JP2004531457A - Methods and compositions for modulating T cell activation and uses thereof - Google Patents

Abstract

The present invention relates generally to the fields of immunology and neuroimmunology. In particular, the present invention provides a method for reducing or inhibiting T cell activation, comprising the step of administering to a mammal an effective amount of an antagonist of NCAM L1 wherein Reduction or inhibition of cell activation is preferred, thereby reducing or inhibiting T cell activation in the mammal. The present invention also provides a method for enhancing T cell activation, the method comprising multimerizing neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding L1. Administering to the mammal an effective amount of a molecule or a functional derivative or fragment thereof, or an agent that enhances L1 production and / or costimulatory function, wherein T cell activation is preferred, Enhances T cell activation in mammals.

Description

改変は当業者には明らかなので、本発明が添付の特許請求の範囲の範囲によってのみ限定されることが意図される。
【図面の簡単な説明】
【図１】
Ｌ１は、臍帯血由来のＤＣによって発現され、そして同種異系ＭＬＲに寄与する。（Ａ）、ＣＤ３４＋細胞を、正常な臍帯血から富化し、そしてＬ１発現について染色する前に２１日間にわたって拡張した。富化したＣＤ３４＋細胞をまた、培養前（０日目）にＬ１について染色した。これらの細胞を、ＦＩＴＣに直接結合体化されたｍＡｂ ５Ｇ３で染色した。（Ｂ）、臍帯血由来のＤＣ（ＤＣ＋）を、一方向ＭＬＲにおいて、異なるドナー由来のＰＢＭＣと共存培養した。細胞を、抗Ｌ１ ｍＡｂ ５Ｇ３の存在下もしくは非存在下においてか、またはコントロール抗体ＵＰＣ１０と共に、培養した。ＣＤ３４＋富化画分と同じ条件下で培養されたＣＤ３４陰性臍帯血細胞をまた、刺激物質として試験した（非ＤＣ）。培養物を、３日間の共存培養中の最後の１８時間の間に、［３Ｈ］−チミジンでパルス（ｐｕｌｓｅ）した。処理を、三連で実施した。エラーバーは、±１ＳＥである。
【図２】
抗Ｌ１抗体５Ｇ３および可溶性Ｌ１は、マイトジェンに対する自己Ｔ細胞応答を阻害する。（Ａ）ＰＢＭＣを、ｍＡｂ ５Ｇ３の非存在下もしくは存在下、またはコントロール抗体ＵＰＣ１０の存在下において、ＰＨＡ（１０ｍｇ／ｍｌ）で処理した。さらなるＰＢＭＣを、可溶性組換えＬ１−ＥＣＤ（ｓＬ１；１００ｍｇ／ｍｌ）の存在下において培養した。（Ｂ）ＰＢＭＣを、ｍＡｂ ５Ｇ３の非存在下もしくは存在下、またはコントロール抗体ＵＰＣ１０の存在下において、一定範囲のＰＨＡ濃度で処理した。培養物を、３日間の共存培養のなかの最後の１８時間の間に、［３Ｈ］−チミジンでパルス（ｐｕｌｓｅ）した。（Ｂ、挿入図）いくつかの細胞をＰＨＡで処理し、そして１８時間のみ、［３Ｈ］−チミジンでパルスした。（Ｃ）富化したＣＤ４＋またはＣＤ８＋Ｔ細胞サブセットを、ｍＡｂ ５Ｇ３の非存在下もしくは存在下、またはコントロール抗体ＵＰＣ１０の存在下において、ＰＨＡで処理した。培養物を、３日間の共存培養のなかの最後の１８時間の間に、［３Ｈ］−チミジンでパルスした。処理を三連で実施した。エラーバーは、±１ＳＥである。
【図３】
精製された固定化Ｌ１は、ＣＤ３連結に応答してＴ細胞増殖を増強する。９６ウェルプレートのウェルを、抗ＣＤ３抗体（ＯＫＴ３：２５Ｕ／ｍｌ）、精製されたＬ１−エクトドメイン（ｅｃｔｏｄｏｍａｉｎ）（４０ｍｇ／ｍｌ）、またはＬ１およびこの抗体の両方の配合物を用いて、前処理した。このウェルを洗浄した後、ＰＢＭＣを７２時間にわたり、プレコーティングされたウェルまたは処理されていないウェルに添加した。ＰＢＭＣを、ｍＡｂ ５Ｇ３の非存在下もしくは存在下、またはコントロール抗体ＵＰＣ１０の存在下において培養した。培養物を、３日間の共存培養のなかの最後の１８時間の間に、［３Ｈ］−チミジンでパルスした。処理を三連で実施した。エラーバーは、±１ＳＥである。
【図４】
Ｌ１のトランスフェクションおよびデノボ発現は、ＭＬＲを増強する。（ＡおよびＢ）照射された野生型（ＷＴ細胞）またはＬ１でトランスフェクトされたＪ５５８Ｌ骨髄腫細胞（Ｌ１＋細胞）を、一方向ＭＬＲにおいて、ＰＢＭＣ（Ａ）または富化されたＣＤ４＋もしくはＣＤ８＋Ｔ細胞サブセット（Ｂ）と共に共存培養した。Ｌ１＋Ｊ５５８Ｌ骨髄腫細胞を、抗Ｌ１ ｍＡｂ ５Ｇ３（８０ｍｇ／ｍｌ）の存在下もしくは非存在下、またはコントロールｍＡｂ ＵＰＣ１０（８０ｍｇ／ｍｌ）の存在下において共存培養した。（Ａ、挿入図）Ｌ１＋Ｊ５５８Ｌ骨髄腫細胞およびＰＢＭＣのさらなる共存培養を、２０〜１６０ｍｇ／ｍｌまで変動する濃度で、ｍＡｂ ５Ｇ３と共にインキュベートした（挿入図）。培養物を、３日間の共存培養のなかの最後の１８時間の間に、［３Ｈ］−チミジンでパルスした。処理を三連で実施した。エラーバーは、±１ＳＥである。
【図５】
阻害抗体５Ｇ３は、Ｌ１でトランスフェクトされた骨髄腫細胞によるＬ１−Ｌ１媒介性接着をブロックする。野生型（ＷＴ）またはＬ１でトランスフェクトされた（Ｌ１＋）骨髄腫細胞は、コントロールｍＡｂ（ＵＰＣ１０）または抗Ｌ１ ｍＡｂ（５Ｇ３）の存在下または非存在下において、固定化された組換えＬ１に接着される。接着細胞を、４０×高倍率の対物レンズを用いて、単位面積あたりで計数した。実験処理を三連で実施し、１ウェルあたり４つの領域を計数した。エラーバーは、±１ＳＥを表す。[0001]
(Technical field)
The present invention relates generally to the field of immunology or neuroimmunology. In particular, the present invention provides methods for reducing or inhibiting T cell activation. The method comprises the step of administering an effective amount of an antagonist of NCAM L1 to a mammal in which reduction or inhibition of T cell activation is desired, thereby reducing or inhibiting T cell activation in the mammal. Include. Further provided are combinations and combinatorial methods for modulating T cell activation. The present invention also provides a method for enhancing T cell activation. The method comprises the steps of: providing an effective amount of a multimerized neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding the L1 or a functional derivative or fragment thereof; Administering an agent that enhances the production and / or costimulatory function of to a mammal in which T cell activation is desired, thereby enhancing T cell activation in the mammal.
[0002]
(Background technology)
The current paradigm for T cell activation is that optimal activation is provided by two signals, the first being occupation of the T cell receptor (TCR) by the MHC / antigen complex, and the second (Provided by one or more costimulatory ligands) (1). A series of molecules on the APC surface can function as costimulatory ligands, including members of the immunoglobulin superfamily (IgSF) such as B7-1, B7-2 and ICAM-1 (1). .
