.

  The DLL-1 deletion cloned into pCRbluntII was cut with HindIII, followed by ApaI partial restriction. This sequence was then gel purified and ligated into the pCONγ vector, which was cloned into TOP10 cells.

  Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The resulting construct (pCONγhDLL1EGF1-3) encoded the following DLL-1 sequence (SEQ ID NO: 24) fused to the IgG Fc domain encoded by the pCONγ vector.

(In the sequence, one underlined bold part sequence is a DSL domain, and two underlined bold part sequences are EGF repeats 1 to 3, respectively)
C) Delta1 DSL domain and EGF repeats 1-4
A human Delta1 (DLL-1) deletion encoding the DSL domain and only the first four natural EGF repeats (ie, deleting EGF repeats 5 through 8) was transferred to the DLL- Produced by PCR from 1EC domain / V5His clone.

PCR conditions are as follows.
18 cycles at 95 ° C / 3 minutes (1 cycle at 95 ° C / 1 minute, 60 ° C / 1 minute, 72 ° C / 2.5 minutes) and 1 cycle at 72 ° C / 10 minutes. It was isolated from a 1% agarose gel in V-SafeTAE (Tris / acetic acid / EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and used as a template for PCR with the following primers.

PCR conditions are as follows.
1 cycle at 94 ° C / 3 min (94 ° C / 1 min, 68 ° C / 1 min, 72 ° C / 2.5 min) and 1 cycle at 72 ° C / 10 min This fragment was ligated to pCRbluntITOPO and TOP10 Cloned into cells (Invitrogen). Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The IgFc fusion vector pCONγ (Lonza Biology, UK) was cut with ApaI and HindIII, then treated with shrimp alkaline phosphatase (Roche) and gel purified.

  The DLL-1 deletion cloned into pCRbluntII was cut with HindIII (and EcoRV to facilitate subsequent selection of the desired DNA product) followed by ApaI partial restriction. This sequence was then gel purified and ligated into the pCONγ vector, which was cloned into TOP10 cells.

  Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The resulting construct (pCONγhDLL1EGF1-4) encoded the following DLL-1 acid sequence (SEQ ID NO: 29) fused to the IgGFc domain encoded by the pCONγ vector.

(In the sequence, one underlined bold part sequence is a DSL domain, and two underlined bold part sequences are EGF repeats 1 to 4, respectively)
D) Delta1 DSL domain and EGF repeat 1-7
A human Delta1 (DLL-1) deletion encoding the DSL domain and the first seven natural EGF repeats (ie, deleting EGF repeat 8) was performed using the following primer pair with the DLL-1EC domain / V5His clone: Produced by PCR.

PCR conditions are as follows.
18 cycles at 95 ° C / 3 minutes (1 cycle at 95 ° C / 1 minute, 68 ° C / 1 minute, 72 ° C / 3 minutes) and 1 cycle at 72 ° C / 10 minutes. Isolated from a 1% agarose gel in SafeTAE (Tris / acetic acid / EDTA) buffer (MWG-Biotech, Ebersberg, Germany) and used as a template for PCR with the following primers.

PCR conditions are as follows.
1 cycle at 94 ° C / 3 min (94 ° C / 1 min, 68 ° C / 1 min, 72 ° C / 3 min) and 1 cycle at 72 ° C / 10 min This fragment was ligated into pCRbluntITOPO and TOP10 cells ( Invitrogen). Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the identity of the PCR product was confirmed by sequencing.

  The IgFc fusion vector pCONγ (Lonza Biology, UK) was cut with ApaI and HindIII, then treated with shrimp alkaline phosphatase (Roche) and gel purified.

  The DLL-1 deletion cloned into pCRbluntII was cut with HindIII (and EcoRV to facilitate subsequent selection of the desired DNA product) followed by ApaI partial restriction. This sequence was then gel purified and ligated into the pCONγ vector, which was cloned into TOP10 cells.

  Plasmid DNA was produced using the Qiagen Minprep kit (QIAprep ™) according to the manufacturer's instructions and the PRC product was sequenced.

  The resulting construct (pCONγhDLL1EGF1-7) encoded the following DLL-1 acid sequence (SEQ ID NO: 34) fused to the IgG Fc domain encoded by the pCONγ vector.

(In the sequence, one underlined bold part sequence is a DSL domain, and two underlined bold part sequences are EGF repeats 1 to 7, respectively)
E) Transfection and expression
i) Transfection and expression of constructs A, C, and D Cos1 cells were individually treated with the expression constructs of Examples 1, 3, and 4 above (ie, pCONγhDLL1EGF1-2, pCONγhDLL1EGF1-4, pCONγhDLL1EGF1-7). ) And pCONγ control were transfected as follows.

In either case, 3 × 10 ⁶ cells were plated in 10 cm dishes of Dulbecco's modified Eagle medium (DMEM) + 10% fetal calf serum (FCS) and the cells were allowed to attach to the plate overnight. The cell monolayer was washed twice with 5 ml phosphate buffered saline (PBS) and the cells were placed in 8 ml OPTIMEM ™ medium (Gibco / Invitrogen). 12 μg of the appropriate construct DNA was diluted in 810 μl of OPTIMEM medium and 14 μl of Lipofectamine 2000 ™ cationic lipid transfection reagent (Invitrogen) was diluted in 810 μl of OPTIMEM medium. This DNA-containing, Lipofectamine 2000 reagent-containing solution was then mixed, incubated at room temperature for a minimum of 20 minutes, added to the cells, and the transfection mixture was evenly distributed on the dish. Cells were incubated with transfection reagent for 6 hours, after which the medium was removed and replaced with 20 ml DMEM + 10% FCS. After 5 days, the supernatant containing the secreted protein was collected from the cells and dead cells suspended in the supernatant were removed by centrifugation (5 minutes at 4500 rpm). The obtained expression product was named as follows. hDLL1EGF1-2Fc (derived from pCONγhDLL1EGF1-2), hDLL1EGF1-4Fc (derived from pCONγhDLL1EGF1-4), and hDLL1EGF1-7Fc (derived from pCONγhDLL1EGF1-7).

  Expression of the Fc fusion protein was assessed by Western blot. The protein in 10 μl of the supernatant was separated by 12% SDS-PAGE and blotted onto a Hybond ™ -ECL (Amersham Pharmacia Biotech) nitrocellulose membrane (17 V, 28 minutes) with a semi-dry apparatus. The presence of the Fc fusion protein was determined by Western blot using JDC14 anti-human IgG4 antibody diluted 1: 500 in blocking solution (Tris buffered saline containing 5% non-fat milk solids and Tween 20 surfactant jTBS-T). Detected by. The blot was incubated in the solution for 1 hour and washed in TBS-T. After washing 3 times for 5 minutes each, the presence of mouse anti-human IgG4 antibody was detected using IgG-HPRT conjugated antiserum diluted 1: 10000 in blocking solution. The blot was incubated in the solution for 1 hour and washed in TBS-T (3 times for 5 minutes each). The presence of the Fc fusion protein was then visualized using ECL ™ detection reagent (Amersham Pharmacia Biotech).

  The amount of protein present in the 10 ml supernatant was assessed by comparison to Kappa chain standards containing 10 ng (7), 30 ng (8), and 100 ng (9) protein.

  The result of the blot is shown in FIG.

ii) Transfection and expression of construct B construct Cos1 cells were transfected with the expression construct of Example 2 above (ie, pCONγhDLL1EGF1-3) as follows.

7.1 × 10 ⁵ cells were plated in T25 flasks of Dulbecco's Modified Eagle Medium (DMEM) + 10% fetal calf serum (FCS) and the cells were allowed to attach to the plates overnight. The cell monolayer was washed twice with 5 ml phosphate buffered saline (PBS) and the cells were placed in 1.14 ml OPTIMEM ™ medium (Gibco / Invitrogen). 2.85 μg of the appropriate construct DNA was diluted in 143 μl of OPTIMEM medium and 14.3 μl of Lipofectamine 2000 ™ cationic lipid transfection reagent (Invitrogen) was diluted in 129 μl of OPTIMEM medium and incubated at room temperature for 45 minutes. The DNA-containing, Lipofectamine 2000 reagent-containing solution was then mixed, incubated at room temperature for 15 minutes, added to the cells to ensure even distribution of the transfection mixture in the flask. Cells were incubated with transfection reagent for 18 hours, after which the medium was removed and replaced with 3 ml DMEM + 10% FCS. After 4 days, the supernatant containing the secreted protein was collected from the cells, and dead cells suspended in the supernatant were removed by centrifugation (1200 rpm for 5 minutes). The obtained expression product was named as follows. hDLL1EGF1-3Fc (derived from pCONγhDLL1EGF1-3).

F) Luciferase reporter assay Fc-tagged Notch ligand expression products (hDLL1EGF1-2Fc, hDLL1EGF1-4Fc, and hDLL1EGF1-7Fc) of A to D described above were respectively biotinylated α-IgG-4 (Pharmingen (catalog number) 555879) clone JDC14, 0.5 mg / ml) and Streptavidin-Dynabeads (Dynal (UK) Ltd CELLection Biotin Binder Dynabeads (Cat. No. 115.21) “Bead”, 4.0 × 10 ⁸ beads / ml) Immobilized individually.

For each sample analyzed, 1 × 10 ⁷ beads (25 μl of beads at 4.0 × 10 ⁸ beads / ml) and 2 μg of biotinylated α-IgG4 were used. PBS was added to the beads to 1 ml and the mixture was spun down at 13000 rpm for 1 minute. After further washing with 1 ml PBS, the mixture was centrifuged again. The beads were then resuspended in a final volume of 100 μl of PBS containing biotinylated α-IgG4 in a sterile Eppendorf tube and placed on a shaker for 30 minutes at room temperature. PBS was added to 1 ml and the mixture was spun down at 13000 rpm for 1 minute, then washed twice more with 1 ml PBS.

The mixture was then spun down at 13000 rpm for 1 minute and the beads were resuspended in 50 μl PBS per sample. 50 μl of biotinylated α-IgG4 coated beads was added to each sample and the mixture was incubated on a rotary shaker at 4 ° C. overnight. The tube was then rotated at 1000 rpm for 5 minutes at room temperature. The beads are washed with 10 ml of PBS, spun down, resuspended in 1 ml of PBS, transferred to a sterile Eppendorf tube, washed with 2 × 1 ml of PBS, spun down and contain 10% (HI) FCS, glutamine, P / S Resuspended in a final volume of 100 μl DMEM, ie 1.0 × 10 ⁵ beads / μl.

Stable N27 # 11 cells (T80 flask) were removed using 0.02% EDTA solution (Sigma), spun down and resuspended in 10 ml DMEM containing 10% (HI) FCS, glutamine, P / S. . 10 μl of cells were counted and the cell density was adjusted to 1.0 × 10 ⁵ cells / ml using fresh DMEM containing 10% (HI) FCS, glutamine, P / S. 1.0 × 10 ⁵ cells per well were plated in 24-well plates in 1 ml of DMEM containing 10% (HI) FCS, glutamine, P / S, placed in an incubator and allowed to stand for at least 30 minutes.

Thereafter, 20 μl of beads were added in duplicate to a pair of wells to give 2.0 × 10 ⁶ beads / well (100 beads / cell). The plate was left overnight in a CO ₂ incubator.

  The supernatant was then removed from all wells, 100 μl of SteadyGlo ™ luciferase assay reagent (Promega) was added and the resulting mixture was allowed to stand at room temperature for 5 minutes.

  The mixture was then pipetted up and down twice to ensure cell lysis and the contents of each well were transferred to a 96-well plate (with V-type wells) and spun at 1000 rpm in a plate holder for 5 minutes at room temperature.

  Thereafter, the bead pellet was left and 175 μl of the clear supernatant was transferred to a white 96-well plate (Nunc).

  Luminescence was then measured with a TopCount ™ counter (Packard). The results are shown in FIG. 31 (for comparison, the activity of a fusion protein containing the complete Dll1EC domain (hDelta1-IgG4Fc) is also shown).

Jagged Short Form A similar series of short forms based on human Jagged 1 containing various numbers of EGF repeats was prepared as follows.