[0003]
Previous studies have identified L1 as a neuronal CAM. It also belongs to IgSF (2). To date, the function of L1 has been almost exclusively associated with neurological processes, including axonal guidance (3, 4). Although such L1-mediated processes were primarily attributed to homophilic L1-L1 binding (5), this CAM also contains axonin1 / TAG1, chondroitin sulfate proteoglycan, It can interact with multiple heterophilic ligands, including laminin and certain integrins (6, 7). L1 has also been shown to support cis interactions with the thermostable antigens CD24 (8) and the tetraspan molecule CD9 (9).
[0004]
Despite its neuronal manifestations, L1 expression has recently been described in cells of both lymphoid and myeloid monocyte origin (10,11). In particular, L1 can be detected in freshly isolated peripheral blood mononuclear cells, and in functionally mature mononuclear cell-derived dendritic cells (DC), and in situ in vesicular DC. (11). Further constitutive expression was evident in a subset of B cells and was described for CD4 + T cells (10, 11). Despite these findings, little is known about the function of L1 in the immune system. One recent study has shown that L1 is important in maintaining lymph node architecture (12). Using various experimental approaches, including one-way MLR and mitogen activation assays, we demonstrate herein that L1 can be recruited as a costimulatory molecule in T cell activation. . In this capacity, L1 contributes to the initiation of the human immune response in normal and disease processes, including those involving the nervous system.
[0005]
Accordingly, it is an object of the present invention to provide a method for modulating T cell activation using NCAM L1 as a regulatory target. It is another object of the present invention to provide formulations and combinatorial methods for modulating T cell activation.
[0006]
(Disclosure of the Invention)
The present invention relates generally to the field of immunology or neuroimmunology. In one aspect, the invention provides a method for enhancing T cell activation. The method comprises the steps of: providing an effective amount of a multimerized neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding the L1 or a functional derivative or fragment thereof; Administering an agent that enhances the production and / or costimulatory function of to a mammal in which T cell activation is desired, thereby enhancing T cell activation in the mammal.
[0007]
Any multimerized (eg, dimerized) NCAM L1, or functional derivative or fragment thereof, that can function as a stimulatory molecule in the activation of T cells, and such NCAM L1, or Any nucleic acid encoding a functional derivative or fragment can be used in the methods of the invention. Preferably, NCAM L1, or a functional derivative or fragment thereof, is capable of performing L1-L1 homophilic interactions (eg, mediates L1-L1 binding between antigen presenting cells (APCs) and T cells. obtain). Also preferably, NCAM L1 or a functional derivative or fragment thereof supports an interaction with an integrin involved in T cell activation (eg, trans- or cis-interaction with an integrin α5β1 or αvβ3). To support). More preferably, NCAM L1, or a functional derivative or fragment thereof, supports an interaction with a ligand involved in costimulation (eg, supports a cis-type interaction with CD9 and / or CD24).
[0008]
Any factor that enhances NCAM L1 production and / or costimulatory function can be used in the methods of the invention. Preferably, the factor used herein is an L1-L1 homophilic interaction between two NCAM L1, or functional derivatives or fragments thereof, or one NCAM L1, or a functional derivative or fragment thereof. And enhances the interaction between integrin involved in T cell activation, or between one NCAM L1, or a functional derivative or fragment thereof, and a ligand involved in costimulation. One exemplary factor is the anti-NCAM L1 monoclonal antibody 557. B6 (Appel et al., J. Neurobiol. 28 (3): 297-312 (1995)).
[0009]
NCAM L1 from any mammalian source, or a functional derivative or fragment thereof, may be used. Preferably, when the mammal to be treated is a human, NCAM L1 of human origin, or a functional derivative or fragment thereof, is used.
[0010]
Using the method of the present invention, CD4 ⁺ T cells, CD8 ⁺ T cells or both can be activated. The methods of the invention can be used to treat a mammal, either prophylactically or therapeutically, having a disease or disorder associated with a defect in T cell activation. Examples of such diseases or disorders include, but are not limited to, tumors, cancers and infections. A mammal (preferably a human) having a tumor, cancer or infection is treated with the method of the invention.
[0011]
In another aspect, the invention relates to a formulation. The formulation comprises: a) an effective amount of the multimerized NCAM L1, or a functional derivative or fragment thereof, or a nucleic acid encoding the L1, or a functional derivative or fragment thereof, or An agent that enhances this L1 production and / or costimulatory function; and b) an effective amount of another costimulatory molecule. Preferably, the formulation is in the form of a pharmaceutical composition. Furthermore, the invention relates to a method for enhancing the activation of T cells. The method comprises the steps of: producing an effective amount of a multimerized NCAM L1, or a functional derivative or fragment thereof, or a nucleic acid encoding the L1 or a functional derivative or fragment thereof, or producing and / or co-stimulating the L1. Administering a function enhancing agent and an effective amount of another costimulatory molecule to a mammal in which T cell activation is desired, thereby enhancing T cell activation in the mammal. I do. Any costimulatory molecule can be used in the formulations and methods described above. Preferably, the costimulatory molecule used is CD28, OX40, 4-1BB or ICOS. Also preferably, the costimulatory molecule is derived from an antigen presenting cell (APC) (eg, LFA-1, LFA-3, ICAM-1, ICAM-2, ICAM-3, CD40 or B7).
[0012]
In yet another aspect, the invention provides a method for reducing or inhibiting T cell activation. The method comprises the step of administering an effective amount of an antagonist of NCAM L1 to a mammal in which reduction or inhibition of T cell activation is desired, thereby reducing or inhibiting T cell activation in the mammal. Include.
[0013]
Any antagonist that reduces or inhibits the production and / or costimulatory function of NCAM L1 can be used in the methods of the invention. The antagonist can be an NCAM L1 antisense oligonucleotide, an anti-NCAM L1 antibody, particularly a monoclonal antibody (eg, mAb 5G3), a soluble NCAM L1, or a derivative or fragment thereof. The antagonist may reduce or inhibit L1-L1 homophilic interactions (eg, L1-L1 binding between antigen presenting cells and T cells). The antagonist may reduce or inhibit L1-L1 binding without simultaneously causing NCAM L1 clustering and signaling. The antagonist may reduce or inhibit NCAM L1 interactions with integrins involved in T cell activation (eg, NCAM L1 trans or cis interactions with integrins α5β1 or αvβ3), or NCAM L1 interactions with ligands involved in costimulation (eg, NCAM L1 interaction with CD9 and / or CD24) can be reduced or inhibited. Preferably, the NCAM L1 antagonist used in the method or formulation is a protein, polypeptide or peptide antagonist. Also preferably, the NCAM L1 antagonist used in the method or formulation is a small molecule antagonist (eg, ethanol) (Bearer et al., J. Biol. Chem. 274 (19): 13264-13270 (1999)). .
[0014]
Using the method of the present invention, CD4 ⁺ T cells, CD8 ⁺ It may reduce or inhibit the activation of T cells or both.
[0015]
The methods of the invention can be used to treat a mammal, either prophylactically or therapeutically, having a disease or disorder associated with unwanted T cell activation. Examples of such diseases or disorders include, but are not limited to, autoimmunity, graft rejection, and neuroimmunological disorders. Mammals (preferably humans) with autoimmunity, graft rejection, and neuroimmunological disorders are treated with the methods of the invention.