  In a manner similar to that described in Example 21, the nucleotide sequence encoding the human Jagged 1 (hJag 1) DSL domain and the first 2, 3, 4, and 16 native Jagged EGF repeats, respectively, was obtained as human Jagged-1 (See, eg, GenBank Accession No. U61276). These sequences were then purified, ligated into a pCONγ expression vector encoding an immunoglobulin Fc domain, expressed, and coated on microbeads. These expressed proteins are the first two (hJag1EGF1-2), three (hJag1EGF1-3), four (hJag1EGF1-4), and sixteen (hJag1EGF1-4), respectively, fused to the IgGFc domain encoded by the pCONγ vector. 16) Jagged EGF repeat as well as DSL domain.

  The beads coated with each expressed protein were then tested for activity in the Notch signaling reporter assay described above (Example 21). The obtained activity data is shown in FIG.

  For easier comparison, a similar assay was performed using the expressed Jagged protein along with the corresponding Delta protein. The results are shown in FIG.

Jagged EGF1-2 assay with high sensitivity In further experiments, beads were coated with a purified protein (hJagged1EGF1 & 2-IgG4Fc) containing the human Jagged1 DSL domain of Example 7 and the first two EGF repeats, with high protein loading to give high sensitivity, Tested for activity in the Notch reporter assay described above. The resulting activity data is shown in FIG. 34 (for comparison, the activity of a fusion protein containing the complete Dll1EC domain (hDelta1-IgG4Fc) is also shown).

  All publications are hereby incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the claimed invention should not be limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. The

It is a figure which shows the outline of Notch / ligand interaction. It is a figure which shows the outline of a Notch signaling path | route. It is a figure which shows the outline of Notch1-4. It is a figure which shows the outline of Notch ligand, Jagged, and Delta. FIG. 2 shows the aligned amino acid sequences of DSL domains from various Drosophila and mammalian Notch ligands. It is a figure which shows the amino acid sequence of human Delta-1, Delta-3, and Delta-4. It is a figure which shows the amino acid sequence of human Jagged-1 and Jagged-2. It is a figure which shows the amino acid sequence of human Notch-1. It is a figure which shows the amino acid sequence of human Notch-2. FIG. 1 shows a schematic of a Notch ligand / IgFc fusion protein suitable for use in the present invention. It is a figure which shows the outline of the nucleic acid expression construct by this invention. 2 is a view showing an amino acid sequence and a domain structure of the fusion protein of Example 1. FIG. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 4. It is a figure which shows the result of Example 5. It is a figure which shows the result of Example 6. It is a figure which shows the result of Example 8. It is a figure which shows the result of Example 9. It is a figure which shows the result of Example 10. It is a figure which shows the result of Example 11. It is a figure which shows the result of Example 12. It is a figure which shows the result of Example 13. It is a figure which shows the result of Example 14. It is a figure which shows the result of Example 15. It is a figure which shows the result of Example 16. It is a figure which shows the result of Example 17. It is a figure which shows the result of Example 18. It is a figure which shows the result of Example 19. It is a figure which shows the result of Example 19. FIG. 10 shows the results of Example 21. FIG. 10 shows the results of Example 21. It is a figure which shows the result of Example 22. It is a figure which shows the result of Example 22. It is a figure which shows the result of Example 23.

Claims (212)

i) an inhibitor of the Notch signaling pathway, or a polynucleotide encoding such an inhibitor, and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant,
Products included as simultaneous, concomitant, individual, or sequential use combination preparations to regulate the immune system.   2. The product of claim 1 wherein the inhibitor of Notch signaling does not act by down-regulating Notch or Notch ligand expression. i) an Notch antagonist, or an inhibitor of Notch signaling in the form of a polynucleotide encoding such an antagonist, and ii) a pathogen antigen or antigenic determinant, or a poly encoding a pathogen antigen or antigenic determinant Nucleotides
Products included as simultaneous, concomitant, individual, or sequential use combination preparations to regulate the immune system. i) an agent that inhibits Notch-Notch ligand interaction, or an inhibitor of Notch signaling in the form of a polynucleotide encoding such an agent, and ii) a pathogen antigen or antigenic determinant, or a pathogen antigen or antigen A polynucleotide encoding the determinant,
Products included as simultaneous, concomitant, individual, or sequential use combination preparations to regulate the immune system.   5. The product of claim 4, wherein the inhibitor of Notch signaling binds to a Notch ligand or Notch receptor so as to prevent Notch-Notch ligand interaction.   The product according to any one of claims 1 to 5, which is in the form of a pharmaceutical composition or a kit.   The product according to any one of claims 1 to 6, which is in the form of a therapeutic vaccine composition or kit for treating an infectious disease.   The product according to any one of claims 1 to 6, which is in the form of a prophylactic vaccine composition or kit for preventing infectious diseases.   The product according to any one of claims 1 to 8, wherein the inhibitor of Notch signaling is a factor capable of inhibiting the activity of Notch receptor or Notch ligand.   The product according to any one of claims 1 to 9, wherein the inhibitor of Notch signaling is a factor capable of inhibiting the activity of a downstream component of the Notch signaling pathway or down-regulating expression.   The product according to any one of claims 1 to 10, wherein the inhibitor of Notch signaling is a protein or polypeptide, or a polynucleotide encoding such a protein or polypeptide.   The product according to any one of claims 1 to 11, wherein the inhibitor of Notch signaling comprises or encodes the extracellular domain of Delta or a fragment thereof.   The product according to any one of claims 1 to 11, wherein the inhibitor of Notch signaling comprises or encodes the Serrate or Jagged extracellular domain, or a fragment thereof.   12. A product according to any one of the preceding claims, wherein the inhibitor of Notch signaling comprises or encodes the extracellular domain of Notch or a fragment thereof. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain, and optionally a Notch ligand N-terminal domain, or a heterologous amino acid sequence, but substantially free of a Notch ligand EGF-like domain;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least one Notch ligand EGF-like domain;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least two Notch ligand EGF-like domains;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain and one or two, but not more than two Notch ligand EGF-like domains;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 50% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 50% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising one or two but not more than two Notch ligand EGF-like domains having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch EGF-like domain having at least 50% amino acid sequence identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least 50% to EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising a NotchEGF-like domain having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 DSL domain, and at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 EGF-like domain A protein or polypeptide comprising at least one Notch ligand EGF-like domain having
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 DSL domain, and at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 EGF-like domain A protein or polypeptide comprising a Notch ligand EGF-like domain having 0, 1, or 2 but no more than 2;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 70% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 70% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 70% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 70% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising a Notch ligand EGF-like domain with 0, 1, or 2 but no more than 2 amino acid sequence identities,
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above. Inhibitors of Notch signaling are
i) an EGF domain having at least 70% amino acid sequence identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least 70% of EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising an EGF domain having amino acid sequence identity;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) polynucleotides encoding such proteins or polypeptides. 12. The product according to any one of 11 above.   26. A product as claimed in any one of claims 12 to 25 wherein the protein or polypeptide is fused to a heterologous amino acid sequence.   27. The product of claim 26, wherein the protein or polypeptide is fused to an immunoglobulin Fc (IgFc) domain.   28. The product of claim 27, wherein the IgFc domain is a human IgG1 or IgG4 Fc domain.   29. A product according to any one of claims 12 to 28, wherein the protein or polypeptide further comprises a Notch ligand N-terminal domain.   The product according to any one of claims 1 to 11, wherein the inhibitor of Notch signaling is an antibody, an antibody fragment, or an antibody derivative, or a polynucleotide encoding the antibody, antibody fragment, or antibody derivative.   32. The product of claim 30, wherein the antibody, antibody fragment, or antibody derivative binds to a Notch receptor or Notch ligand so as to prevent Notch ligand-receptor interaction.   Use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use as an immunostimulator, wherein the medicament is not used for reversing bacterial, infection, or tumor-induced immunosuppression, or for treating a tumor .   Use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use as an immunostimulatory agent, wherein the inhibitor does not act by down-regulating Notch or Notch ligand expression.   Use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use in vaccination against pathogens.   Use of an inhibitor of the Notch signaling pathway in the manufacture of a medicament for use as an adjuvant for vaccination against pathogens.   36. Use according to any one of claims 30 to 35, wherein the inhibitor of the Notch signaling pathway is a Notch signaling repressor or a factor that increases the expression or activity of a Notch signaling repressor.   36. Use according to any one of claims 30 to 35, wherein the inhibitor of the Notch signaling pathway is a factor capable of inhibiting the activity of the Notch receptor or Notch ligand.   36. Use according to any one of claims 30 to 35, wherein the inhibitor of the Notch signaling pathway is a factor capable of inhibiting the activity of a downstream component of the Notch signaling pathway or down-regulating expression.   36. Use according to any one of claims 30 to 35, wherein the inhibitor of the Notch signaling pathway is a factor that binds to the Notch receptor or Notch ligand so as to prevent Notch ligand-receptor interaction.   40. Use according to any one of claims 30 to 39, wherein the inhibitor of the Notch signaling pathway is a protein or polypeptide, or a polynucleotide encoding such a protein or polypeptide.   41. Use according to claim 40, wherein the factor comprises or encodes the extracellular domain of Delta or a fragment thereof.   41. Use according to claim 40, wherein the factor comprises or encodes the Serrate or Jagged extracellular domain, or a fragment thereof.   41. Use according to claim 40, wherein the factor comprises or encodes the extracellular domain of Notch, or a fragment thereof. An inhibitor of the Notch signaling pathway,
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least one Notch ligand EGF-like domain;
41. Use according to any one of claims 30 to 40, comprising ii) a multimer of such a protein or polypeptide, or iii) a polynucleotide encoding such a protein or polypeptide. An inhibitor of the Notch signaling pathway,