[0016]
In yet another aspect, the invention relates to a formulation. The formulation comprises: a) an effective amount of an antagonist of NCAM L1; and b) an effective amount of another costimulatory inhibitor molecule. Preferably, the formulation is in the form of a pharmaceutical composition. Further, the invention relates to a method for reducing or inhibiting T cell activation. The method comprises administering an effective amount of an antagonist of NCAM L1 and an effective amount of another costimulatory inhibitor molecule to a mammal in which T cell reduction or inhibition is desired, whereby the T cells in the mammal are reduced. Reducing or inhibiting activation. Any costimulatory inhibitor molecule can be used in the formulations and methods described above. Preferably, the costimulatory inhibitor molecule used is T lymphocyte-associated antigen 4 (CTLA-4).
[0017]
(Execution mode of the present invention)
(A. Definition)
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications, and sequences from GenBank and other databases cited herein are incorporated by reference in their entirety.
[0018]
As used herein, "T cell activation" refers to the cellular activation of resting T cells that exhibits a variety of responses including T cell proliferation, cytokine secretion and / or effector functions. T cell activation can be induced by stimulation of the T cell receptor (TCR) by the antigen / MHC complex. Alternatively, T cell activation can be induced by certain lectins, such as phytohemagglutinin or anti-TCR monoclonal antibodies.
[0019]
As used herein, "costimulatory molecule" refers to a molecule that modulates the consequences of TCR pre-binding to enhance T cell activation phenomena, including T cell proliferation and effector functions. Signals by costimulatory molecules are not antigen-specific and are not MHC-restricted. And the signal alone, ie, in the absence of TCR binding, cannot induce a significant response in T cells. In the absence of a costimulatory molecule, binding of the TCR results in no non-immune response or reduced reactivity.
[0020]
As used herein, "neural cell adhesion molecule L1 (NCAM L1)" refers to a nerve cell adhesion molecule that belongs to the IgSF superfamily and can function as a costimulatory molecule in T cell activation. Integrins (eg, mediating L1-L1 binding between APCs and T cells) via L1-L1 homophilic interactions or involved in T cell activation (eg, integrin α5β1 or integrin αvβ3) NCAM L1 may exert a costimulatory molecule function through its interaction with, or through its interaction with ligands involved in costimulation (eg, CD9 and / or CD24). Preferably, NCAM L1 has six immunoglobulin-like regions and five type III fibronectin domains, and a sufficient number (eg, 23) of hydrophobic side chains starting with small amino acids (eg, glycine) It is a transmembrane glycoprotein predicted to penetrate cell membranes in regions having amino acid residues (EP 0,572,664 A1; and Moos et al., Nature 334: 701-703 (1988)). NCAM L1 is intended to include variants containing conservative amino acid substitutions that do not substantially alter its costimulatory activity. Suitable conservative substitutions of amino acids are well known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. In general, those skilled in the art will recognize that a single amino acid substitution in a non-critical region of a polypeptide will not substantially alter biological activity (see, eg, Watson et al., Molecular Biology of the Gene, No. 4th Edition, 1987, The Bejacmin / Cummings Pub.co., p.224).
[0021]
As used herein, the stringency of hybridization in determining the percentage of mismatch is as follows:
1) High stringency: 0.1 × SSPE, 0.1% SDS, 65 ° C.
2) Medium stringency: 0.2 × SSPE, 0.1% SDS, 50 ° C.
3) Low stringency: 1.0 × SSPE, 0.1% SDS, 50 ° C.
It is understood that equivalent stringency can be achieved with alternative buffers, salts and temperatures.
[0022]
As used herein, “a functional derivative or functional fragment of NCAM L1” refers to a functional derivative or functional fragment of NCAM L1 that still substantially retains its function as a costimulatory molecule. . Usually, a functional derivative or functional fragment will retain at least 1%, 10%, 20%, 30%, 40%, 50% of the costimulatory activity of NCAM L1. Preferably, the functional derivative or functional fragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and 100% of the costimulatory activity of NCAM L1. A functional derivative or functional fragment of NCAM L1 also includes a peptide or polypeptide derivative or peptide fragment or polypeptide fragment of NCAM L1 that substantially retains its function as a costimulatory molecule.
[0023]
As used herein, “an agent that enhances the production of NCAM L1” refers to a substance that increases the transcription and / or translation of the NCAM L1 gene, or a post-translational modification of NCAM L1 precursor and / or intracellular. Refers to a substance that increases transport or prolongs the half-life of the NCAM L1 protein.
[0024]
As used herein, “an agent that enhances the costimulatory function of NCAM L1” refers to a substance that increases the efficacy of the costimulatory activity of NCAM L1, or the natural activity of NCAM L1 in the costimulatory signaling pathway. Refers to a substance that increases the sensitivity of a ligand or decreases the potency of an antagonist of NCAM L1.
[0025]
As used herein, “integrin” refers to a family of cell membrane glycoproteins that are heterodimers composed of α and β chain subunits. Integrins are involved in cell-cell or cell-substrate adhesion (eg, mediating neutrophil adhesion to endothelial cells or neutrophil adhesion to extracellular matrix such as collagen). Serves as a glycoprotein receptor.
[0026]
As used herein, "neoplasia (neoplasia)" refers to abnormal new growth, and thus is synonymous with a tumor and may be benign or malignant. Unlike hyperplasia, neoplastic growth persists in the absence of the original stimulus.
[0027]
As used herein, "cancer" refers to a general phrase relating to a disease caused by any type of malignancy.
[0028]
As used herein, “antagonist of NCAM L1 (or NCAM L1 antagonist)” refers to a substance that decreases the production and / or costimulatory function of NCAM L1. Such antagonists reduce transcription and / or translation of the NCAM L1 gene, or reduce post-translational modification and / or intracellular trafficking of the NCAM L1 precursor, or reduce the half-life of the NCAM L1 protein. This may reduce the production of NCAM L1. Such antagonists reduce the potency of the NCAM L1 costimulatory activity, or reduce the sensitivity of the natural ligand of NCAM L1 in the costimulatory signaling pathway, or increase the potency of the NCAM L1 antagonist. Can reduce the costimulatory function of NCAM L1. NCAM L1 antagonists can be various types of substances, including protein antagonists, polypeptide antagonists, peptide antagonists, or small molecule antagonists.
[0029]
As used herein, "antibody" includes an antibody fragment (eg, a Fab region composed of a light chain and various heavy chain variable regions).
[0030]
As used herein, "formulation" refers to any bond between two or more items.
[0031]
As used herein, "composition" refers to any mixture of two or more products or two or more compounds. The composition can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or a combination thereof.
[0032]
As used herein, “antisense polynucleotide” refers to a synthetic sequence of nucleotide bases that is complementary to the sense strand of mRNA or double-stranded DNA. Mixing of the sense and antisense polynucleotides under appropriate conditions results in binding of the two molecules, ie, hybridization. When these polynucleotides bind to (hybridize) mRNA, protein synthesis (translation) is inhibited. When these polynucleotides bind to double-stranded DNA, inhibition of RNA synthesis (transcription) occurs. The resulting inhibition of translation and / or transcription results in inhibition of the synthesis of the protein encoded in the sense strand.
[0033]
As used herein, "NCAM L1 antisense polynucleotide" refers to any oligomer that interferes with the production or expression of a NCAM L1 polypeptide. The size of such an oligomer can be any length that is effective for this purpose. Generally, the antisense oligomer is prepared to match the nucleotide sequence of the transcribed portion of NCAM L1, including the translation initiation codon, and contains a sufficient number of complementary nucleotides to inhibit translation.
[0034]
As used herein, "autoimmunity" refers to a specific humoral or cellular immune response to the body's own tissues.
[0035]
For clarity of disclosure and not for limitation, the detailed description of the present invention is divided into the following subsections.