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least two Notch ligand EGF-like domains;
41. Use according to any one of claims 30 to 40, comprising ii) a multimer of such a protein or polypeptide, or iii) a polynucleotide encoding such a protein or polypeptide. An inhibitor of the Notch signaling pathway,
i) a protein or polypeptide comprising a Notch ligand DSL domain and 0, 1 or 2 but not more than 2 Notch ligand EGF-like domains;
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 50% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having the amino acid sequence identity of:
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 50% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising one or two but not more than two Notch ligand EGF-like domains having the amino acid sequence identity of:
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) an EGF domain having at least 50% amino acid sequence identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least 50% of EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising an EGF domain having amino acid sequence identity;
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 DSL domain, and at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 EGF-like domain A protein or polypeptide comprising at least one Notch ligand EGF-like domain having
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 DSL domain, and at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 EGF-like domain A protein or polypeptide comprising one or two but not more than two Notch ligand EGF-like domains,
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) Notch ligand DSL domain with at least 70% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 70% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having the amino acid sequence identity of:
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) Notch ligand DSL domain with at least 70% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 70% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising a Notch ligand EGF-like domain with one or two but no more than two amino acid sequence identities,
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40. An inhibitor of the Notch signaling pathway,
i) an EGF domain having at least 70% amino acid sequence identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least 70% of EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising an EGF domain having amino acid sequence identity;
31. comprising a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 41. Use according to any one of 40.   55. Use according to any one of claims 40 to 54, wherein the protein or polypeptide is fused to a heterologous amino acid sequence.   56. The use of claim 55, wherein the protein or polypeptide is fused to an immunoglobulin Fc (IgFc) domain.   57. Use according to claim 56, wherein the IgFc domain is a human IgGl or IgG4 Fc domain.   40. Use according to any one of claims 32 to 39, wherein the inhibitor of Notch signaling pathway is an antibody, antibody fragment, or antibody derivative, or a polynucleotide encoding an antibody, antibody fragment, or antibody derivative.   59. The use of claim 58, wherein the antibody, antibody fragment, or antibody derivative binds to a Notch receptor or Notch ligand so as to prevent Notch ligand-receptor interaction.   Use of a binding agent that binds to a Notch ligand or a polynucleotide encoding such a binding agent so as to prevent binding of the ligand to the Notch receptor in the manufacture of a medicament for use as an immunostimulant.   Use of an antibody or antibody derivative that binds to a Notch receptor or Notch ligand, or a polynucleotide encoding such an antibody or antibody derivative, in the manufacture of a medicament for use as an immunostimulatory agent.   A method of stimulating the immune system by administering an inhibitor of the Notch signaling pathway, which does not involve reversal of bacterial, infection, or tumor-induced immunosuppression, or treatment of a tumor.   A method of stimulating the immune system by administering an inhibitor of the Notch signaling pathway, wherein the inhibitor does not act by down-regulating Notch or Notch ligand expression.   A method of stimulating the immune system to treat or prevent infection by administering an inhibitor of Notch signaling pathway, the method comprising no reversal of bacterial, infection, or tumor-induced immune suppression, or treatment of a tumor .   A method of stimulating the immune system to treat or prevent infection by administering an inhibitor of Notch signaling pathway, wherein the inhibitor of Notch signaling pathway down-regulates expression of Notch or Notch ligand How does not work by.   A method of vaccinating against a pathogen by administering an inhibitor of the Notch signaling pathway.   A method of enhancing vaccination against pathogens by administering an inhibitor of Notch signaling pathway.   A method of treating chronic pathogen infection by administering an inhibitor of Notch signaling pathway.   A method for increasing the immune response of a subject to a vaccine antigen or antigenic determinant, wherein the polynucleotide encodes the vaccine antigen or antigenic determinant simultaneously, separately or sequentially with the vaccine antigen or antigenic determinant Administering an effective amount of an inhibitor of Notch signaling pathway to said subject simultaneously, separately or sequentially.   70. The method of any one of claims 62 to 69, wherein the Notch signaling pathway inhibitor comprises a protein or polypeptide, or a polynucleotide encoding such a protein or polypeptide.   70. The method according to any one of claims 62 to 69, wherein the factor comprises or encodes the extracellular domain of Delta or a fragment thereof.   70. The method of any one of claims 62 to 69, wherein the Notch signaling pathway inhibitor comprises or encodes a Serrate or Jagged extracellular domain, or a fragment thereof.   70. The method of any one of claims 62 to 69, wherein the inhibitor of the Notch signaling pathway comprises or encodes the extracellular domain of Notch, or a fragment thereof. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain and at least one Notch ligand EGF-like domain;
70. A method according to any one of claims 62 to 69 comprising ii) a multimer of such a protein or polypeptide, or iii) a polynucleotide encoding such a protein or polypeptide. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain, and at least two Notch ligand EGF-like domains;
70. A method according to any one of claims 62 to 69 comprising ii) a multimer of such a protein or polypeptide, or iii) a polynucleotide encoding such a protein or polypeptide. Inhibitors of Notch signaling are
i) a protein or polypeptide comprising a Notch ligand DSL domain and 0, 1 or 2 but not more than 2 Notch ligand EGF-like domains;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4 and at least 50% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having the amino acid sequence identity of:
from ii) a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 50% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising 0, 1 or 2 but not more than 2 Notch ligand EGF-like domains having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) an EGF domain having at least 50% amino acid sequence identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least 50% of EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising an EGF domain having amino acid sequence identity;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 DSL domain, and at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 EGF-like domain A protein or polypeptide comprising at least one Notch ligand EGF-like domain having
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 DSL domain, and at least 50% amino acid sequence identity to the human Jagged 1 or Jagged 2 EGF-like domain A protein or polypeptide comprising a Notch ligand EGF-like domain, having 0, 1 or 2 but no more than 2;
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 70% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 70% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising at least one Notch ligand EGF-like domain having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) Notch ligand DSL domain with at least 70% amino acid sequence identity to the DSL domain of human Delta1, Delta3, or Delta4, and at least 70% to the EGF-like domain of human Delta1, Delta3, or Delta4 A protein or polypeptide comprising one or two but not more than two Notch ligand EGF-like domains having the amino acid sequence identity of:
from ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69. Inhibitors of Notch signaling are
i) an EGF domain having at least 70% amino acid sequence identity to EGF11 of human Notch1, Notch2, Notch3, or Notch4, and at least 70% of EGF12 of human Notch1, Notch2, Notch3, or Notch4 A protein or polypeptide comprising an EGF domain having amino acid sequence identity;
from ii) a multimer of such proteins or polypeptides (each monomer may be the same or different), or iii) a polynucleotide encoding such a protein or polypeptide. 70. The method according to any one of 69.   70. The method according to any one of claims 62 to 69, wherein the protein or polypeptide is fused to a heterologous amino acid sequence.   86. The method of claim 85, wherein the protein or polypeptide is fused to an immunoglobulin Fc (IgFc) domain.   90. The method of claim 86, wherein the IgFc domain is a human IgG4 Fc domain.   70. The method according to any one of claims 62 to 69, wherein the inhibitor of the Notch signaling pathway is a Notch signaling repressor or a factor that increases Notch signaling repressor expression or activity.   70. The method according to any one of claims 62 to 69, wherein the inhibitor of Notch signaling pathway is a factor capable of inhibiting the activity of Notch receptor or Notch ligand.   70. The method according to any one of claims 62 to 69, wherein the inhibitor of the Notch signaling pathway is a factor capable of inhibiting the activity of a downstream component of the Notch signaling pathway or down-regulating expression.   70. The method according to any one of claims 62 to 69, wherein the inhibitor of the Notch signaling pathway is a factor that binds to a Notch receptor or a Notch ligand so as to prevent Notch-Notch ligand interaction.   92. The method of claim 91, wherein the factor is a protein or polypeptide, or a polynucleotide encoding such a protein or polypeptide.   70. The method according to any one of claims 62 to 69, wherein the inhibitor of the Notch signaling pathway is an antibody, antibody fragment, or antibody derivative, or a polynucleotide encoding an antibody, antibody fragment, or antibody derivative.   94. The method of claim 93, wherein the antibody, antibody fragment, or antibody derivative binds to a Notch receptor or Notch ligand so as to prevent Notch-Notch ligand interaction.   A method of stimulating the immune system by administering an antibody or antibody derivative that binds to a Notch receptor or Notch ligand, or by administering a polynucleotide encoding such an antibody or antibody derivative.   An adjuvant composition comprising an inhibitor of Notch signaling pathway.   99. The composition of claim 96, wherein the Notch signaling pathway inhibitor is a Notch signaling repressor, or a factor that increases Notch signaling repressor expression or activity.   99. The composition of claim 96, wherein the Notch signaling pathway inhibitor is a factor capable of inhibiting the activity of a Notch receptor or Notch ligand.   99. The composition of claim 96, wherein the Notch signaling pathway inhibitor is a factor capable of inhibiting the activity of a downstream component of the Notch signaling pathway or downregulating expression.   99. The composition of claim 96, wherein the inhibitor of the Notch signaling pathway is a factor that binds to a Notch receptor or Notch ligand so as to prevent Notch-Notch ligand interaction.   99. The composition of claim 96, wherein the factor is a protein or polypeptide, or a polynucleotide encoding such a protein or polypeptide.   102. The composition of claim 101, wherein the Notch signaling pathway inhibitor is an antibody, antibody fragment, or antibody derivative, or a polynucleotide encoding an antibody, antibody fragment, or antibody derivative.   105. The composition of claim 102, wherein the antibody, antibody fragment, or antibody derivative binds to a Notch receptor or Notch ligand so as to prevent Notch-Notch ligand interaction.   99. The composition of claim 96, wherein the factor comprises or encodes the extracellular domain of Delta or a fragment thereof.   99. The composition of claim 96, wherein the factor comprises or encodes a Serrate or Jagged extracellular domain, or a fragment thereof.   99. The composition of claim 96, wherein the factor comprises or encodes the extracellular domain of Notch, or a fragment thereof.   107. A vaccine composition comprising the adjuvant composition according to any one of claims 94 to 106 and a pathogen antigen or antigenic determinant, or a polynucleotide encoding the pathogen antigen or pathogen determinant.   108. The vaccine composition of claim 107, comprising a polynucleotide encoding a viral, fungal, parasitic or bacterial antigen or antigenic determinant, or a viral, fungal, parasitic or bacterial antigen or antigenic determinant. i) an inhibitor of the Notch signaling pathway, and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant,
Products included as simultaneous, concomitant, individual, or sequential use combination preparations to regulate the immune system. i) a Notch antagonist, and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant,
Products included as simultaneous, concomitant, individual, or sequential use combination preparations to regulate the immune system.   111. A product according to claim 109 or 110 for increasing effector T cell activity. A method of regulating the immune system in a mammal, comprising:
i) administering an effective amount of an inhibitor of Notch signaling pathway, and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant, simultaneously, simultaneously, separately or sequentially. Including methods. For simultaneous, simultaneous, separate or sequential use in the regulation of the immune system,
i) an inhibitor of the Notch signaling pathway, and ii) a pathogen antigen or antigenic determinant, or a combination of polynucleotides encoding a pathogen antigen or antigenic determinant.   Inhibitors of the Notch signaling pathway for use in modulating the immune system in the same, simultaneous, separate or sequential combination with a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant. In the manufacture of drugs that regulate the immune system,
i) use of Notch signaling pathway inhibitors, and ii) pathogen antigens or antigenic determinants, or combinations of polynucleotides encoding pathogen antigens or antigenic determinants.   Inhibition of the Notch signaling pathway in the manufacture of agents that modulate the immune system, simultaneously, contemporaneously, individually or sequentially in combination with a pathogen antigen or antigenic determinant or a polynucleotide encoding a pathogen antigen or antigenic determinant Agent use.   A pharmaceutical kit comprising an inhibitor of Notch signaling pathway and a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant.   A conjugate comprising first and second sequences, wherein the first sequence comprises a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant, wherein the second sequence comprises A conjugate comprising a polypeptide or polynucleotide for modulating Notch signaling.   119. The conjugate of claim 118, in the form of a vector comprising a first polynucleotide sequence encoding a modulator of the Notch signaling pathway and a second polynucleotide sequence encoding a pathogen antigen or antigenic determinant. .   120. The conjugate of claim 119, which is in the form of an expression vector.   121. A conjugate according to any one of claims 118 to 120, wherein the first polynucleotide sequence encodes a Notch ligand, or a fragment, derivative, homologue, analogue or allelic variant thereof.   122. The conjugate of claim 121, wherein the first polynucleotide sequence encodes a Delta or Serrate / Jagged protein, or a fragment, derivative, homologue, analog, or allelic variant thereof.   123. A conjugate according to any one of claims 118 to 122, wherein the first polynucleotide sequence encodes a protein or polypeptide comprising a Notch ligand DSL domain and at least one Notch ligand EGF-like domain.   124. The conjugate of claim 123, wherein the first polynucleotide sequence encodes a protein or polypeptide comprising a Notch ligand DSL domain and at least two Notch ligand EGF-like domains.   124. The conjugate of claim 123, wherein the first polynucleotide sequence encodes a protein or polypeptide comprising a Notch ligand DSL domain and one or two, but not more than two Notch ligand EGF-like domains.   126. A conjugate according to any one of claims 118 to 125, wherein the first and second polynucleotide sequences are operably linked to one or more promoters. i) inhibitors of Notch signaling pathways; and ii) pathogen antigens or antigenic determinants, or polynucleotides encoding pathogen antigens or antigenic determinants, in any order, against pathogen antigens or antigenic determinants A method of increasing the immune response. i) an agent that binds to Notch or a Notch ligand and inhibits Notch-Notch ligand interaction; and ii) a pathogen antigen or antigenic determinant, or a polynucleotide encoding a pathogen antigen or antigenic determinant, in any order To increase the immune response to a pathogen antigen or antigenic determinant.   129. A method according to claim 127 or 128 for treating an infection.   129. A method according to claim 127 or 128 for treating a chronic infection.   129. A method according to claim 127 or 128 for prophylactic vaccination.   132. The method of claim 131, which provides protective immunity. i) a protein or polypeptide comprising a Notch ligand DSL domain and 0, 1 or 2 but not more than 2 Notch ligand EGF-like domains;