[0036]
(B. Method for enhancing T cell activation)
In one aspect, the invention provides a method for enhancing T cell activation, comprising: an effective amount of a multimerized neuronal adhesion molecule L1 (NCAM L1) or a functional derivative or A functional fragment, or a nucleic acid encoding NCAM L1 or a functional derivative or functional fragment thereof, or a factor that enhances the production and / or costimulatory function of NCAM L1 can be transferred to a mammal in which T cell activation is desired. Administration, thereby enhancing T cell activation in the mammal.
[0037]
Any multimeric (eg, dimeric) NCAM L1 or functional derivative or functional fragment thereof, and such NCAM L1 or functional derivative or functional derivative thereof that can function as a stimulatory molecule in T cell activation Any nucleic acid encoding a fragment can be used in the present method.
[0038]
For example, the NCAM L1 protein with the following GenBank accession number may be used: T30532 (Fugu rubripes); T30581 (Zebra Danio); S36126 (rat); A43425 (chicken); S05479 (mouse); A41060 (human); (Mus musculus); NP_006605 (homogeneous homologue of L1); NP_000416 (Homo sapiens); AAF22153 (Mus musculus); CAB57301 (Mus musculus); P32004 (human); Q05695 (rat); (Cercopithecus aethios); CAB37831 (Homo sapiens); A C51746 (Homo sapiens); AAC15580 (Fugu rubripes); AAC14352 (Homo sapiens); CAA96469 (Fugu rubripes); CAA82564 (Homo sapiens); CAA41576 (Homo sapiens); 1411301A; CAA42508 (Homo sapiens); CAA41860 (Rattus norvegicus); AAA99159 (Carassius auratus); CAA61491 (Danio rerio); CAA61490 (Danio rerio); AAA59476 (Homo sapiens); AAA36353 (Homo sapiens). In addition, any protein derived from the NCAM L1 protein or a portion of such NCAM L1 protein that still substantially retains costimulatory activity can be used. Preferably, such an NCAM L1 derivative or NCAM L1 fragment can be recognized by an antibody that specifically recognizes the NCAM L1 protein from which the derivative or fragment originated.
[0039]
Similarly, nucleic acids encoding the NCAM L1 protein with the following GenBank accession numbers can be used: AC005775 (Homo sapiens); AC 004690 (Homo sapiens); M28231 (Drosophila melanogaster glia progenitor cells); AF050085 (Drosophila melanogaster glia (nrg) gene); AF172277 (Homo sapiens); AF1333093 (Mus musculus); AJ011930 (Homo sapiens); U52112 (Homo sapiens); M97161 (Rattus norvegicus); AC005626 (Homo sapants); . In addition, any nucleic acid or a portion thereof derived from the nucleic acid encoding NCAM L1 protein that still substantially retains costimulatory activity can be used. Preferably, such a NCAM L1 nucleic acid derivative or NCAM L1 fragment is capable of hybridizing under low, medium, or high stringency conditions to the NCAM L1 nucleic acid from which the derivative or fragment originated.
[0040]
Preferably, NCAM L1 or a functional derivative or functional fragment thereof can mediate L1-L1 homophilic interactions (eg, L1-L1 binding between antigen presenting cells (APCs) and T cells). . Also preferably, NCAM L1 or a functional derivative or functional fragment thereof supports an interaction with an integrin involved in T cell activation. (Eg, integrin α5β1 (Rupert et al., J. Cell Biol., 131: 1881-11891 (1995)), or integrin α5β3, (Sturmhofel et al., J. Immunol., 154 (5): 2104-11 (1995); And Poul et al., Mol. Immunol., 32 (2) 101-16 (1995)), or CD11c, β2 integrin (Meunier et al., J. Invest. Dermatol., 103 (6): 775-9 (1994)), Or a trans- or cis-interaction with the VLA integrin family (Dang et al., J. Exp. Med., 172 (2): 649-52 (1990)), and more preferably the NCAM L1 or a thereof. Functional derivatives Alternatively, a functional fragment supports interaction with a ligand involved in costimulation, for example, cis-type interaction with CD9 and / or CD24 (Liu et al., J. Exp. Med., 175: : 437-445 (1992); and Lagaudrie-Gesbert et al., Cell. Immunol., 182: 105-112 (1997)).
[0041]
NCAM L1 or a functional derivative or functional fragment thereof, or a nucleic acid encoding NCAM L1 or a functional derivative or functional fragment thereof, can be administered to a mammal by any method known to those skilled in the art. For example, NCAM L1 or a functional derivative or functional fragment thereof, or a nucleic acid encoding NCAM L1 or a functional derivative or functional fragment thereof, can be administered directly to a mammal. Alternatively, NCAM L1 or a functional derivative or functional fragment thereof, or a nucleic acid encoding NCAM L1 or a functional derivative or functional fragment thereof, can be delivered to antigen presenting cells (eg, macrophages and dendritic cells), and Thereafter, the antigen-presenting cells containing the NCAM L1 or nucleic acid are administered to a mammal.
[0042]
Any factor that enhances NCAM L1 generation and / or costimulatory function can be used in the present methods. Preferably, the agent used in the method is an L1-L1 homophilic interaction between two NCAM L1 or a functional derivative or functional fragment thereof, or NCAM L1 or a functional derivative or functional fragment thereof. And the interaction between integrin involved in T cell activation or between NCAM L1 or a functional derivative or functional fragment thereof and a ligand involved in costimulation.
[0043]
NCAM L1 or a functional derivative or functional fragment thereof may be derived from any mammal. Preferably, when the mammal to be treated is a human, NCAM L1 of human origin or a functional derivative of NCAM L1 or a fragment thereof is used.
[0044]
The method of the present invention uses CD4 ⁺ T cells, CD8 ⁺ It can be used to activate T cells or both. The methods of the present invention may preventively or therapeutically treat a mammal having a disease or disorder associated with defective T cell activation. Examples of such diseases or disorders include, but are not limited to, tumors, cancers or infections. Examples of tumors or cancers that can be treated using the present invention include breast cancer, Burkitt's lymphoma, colon cancer, small cell lung cancer, melanoma, multiple endocrine adenoma (MEN), neurofibroblast cell types, p53-associated tumors, Pancreatic cancer, prostate cancer, Ras-related tumors, retinoblastoma and von Hippel-Lindau disease (VHL). Preferably, tumors or cancers arising from the immune and / or nervous system can be treated.
[0045]
Any mammal having a tumor, cancer, or infection, such as a mouse, rat, rabbit, cat, dog, pig, cow, bull, sheep, goat, horse, monkey and other non-human primates, , Can be treated in the present method. Preferably, a human having a tumor or cancer is treated in the present method.
[0046]
(C. Method for inhibiting T cell activation)
In another aspect, the invention is directed to a method of reducing or inhibiting T cell activation. The method comprises administering to a mammal in which reduction or inhibition of T cells is desired, an effective amount of an antagonist of NCAM L1, thereby reducing or inhibiting T cell activation in the mammal.
[0047]
Any antagonist that reduces or inhibits the production and / or costimulatory function of NCAM L1 can be used in the present methods. Preferably, the antagonist used herein is an antisense oligonucleotide of NCAM L1, an anti-NCAM L1 antibody (in particular, mAb 5G3 (Balaian et al., Eur. J. Immunol., 30 (3): 938-43 (2000)). ), Or a derivative or fragment thereof, and preferably the NCAM L1 antagonist used is, for example, an L1-L1 linkage between an antigen presenting cell and a T cell. More preferably, these antagonists reduce or inhibit L1-L1 ligation without simultaneously causing NCAM L1 clustering and signal transduction. Preferably, the NCAM used Antagonists of L1 may inhibit NCAM L1 interactions with integrins involved in T cell activation (eg, NCAM L1 trans or cis interactions with the integrin α5β1, αvβ3, CD11c, β2 integrin, or VLA integrin family). ) Or reduce or inhibit the interaction of NCAM L1 with a ligand involved in costimulation (eg, the interaction of NCAM L1 with CD9 and / or CD24).
[0048]
In a preferred embodiment, the NCAM L1 antagonist used in the present methods or formulations is a protein, polypeptide or peptide antagonist. In another preferred embodiment, the NCAM L1 antagonist used in the present methods or formulations is a small molecule antagonist (eg, ethanol) (Bearer et al., J. Biol. Chem., 274 (19): 13264-13270). (1999)).
[0049]
The method uses CD4 ⁺ T cells, CD8 ⁺ It can be used to reduce or inhibit the activation of T cells or both.
[0050]
The method is used to preventably or therapeutically treat a mammal having a disease or disorder associated with unwanted T cell activation. Examples of such diseases or disorders include, but are not limited to, autoimmunity, graft rejection, and neurological immune disorders. Mammals (preferably humans) with autoimmunity, graft rejection and neurological immunity disorders are treated in the present method.
[0051]
D. Formulations and combinatorial treatments
In yet another aspect, the invention relates to a formulation. The formulation comprises: a) an effective amount of multimeric NCAM L1, or a functional derivative or fragment thereof, or a nucleic acid encoding L1 or a functional derivative or fragment thereof, or production of L1 and And / or an agent that enhances costimulatory function; or b) an effective amount of another costimulatory molecule, or an immunostimulant (eg, an agonist of a costimulatory molecule), or a particular cytokine (eg, IL-2). Preferably, the formulation is in the form of a pharmaceutical composition. Further, the invention relates to a method for enhancing T cell activation. The method comprises providing an effective amount of multimerized NCAM L1 or a derivative or fragment thereof, or a nucleic acid encoding NCAM L1 or a functional derivative or fragment thereof, or an agent that enhances L1 production and / or costimulatory function. And the administration of an effective amount of another costimulatory molecule or immunostimulatory agent to a mammal in which T cell activation is desired, thereby enhancing T cell activation in said mammal. Any co-stimulatory molecule can be used in the formulations and methods described above. Preferably, the costimulatory molecule is CD28, OX40, A-1BB or ICOS.
[0052]
CD28 is a major positive T cell costimulatory molecule and is defined by its ability to enhance T cell activation in the presence of insufficient TCR stimulation for T cell proliferation (Generally, Chambers and Allison, Curr. Opin.Cell.Biol., 11 (2): 203-10 (1999)). CD28 is an immunoglobulin superfamily glycoprotein expressed in T cells in a homodimer. CD28 binds to ligands B7.1 and B7.2 via the MYPPPY (single letter amino acid) motif in the immunoglobulin region. The cytoplasmic tail of CD28 has a tyrosine-containing motif that is presumed to be involved in signal transduction and protein transport. In certain embodiments, the CD28 protein with the following Genbank accession numbers may be used in formulations and combinatorial treatments: NP_006130 (Homo sapiens); B45895 (human); I49584 (mouse); I46689 (rabbit); S24413 ( A43523 (mouse); RWHU28 (human); AAF45150 (Mus musculus); BAA92349 (Felis catus); AAF36501 (Marmota monax); (Homo sapiens); BAA08641 (Or NP_031668 (Mus musculus); NP_008820 (Homo sapiens); NP_005182 (Homo sapiens); Q28071 (Bovin); P42069 (rabbit); P31042 (rat); P31042 (rat); P31042 (rat); ) AD04379 (Ovis arie gi); AAB53574 (Felis catus); CAA63707 (Bos taurus); CAA39003 (Rattus norvegicus); sculus). Similarly, nucleic acids encoding the CD28 protein with the following GenBank accession numbers can be used in formulations and combinatorial treatment methods: AB025316 (Felis catus); AF130427 (Marmota monax); AF222343 (Homo sapiens); AF222342 (Homo). AF222341 (Homo sapiens); D49841 (rabbit); AF092739 (ovis arees); Al528690 (mouse); AI386609 (human); AI327267 (mouse); AI324382 (mouse); U57754 (Felis catus); AA17418 (human); AA163825 (Mouse); J02988 (human); M34563 (mouse).
[0053]
In another embodiment, OX40 protein with the following GenBank accession numbers may be used in the formulations and combinatorial treatment methods: 137552 (OX40 human homolog); JE0351 (rat); 148700 (mouse); S48290 (mouse); S12783 (rat); 1D0AL 1D0AK (L chain, human); 1D0AJ (J chain, human); 1D0AI (I chain, human); 1D0AH (H chain, human); 1D0AG (G chain, human); 1D0AF (F chain 1D0AE (E chain, human); 1D0AD (D chain, human); 1D0AC (C chain, human); 1D0AB (B chain, human); 1D0AA (A chain, human); NP 003318 (Homo sapiens) CAA18438 (Homo sapiens); 002765 (C G); P47741 (mouse); P43488 (mouse); P15725 (rat); P23510 (human); AAC67236 (Rattus norvegicus); BAA20060 (Oryctolagus cuniculus); sapiens); CAA79772 (Mus musculus); CAA59476 (Mus musculus); AAA21871 (Mus musculus). Similarly, nucleic acids encoding OX40 protein with the following GenBank accession numbers can be used in formulations and combinatorial treatment methods: AL022310 (human); AF037067 (Rattus norvegicus); AB003912 (rabbit); U12763 (Mus musculus) .
[0054]
In yet another specific embodiment, a 4-1BB protein having the following GenBank accession number may be used in the formulations and combinatorial treatment methods: 138427 (human); 138426 (human); 153384 (mouse); B32393 ( P41273 (human); P41274 (mouse); Q07011 (human); P20334 (mouse); AAA93113 (mus musculus); AAA53134 (Homo sapiens); AAA53133 (Homo sapiens); musculus). Similarly, nucleic acids encoding 4-1BB proteins having the following GenBank accession numbers can be used in formulations and combinatorial treatment methods: AI664286; AII58772; AA109726; AA3899045; AA155147; AA87107; W62906; U03398 (human); U03397 (human); J04492 (mouse); L15435 (Mus musculus).
[0055]
Recently, a CD28 / CTLA-4 homolog called ICOS was isolated from activated human T cells. ICOS has a similar structure to CD28 and CTLA-4, but has no conserved MYPPY motif, suggesting that ICOS binds to its own ligand. Antibodies cross-linking to ICOS enhance anti-CD3-mediated T cell proliferation and cytokine production, but, unlike CD28, do not enhance IL-2 production. ICOS proteins and nucleic acids encoding ICOS proteins with the following GenBank accession numbers can be used for formulation and combinatorial processing: S78540 (human) and AJ250559 (Mus musculus).
[0056]
Also preferably, costimulatory molecules such as, for example, LFA-1, LFA-3, ICAM-1, ICAM-2, ICAM-3, CD40 or B7 are derived from antigen presenting cells (APC).
[0057]
In yet another aspect, the invention relates to a formulation. The formulation comprises: a) an effective amount of an NCAM L1 antagonist; and b) an effective amount of another costimulatory inhibitor molecule. Preferably, the formulation is in the form of a pharmaceutical composition. Further, the invention relates to a method of reducing or inhibiting T cell activation. The method comprises the step of administering an effective amount of an antagonist of NCAM L1 and an effective amount of another costimulatory inhibitor molecule to a mammal in which T cell reduction or inhibition is desired, whereby the T cell Reduces or inhibits activation.