ii) multimers of such proteins or polypeptides (each monomer may be the same or different), or iii) a pharmaceutical composition comprising a polynucleotide encoding such a protein or polypeptide. By administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a method of altering an immune response by administering a polynucleotide encoding such a Notch ligand protein or polypeptide. By administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a method of increasing an immune response by administering a polynucleotide encoding such a Notch ligand protein or polypeptide. By administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a method of reducing immune tolerance by administering a polynucleotide encoding such a Notch ligand protein or polypeptide. By administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a method of altering T cell activity by administering a polynucleotide encoding such a Notch ligand protein or polypeptide. By administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a method of increasing helper (T _H ) or cytotoxic (T _C ) T cell activity by administering a polynucleotide encoding such a Notch ligand protein or polypeptide. By administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally one or two EGF repeat domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a method of reducing the activity of regulatory T cells by administering a polynucleotide encoding such a Notch ligand protein or polypeptide.   140. The method of claim 138 or 139, wherein the regulatory T cell is a Tr1 regulatory T cell. Administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
141. The method of any one of claims 1-140, comprising administering a polynucleotide encoding such a Notch ligand protein or polypeptide. Administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) one Notch ligand EGF domain,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
144. The method of any one of claims 134 to 141, comprising administering a polynucleotide encoding such Notch ligand protein or polypeptide. Administering a Notch ligand protein or polypeptide consisting essentially of the following components:
i) Notch ligand DSL domain,
ii) two Notch ligand EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
143. A method according to any one of claims 134 to 142, comprising administering a polynucleotide encoding such Notch ligand protein or polypeptide.   145. The method of any one of claims 134 to 143, comprising administering a Notch ligand protein or polypeptide that is not bound to a cell or a portion of a cell.   145. The method of any one of claims 134 to 143, comprising administering a Notch ligand protein or polypeptide that is bound to the cell or a portion of the cell.   146. The method according to any one of claims 134 to 145, wherein the Notch ligand protein or polypeptide is a Notch receptor antagonist.   147. The method of any one of claims 134 to 146, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes a heterologous amino acid sequence that corresponds to all or part of an immunoglobulin Fc domain.   148. The method of any one of claims 134 to 147, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a mammalian Notch ligand sequence.   149. The method of any one of claims 134 to 148, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a human Notch ligand sequence.   The Notch ligand protein, polypeptide, or polynucleotide comprises or encodes a Delta, Serrate, or Jagged Notch ligand domain, or a domain having at least 30% amino acid sequence similarity or identity thereto. 150. The method according to any one of 134 to 149.   The Notch ligand protein, polypeptide or polynucleotide comprises a Delta1, Delta3, Delta4, Jagged1 or Jagged2 Notch ligand domain, or a domain having at least 30% amino acid sequence similarity or identity thereto, or 151. A method according to any one of claims 134 to 150, which codes.   152. The method of any one of claims 134 to 151, wherein the protein, polypeptide, or polynucleotide is administered to the patient in vivo.   152. The method according to any one of claims 134 to 151, wherein the protein, polypeptide or polynucleotide is administered to the patient's cells ex vivo.   154. The method of claim 153, wherein the cell is administered to the patient after administration of the protein, polypeptide, or polynucleotide. Notch ligand protein or polypeptide consisting essentially of the following components for use in the treatment of disease:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a polynucleotide encoding such a Notch ligand protein or polypeptide. A Notch ligand protein or polypeptide or polynucleotide for use according to claim 155, wherein the Notch ligand protein or polypeptide consists essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
Alternatively, the polynucleotide encodes such a Notch ligand protein or polypeptide, or a Notch ligand protein or polypeptide, or polynucleotide. A Notch ligand protein or polypeptide or polynucleotide for use according to claim 155, wherein the Notch ligand protein or polypeptide consists essentially of the following components:
i) Notch ligand DSL domain,
ii) one Notch ligand EGF domain,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the polynucleotide encodes such a Notch ligand protein or polypeptide, or a Notch ligand protein or polypeptide, or polynucleotide. A Notch ligand protein or polypeptide or polynucleotide for use according to claim 155, wherein the Notch ligand protein or polypeptide consists essentially of the following components:
i) Notch ligand DSL domain,
ii) two Notch ligand EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the polynucleotide encodes such a Notch ligand protein or polypeptide, or a Notch ligand protein or polypeptide, or polynucleotide.   159. Notch ligand protein or polypeptide or polynucleotide for use according to any one of claims 155 to 158, wherein the Notch ligand protein or polypeptide is not bound to a cell or part of a cell.   159. Notch ligand protein or polypeptide or polynucleotide for use according to any one of claims 155 to 158, wherein the Notch ligand protein or polypeptide is bound to a cell or a portion of a cell.   170. A Notch ligand protein or polypeptide or polynucleotide for use according to any one of claims 155 to 160, wherein the Notch ligand protein or polypeptide activates a Notch receptor.   164. For use according to any one of claims 155 to 161, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes a heterologous amino acid sequence corresponding to all or part of an immunoglobulin Fc segment. Notch ligand protein or polypeptide, or polynucleotide.   165. Notch ligand protein or polypeptide for use according to any one of claims 155 to 162, wherein the Notch ligand protein, polypeptide or polynucleotide comprises or encodes at least part of a mammalian Notch ligand sequence. Or a polynucleotide.   164. Notch ligand protein or polypeptide for use according to any one of claims 155 to 163, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a human Notch ligand sequence, Or a polynucleotide.   165-164, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes a Delta, Serrate, or Jagged Notch ligand domain, or a domain having at least 30% amino acid sequence similarity thereto. A Notch ligand protein or polypeptide, or polynucleotide for use according to any one of.   A claim wherein the Notch ligand protein, polypeptide or polynucleotide comprises or encodes a Delta1, Delta3, Delta4, Jagged1 or Jagged2 Notch ligand domain, or a domain having at least 30% amino acid sequence similarity thereto 164. Notch ligand protein or polypeptide or polynucleotide for use according to any one of clauses 155 to 165. Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that modifies the immune response:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that modifies the immune response:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains, and iii) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that increases the immune response:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a drug that reduces immune tolerance:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that modifies T cell activity:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide. Helper (T _H) or cytotoxic (T _C) in the manufacture of a medicament for increasing the _T cell activity, Notch ligand protein or polypeptide consisting essentially of the following ingredients,