[0058]
Any costimulatory inhibitor molecule can be used in the formulations and methods. For example, a costimulatory inhibitor molecule can be an antagonist of a costimulatory molecule, including a costimulatory molecule described above (eg, CD28, OX40, 4-1BB or ICOS), and the costimulatory molecule can be an antigen presenting cell (APC) (Eg, LFA-1, LFA-3, ICAM-1, ICAM-2, ICAM-3, CD40 or B7).
[0059]
In another example, the costimulatory inhibitor molecule used is T lymphocyte-associated antigen 4 (CTLA-4) (Chambers and Allison, Curr. Opin. Cell. Biol., 11 (2): 203-10 ( 1999)). CTLA-4 is an immunoglobulin superfamily glycoprotein expressed in homodimers on T cells. CTLA-4 binds to ligands B7.1 and B7.2 via the MYPPPY (single letter amino acid) motif of the immunoglobulin domain. CTLA-4 has a 10-fold higher affinity and 100-fold higher avidity for B7 ligand compared to CD28, and shows alternative binding kinetics. The cytoplasmic tail of CTLA-4 has a motif that includes tyrosine, which is presumed to be involved in signal transduction and protein trafficking. In another specific embodiment, CTLA-4 protein with the following GenBank accession numbers may be used in the formulations and combinatorial treatment methods: I46669 (rabbit); BAA08644 (oryctolagus cuniculus); P42081 (human); P42072; AAD50988 (Felis catus); AAD00698; AAD00697; (Rattus norvegicus); AD00696; (Mus musculus); 1AH1; 2207257A; 1309302A; ). Similarly, nucleic acids encoding CTLA-4 with the following GenBank accession numbers can be used in formulations and combinatorial treatment methods: AF130428 (Marmota monax); D49844 (rabbit); AF143204 (canis familialis). AF1701725 (Felis catus); AF092740 (Ovis arees); AF153202 (Felix catus); U90271 (Rattus norvegicus); U37121 (Rattus norvegicus); L1500 (U1700);
[0060]
The formulation, dosage, and route of administration of the compositions, formulations (preferably in the form of pharmaceutical compositions) described above are determined by methods known to those of skill in the art (eg, Remington: The Science and Practice of Pharmacy). , Alfonso R.Gennaro ed., Mack Publishing Company, April 1997; Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Banga, 1999; and Pharmaceutical Formulation Development of Peptides and Proteins, Hovgaard and Frkjr eds., Taylor & Francis, Inc., 2000; Medical Applications of Liposomes, Lasic and Papahadjopoulos eds., Elsevier Science, 1998; Textbook of Gene Therapy, Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology to Gene Therapy, Vol.15, Seth, Landes Bioscience, 1999; Biopharmaceutical Drug Design and Development, Wu-Pong and Rojanasakul, eds., Humana Press, 1999; Therapeutics Angigen is: From Basic Science to the Clinic, Vol.28, Dole et al., eds., see Springer-Verlag New York, 1999). The composition, formulation or pharmaceutical composition can be administered orally, rectally, topically, by inhalation, buccally (eg, sublingually), parenterally (eg, subcutaneously, intramuscularly, intradermally). Or intravenous administration), transdermal administration, or any other suitable route of administration. The most appropriate route in any given case will depend on the nature and severity of the condition being treated, and the nature of the particular composition, formulation or pharmaceutical composition used.
[0061]
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
[0062]
(E. Example)
(L1 connection is required for optimal allostimulation)
DCs are characterized as the best APCs in the immune system and are recognized as major stimulators of primary MLR cells. A population of "stimulatory" DCs is generated from CD34 + rich stem cells using a blend of GM-CSF, SCF, IL-3, TNF-a, IL-4 (> 76% purity). After 14-21 days, these cells acquire the expected DC morphology and phenotype (CD1a +, CD80 +, CD86 +, CD3-, CD14-, CD19-, CD56-) (data not shown). Importantly, this acquisition of the DC phenotype is also recorded by induction of L1 expression (FIG. 1A). L1 expression in these DCs is consistent with our previous report describing L1 expression in DC precursors and monocyte-derived DC cells (11). To establish allogeneic MLR, L1 + DCs were co-cultured with PBMC from different donors. The contribution of L1 to this allogeneic MLR was determined by including an L1-specific mAb (mAb 5G3; reference 11). Importantly, the mAb 5G3 antibody significantly reduced allogeneic PBL response, even though the isotype compatible control antibody (UPC10) had no significant effect (FIG. 1B). Using PBMC from many different donors, we observed levels of inhibition between 25% and 40% (not shown).
[0063]
(L1 ligation enhances T cell responses to PHA and CD3)
To further confirm the costimulatory function in the autoreactive system, we determined whether the anti-L1 antibody 5G3 also reduced the proliferative response to the T cell mitogen PHA. Importantly, L1 suppression by this mAb significantly reduced T cell proliferation within the PBMC fraction (FIGS. 2A and B) and reduced the proliferation of T cell rich subpopulations, especially CD4 + cells. (FIG. 2C). Inhibition by antibody 5G3 was observed over a range of PHA concentrations and was as early as 18 hours after PHA stimulation (Fig. 2B inset).
[0064]
Since the inventors have previously demonstrated that mAb 5G3 recognizes monocytes in newly isolated PBMCs [11], this mAb 5G3 antibody probably blocks L1 expressed by these accessory cells. By doing so, it appears that mitogen-induced T cell proliferation can be inhibited. In this regard, it is known that either the removal of accessory cells or the blockade of costimulatory molecules expressed by these cells abolishes or reduces the T cell response to PHA. The importance of accessory cell function was confirmed in this study by the reduced mitogenic response of isolated CD4 + and CD8 + T cell subpopulations enriched to about 95% purity ( (Fig. 2). It is important to note that finally, purified recombinant L1 (L1-ECD) was also found to reduce T cell proliferation when provided as a soluble inhibitor (sL1: FIG. 2A).
[0065]
To further demonstrate costimulatory function, it was determined whether purified immobilized L1 could enhance proliferation of polyclonal T cells in response to ligation with the CD3 receptor (mAb OKT3). PBMCs were added to wells previously covered with mAb OKT3 alone or together with the purified L1 extracellular domain (L1-ECD). Wells covered with L1 alone did not elicit a significant response, but L1 actually greatly enhanced the response to anti-CD3 mAb (FIG. 3). The specificity of this coordinated response was confirmed by inhibition with mAb 5G3 (FIG. 3). Note that mAb 5G3 blocks homophilic L1-mediated adhesion to purified L1 ECD but does not block integrin-dependent adhesion to similar L1-ECD preparations (not shown). It is important to. This data confirms that L1 can function as a potent costimulatory molecule, and indicates that allophilic L1-L1 binding rather than direct L1-integrin binding is the stimulatory mechanism.
[0066]
(L1 transfection into myeloma cells promotes adhesion via an allophilic mechanism and enhances MLR)
A direct demonstration of costimulatory function is possible by showing that transfection and novel expression of L1 enhances the ability of transfected cells to promote T cell activation. Thus, the present inventors, in a one-way heterologous MLR, compared to either wild-type or L1 transfected mouse myeloma cells (J558L), human PBMCs or enriched T cells. Responses were compared. Importantly, irradiated myeloma cells engineered to express high levels of human L1 (13) were found to be significantly more effective at inducing PBMC proliferation than their wild-type counterparts (Figure 13). 4A). This specific L1 contribution to the enriched MLR was confirmed by inhibition with mAb 5G3 (FIG. 4A). Both CD4 + and CD8 + T cells were found to respond differentially to L1-transfected myeloma cells, but were found to outperform the CD4 + cells (FIG. 4B). The ability of L1 transfected myeloma cells to induce T cell proliferation was lost when these myeloma cells were co-cultured with mononuclear cells from BALB / c mice (not shown). Since the J558L myeloma line was originally derived from BALB / c mice, this suggests that the ability of L1 to stimulate human T cell proliferation depends on the simultaneous identification and response of mouse heterologous antigens. In this regard, we observed some T cell proliferation in response to wild-type L1 negative J558L cells (FIG. 4B).
[0067]
L1 transduction on myeloma cell surfaces was also found to promote adhesion to purified recombinant L1 (L1-ECD) through a homophilic mechanism (FIG. 5). These data suggest that the enhanced MLR observed in L1 transfectants is due to a novel ability for L1-L1 interaction and adhesion. The complete elimination of the observed L1-mediated adhesion by mAb 5G3 (FIG. 5) suggests that this antibody may inhibit T cell activation by its ability to inhibit L1-L1 homophilic interactions. It is important to keep this in mind.
[0068]
It should be noted that inhibition of T cell activation may depend on the use of antagonists that can block L1 ligation without concomitant L1 clustering and signaling. In support of this concept, we did not observe any significant inhibition of T cell activation using anti-L1 polyclonal antibodies (not shown). In this regard, polyclonal antibodies to L1 (unlike most mAbs and isolated soluble L1) can lead to activation of L1-dependent signaling pathways and significantly increase intracellular cAMP levels. It is described (Reference 14). Finally, it was also observed that the 5G3 antibody preparation was critically dependent on the removal of antibody aggregates by ultracentrifugation, and that the inhibitory activity of the 5G3 antibody preparation was inactivated on both freezing and concentration.
[0069]
(Summary of opinions)
L1 by "professional" APCs of both myeloid and lymphoid origin, described as neural CAMs but including B cells, activated monocytes, monocyte-derived DCs, and follicular DC cells We recently reported expression (11). L1 expression on monocyte-derived DCs was induced after LPS treatment (Reference 11). This LPS treatment is known to promote functional maturation or acquisition of optimal costimulatory capacity. Based on these findings, and on what was sourced in this study, we found that L1 expressed by such "professional" APCs could function as a costimulatory molecule in T cell activation. suggest. A recent report (10) reporting on L1 expression by isolated peripheral blood T cells describes a possible costimulatory mechanism based on allophilic L1-L1 ligation between APC and T cells. Suggest. To further support this mechanism, we show that anti-L1 mAb, which also effectively blocks L1-L1 ligation, inhibits T cell activation.
[0070]
More detailed work is needed to define how L1-mediated signaling enhances T cell costimulation and to determine how to rank L1 alongside other costimulatory molecules. Comparison with previous studies suggests that L1 is less important for T cell costimulation than, for example, members of the B7 family. For example, inhibition of B7.1 / B7.2 has been shown to reduce T cell proliferation to PHA and allogeneic DC by 75% and 95%, respectively (15, 16, 17). However, L1-mediated costimulation can be compared to other well-reported costimulatory molecules such as CD58. For example, blockade of CD2: CD58 binding has been shown to inhibit T cell proliferation against PHA and allogeneic DC by 30% -35% and 45% -50%, respectively (16, 17). .
[0071]
Although our findings indicate that allophilic L1-L1 ligation is required for co-stimulation of T cells, L1 undergoes numerous cis and trans interactions with other heterophilic ligands. It is important to note that they do (Reference 6). For example, recently L1 has been shown to support trans-interaction with integrin a5b1 (Reference 18); and to support integrins that are also involved in T cell activation (Reference 19). It is believed that L1-integrin binding may contribute to costimulation following the initial T cell activation event required for subsequent integrin activation and binding. Furthermore, it should be noted that L1 can undergo cis-form interactions with both CD9 and CD24, which are known to have important functions for costimulation (20, 21).
[0072]
Several important turning points arise from the findings listed here. Like other costimulatory molecules, L1 may contribute to the development of autoimmunity, transplant rejection and anti-tumor responses, and in this context may prove to be a valuable and novel target of immunotherapy intervention. The finding that soluble L1 can inhibit T cell activation may prove an important existing report that active neuroectodermal tumors can secrete large amounts of L1 (13,22). Finally, high levels of L1 expression in post-mitotic neurons and Schwann cells (Refs. 2, 6) indicate that this CAM may cause a gap between the nervous and immune systems, especially in the development of neuroimmunological disorders Suggest that it may function as an important intermediate factor.
[0073]
(Materials and methods)
(Reagents and cell lines)
The anti-human L1 mAb 5G3 was produced in our laboratory (Reference 19). Purified recombinant L1 (L1-ECD), consisting of all of the extracellular region of human L1, was kindly provided by Dr. William Stallcup (The Burnham Institute, La Jolla, CA). J558L myeloma cells stably transfected with full-length human cDNA encoding human L1 (J558L-L1; reference 21) were kindly provided by Dr. Vance Lemmon (Case-Western Reserve University, OH).
[0074]
(Generation of DC and enrichment of CD4T lymphocytes and CD8T lymphocytes)
CD34 + cells were purified from normal cord blood using M-450 Dynabeads coated with anti-CD34 mAb according to the manufacturer's protocol. The CD34 + enriched (> 76%) or CD34-negative cell population was then converted to granulocyte macrophage colony stimulating factor (10 ng / ml), human stem cell factor (40 ng / ml), human interleukin-3 (10 ng / ml). And cultured in the presence of human tumor necrosis factor-a (100 U / mL) and human interleukin-4 (400 U / mL). After 7-21 days of development, the level of L1 expression on the cells was determined using an anti-L1 mAb 5G3 to which fluorescein isothiocyanate (FITC) was directly conjugated.
[0075]
CD4 + and CD8 + cells were isolated from PBMC using anti-CD4 mAb or M-450 Dynabeads coated with anti-CD8 mAb. Isolation was according to the manufacturer's recommendations (Dynal, Fort Lee, NJ). This method did not induce T cell activation and resulted in a purity of approximately 95% or more.
[0076]
(MLR and mitogen assay)
For the mitogen assay, PBMCs or enriched CD4 + or CD8 + cells were plated at 1 × 10 6 in 96-well round bottom plates. ⁵ Cells / well were cultured with or without PHA (Sigma; 20 mg / ml). For MLR assays, irradiated wild type J558L cells or L1 transfected J558L cells (1 × 10 ⁴ Cells / well) or irradiated cord blood-derived dendritic cells (1 × 10 ⁴ PBMC (1 × 10 6 cells / well) ⁵ Cells / well) or enriched CD4 + cells, or enriched CD8 + cells were co-cultured. The mitogen-treated cells and co-cultures are maintained for 3 days and during the last 18 hours of the 3-day culture, the cultures receive [ ³ [H] thymidine (1 mCi / well) was applied. The contribution of L1 to both mitogen and MLR assays was assessed by incorporation of anti-L1 mAb 5G3 (80 mg / ml). If appropriate, a control antibody matching the IgG2a isotype (UPC10; 80 mg / ml) was also added.
[0077]
(Co-stimulation of CD3-mediated T cell activation)
Wells of a 96-well plate were pre-coated with anti-CD3 antibody OKT3 (25 U / ml) or with purified L1 extracellular domain (40 mg / ml) or with a combination of both L1 and L1 antibody. . After washing the wells, PBMC was added to pre-coated or untreated wells for 72 hours. In the absence or presence of mAb 5G3, or in the presence of control antibody UPC10, 1 × 10 ⁵ PBMC was added at cells / well. During the last 18 hours of the 3 day co-culture, ³ [H] thymidine was applied. Treatments were performed in triplicate. Error bars are ± 1 SE.