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components in the manufacture of a medicament that reduces the activity of regulatory T cells:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, the use of a polynucleotide encoding such a Notch ligand protein or polypeptide.   178. Use according to claim 173 for reducing the activity of Tr1 or Th3 regulatory T cells. A Notch ligand protein or polypeptide consists essentially of the following components:
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
175. Use according to any one of claims 167 to 174, wherein the polynucleotide encodes such a Notch ligand protein or polypeptide. A Notch ligand protein or polypeptide consists essentially of the following components:
i) Notch ligand DSL domain,
ii) one EGF domain iii) optionally all or part of the Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
175. Use according to any one of claims 167 to 174, wherein the polynucleotide encodes such a Notch ligand protein or polypeptide. A Notch ligand protein or polypeptide consists essentially of the following components:
i) Notch ligand DSL domain,
ii) two EGF domains iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
175. Use according to any one of claims 167 to 174, wherein the polynucleotide encodes such a Notch ligand protein or polypeptide.   178. Use according to any one of claims 167 to 177, wherein the Notch ligand protein or polypeptide is not bound to a cell or a part of a cell.   178. Use according to any one of claims 167 to 177, wherein the Notch ligand protein or polypeptide is bound to a cell or a part of a cell.   179. Use according to any one of claims 167 to 179, wherein the Notch ligand protein or polypeptide inhibits the Notch receptor.   181. Use according to any one of claims 167 to 180, wherein the Notch ligand protein, polypeptide or polynucleotide comprises or encodes a heterologous amino acid sequence corresponding to all or part of an immunoglobulin Fc segment.   184. Use according to any one of claims 167 to 181 wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a mammalian Notch ligand sequence.   183. Use according to any one of claims 167 to 182, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a human Notch ligand sequence.   The Notch ligand protein, polypeptide, or polynucleotide comprises or encodes a Delta, Serrate, or Jagged Notch ligand domain, or a domain having at least 30% amino acid sequence similarity or identity thereto. 167. Use according to any one of 167 to 183.   The Notch ligand protein, polypeptide or polynucleotide comprises a Delta1, Delta3, Delta4, Jagged1 or Jagged2 Notch ligand domain, or a domain having at least 30% amino acid sequence similarity or identity thereto, or 185. Use according to any one of claims 167 to 184 encoding. Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) optionally one or two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) optionally all or part of a Notch ligand N-terminal domain, and iii) optionally one or more heterologous amino acid sequences,
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) one EGF domain,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of the following components, optionally in combination with a pharmaceutically acceptable carrier:
i) Notch ligand DSL domain,
ii) two EGF domains,
iii) optionally all or part of a Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Alternatively, a pharmaceutical composition comprising a polynucleotide encoding such a Notch ligand protein or polypeptide.   189. The pharmaceutical composition according to any one of claims 186 to 189, wherein the Notch ligand protein or polypeptide is not bound to a cell or part of a cell.   190. The pharmaceutical composition according to any one of claims 186 to 189, wherein the Notch ligand protein or polypeptide is bound to a cell or a part of a cell.   196. The pharmaceutical composition according to any one of claims 186 to 191, wherein the Notch ligand protein or polypeptide inhibits the Notch receptor.   195. The pharmaceutical composition according to any one of claims 186 to 192, wherein the Notch ligand protein, polypeptide or polynucleotide comprises or encodes a heterologous amino acid sequence corresponding to all or part of an immunoglobulin Fc segment.   194. The pharmaceutical composition of any one of claims 186 to 193, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a mammalian Notch ligand sequence.   195. The pharmaceutical composition according to any one of claims 186 to 194, wherein the Notch ligand protein, polypeptide, or polynucleotide comprises or encodes at least a portion of a human Notch ligand sequence.   The Notch ligand protein, polypeptide, or polynucleotide comprises or encodes a Delta, Serrate, or Jagged Notch ligand domain, or a domain having at least 30% amino acid sequence similarity or identity thereto. 186. A pharmaceutical composition according to any one of 186 to 195.   A claim wherein the Notch ligand protein, polypeptide or polynucleotide comprises or encodes a Delta1, Delta3, Delta4, Jagged1 or Jagged2 Notch ligand domain, or a domain having at least 30% amino acid sequence similarity thereto The pharmaceutical composition according to any one of Items 186 to 196. Notch ligand protein or polypeptide consisting essentially of:
i) Notch ligand DSL domain,
ii) optionally all or part of the Notch ligand N-terminal domain,
iii) an immunoglobulin Fc domain, and iv) optionally one or more additional heterologous amino acid sequences,
Or a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of:
i) Notch ligand DSL domain,
ii) one EGF domain iii) optionally all or part of the Notch ligand N-terminal domain, and iv) optionally one or more heterologous amino acid sequences,
Or a polynucleotide encoding such a Notch ligand protein or polypeptide. Notch ligand protein or polypeptide consisting essentially of:
i) Notch ligand DSL domain,
ii) two EGF domains, and iii) optionally one or more heterologous amino acid sequences,
Or a polynucleotide sequence encoding such a Notch ligand protein or polypeptide.   The Notch ligand protein or polypeptide according to any one of claims 198 to 200, which is not bound to a cell or a part of a cell.   The Notch ligand protein or polypeptide according to any one of claims 198 to 200, which is bound to a cell or a part of a cell.   The Notch ligand protein or polypeptide or polynucleotide according to any one of claims 198 to 202, wherein the Notch ligand protein or polypeptide activates a Notch receptor.   204. A Notch ligand protein or polypeptide, or polynucleotide according to any one of claims 198 to 203, comprising or encoding a heterologous amino acid sequence corresponding to all or part of an immunoglobulin Fc segment.   205. A Notch ligand protein or polypeptide, or polynucleotide according to any one of claims 198 to 204, comprising or encoding at least a portion of a mammalian Notch ligand sequence.   206. A Notch ligand protein or polypeptide, or polynucleotide according to any one of claims 198 to 205, comprising or encoding at least a portion of a human Notch ligand sequence.   205. A Notch ligand protein according to any one of claims 198 to 206, comprising or encoding a Delta, Serrate or Jagged Notch ligand domain, or a domain having at least 30% amino acid sequence similarity thereto. Polypeptide or polynucleotide.   207. The Delta1, Delta3, Delta4, Jagged1 or Jagged2 Notch ligand domain, or comprising or encoding a domain having at least 30% amino acid sequence similarity thereto. Notch ligand protein or polypeptide, or polynucleotide.   A vector comprising a polynucleotide encoding the Notch ligand protein or polypeptide of any one of claims 196 to 208.   209. A host cell transformed or transfected with the vector of claim 209. 209. A cell transfected with a polynucleotide encoding Notch ligand protein or polypeptide according to any one of claims 196 to 208 and / or encoding such protein or polypeptide on its surface.