[0078]
(Adhesion assay)
Purified L1-ECD fusion protein (30 mg / ml) in PBS was coated on the bottom of a 96-well Titertek plate essentially as described (13). Wells were then blocked with 5% BSA and wild-type or L1 transfected J558L myeloma cells (1 × 10 ⁵ Cells / well) and allowed to adhere for 60 minutes at 37 ° C. Cells were added to HBSS supplemented with 10 mM Hepes, BSA (0.5%) and CaCl2 (0.5 mM) (pH 7.4). For inhibition studies, cells were pre-treated with 80 mg / ml of the control mAb UPC10, which matches the anti-L1 mAb 5G3 or its isotype, and then added to wells pre-treated with both cells and inhibitor. Unattached cells were removed under constant vacuum as described (13) and the remaining adherent cells were counted using a 40 × objective.
[0079]
(References)
[0080]
(Equation 1)

Since modifications will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the appended claims.
[Brief description of the drawings]
FIG.
L1 is expressed by cord blood-derived DCs and contributes to allogeneic MLR. (A), CD34 + cells were enriched from normal cord blood and expanded for 21 days before staining for L1 expression. Enriched CD34 + cells were also stained for L1 before culture (day 0). These cells were stained with mAb 5G3 conjugated directly to FITC. (B), Cord blood-derived DCs (DC +) were co-cultured with PBMCs from different donors in one-way MLR. Cells were cultured in the presence or absence of anti-L1 mAb 5G3 or with the control antibody UPC10. CD34-negative cord blood cells cultured under the same conditions as the CD34 + enriched fraction were also tested as stimulants (non-DC). Cultures were pulsed with [3H] -thymidine during the last 18 hours during the 3-day co-culture. The treatment was performed in triplicate. Error bars are ± 1 SE.
FIG. 2
Anti-L1 antibody 5G3 and soluble L1 inhibit autologous T cell responses to mitogens. (A) PBMC were treated with PHA (10 mg / ml) in the absence or presence of mAb 5G3, or in the presence of control antibody UPC10. Additional PBMC were cultured in the presence of soluble recombinant L1-ECD (sL1; 100 mg / ml). (B) PBMC were treated with a range of PHA concentrations in the absence or presence of mAb 5G3, or in the presence of control antibody UPC10. Cultures were pulsed with [3H] -thymidine during the last 18 hours of the 3-day co-culture. (B, inset) Some cells were treated with PHA and pulsed with [3H] -thymidine for only 18 hours. (C) Enriched CD4 + or CD8 + T cell subsets were treated with PHA in the absence or presence of mAb 5G3, or in the presence of control antibody UPC10. Cultures were pulsed with [3H] -thymidine during the last 18 hours of the 3-day co-culture. Processing was performed in triplicate. Error bars are ± 1 SE.
FIG. 3
Purified immobilized L1 enhances T cell proliferation in response to CD3 ligation. The wells of a 96-well plate are pre-treated with anti-CD3 antibody (OKT3: 25 U / ml), purified L1-ectodomain (40 mg / ml), or a combination of both L1 and this antibody. did. After washing the wells, PBMC was added to the precoated or untreated wells for 72 hours. PBMC were cultured in the absence or presence of mAb 5G3, or in the presence of control antibody UPC10. Cultures were pulsed with [3H] -thymidine during the last 18 hours of the 3-day co-culture. Processing was performed in triplicate. Error bars are ± 1 SE.
FIG. 4
Transfection and de novo expression of L1 enhances MLR. (A and B) Irradiated wild-type (WT cells) or L558 transfected J558L myeloma cells (L1 + cells) were converted to PBMC (A) or enriched CD4 + or CD8 + T cell subsets in one-way MLR. The cells were co-cultured with (B). L1 + J558L myeloma cells were co-cultured in the presence or absence of anti-L1 mAb 5G3 (80 mg / ml) or control mAb UPC10 (80 mg / ml). (A, inset) Additional co-cultures of L1 + J558L myeloma cells and PBMC were incubated with mAb 5G3 at concentrations varying from 20 to 160 mg / ml (inset). Cultures were pulsed with [3H] -thymidine during the last 18 hours of the 3-day co-culture. Processing was performed in triplicate. Error bars are ± 1 SE.
FIG. 5
Inhibitory antibody 5G3 blocks L1-L1-mediated adhesion by myeloma cells transfected with L1. Myeloma cells transfected with wild-type (WT) or L1 (L1 +) adhere to the immobilized recombinant L1 in the presence or absence of control mAb (UPC10) or anti-L1 mAb (5G3). Is done. Adherent cells were counted per unit area using a 40 × high power objective. Experimental treatments were performed in triplicate and four regions were counted per well. Error bars represent ± 1 SE.

Claims (34)

A method for enhancing T cell activation, said method comprising multimerizing neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding said L1 or a functional derivative thereof. Administering an effective amount of a derivative or fragment to a mammal in need thereof, wherein said NCAM L1, or a functional derivative or fragment thereof, interacts with an integrin involved in T cell activation. A method of promoting, directly or indirectly, 2. The method of claim 1, wherein the NCAM Ll, or a functional derivative or fragment thereof, directly or indirectly promotes a trans or cis interaction with an integrin α5β1 or αvβ3. A method for enhancing T cell activation, said method comprising multimerizing neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding said L1 or a functional derivative thereof. Administering an effective amount of a derivative or fragment to a mammal in need thereof, wherein the NCAM L1, or functional derivative or fragment thereof, directly interacts with a ligand involved in costimulation. Method that promotes, indirectly or indirectly. 4. The method according to claim 1 or 3, wherein the NCAM L1, or a functional derivative or fragment thereof, promotes, directly or indirectly, a cis-type interaction with CD9 and / or CD24. A method for enhancing T cell activation, said method comprising multimerizing neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding said L1 or a functional derivative thereof. Administering to a mammal in need thereof an effective amount of a derivative or fragment, or an agent that enhances the production and / or costimulatory function of said L1, wherein said agent comprises two NCAM L1 or L1-L1 homophilic interaction between functional derivatives or fragments thereof, or interaction between NCAM L1 or a functional derivative or fragment thereof and an integrin involved in T cell activation, or NCAM L1 or a derivative thereof. Between a functional derivative or fragment and a ligand involved in costimulation To strengthen the interaction, way. 6. The method of claim 1,3, or 5, wherein the mammal is a human and the NCAM LI, or a functional derivative or fragment thereof, is of human origin. 6. The method according to claim 1,3, or 5, wherein the activated T cells are CD4 ⁺ cells, CD8 ⁺ cells or both. 6. The method of claim 1,3, or 5, wherein said mammal has a tumor, cancer or infection. A method for enhancing T cell activation, said method comprising multimerizing neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding said L1 or a functional derivative thereof. Administering an effective amount of a derivative or fragment to a mammal in need thereof, wherein said NCAM L1, or a functional derivative or fragment thereof, or a nucleic acid encoding said NCAM L1, or a functional derivative thereof. A derivative or fragment is delivered to an antigen presenting cell, and then the NCAM L1 or the antigen presenting cell containing the nucleic acid is administered to the mammal, or a factor is added to the L1 production and / or costimulatory function. Enhance the way. A method for reducing or inhibiting T cell activation, said method comprising administering to a mammal in need thereof an effective amount of an antagonist of NCAM L1, wherein said NCAM L1 The method, wherein the antagonist is a small molecule antagonist. 11. The method of claim 10, wherein the NCAM L1 antagonist is a protein, polypeptide or peptide antagonist. 11. The method of claim 10, wherein the NCAM L1 antagonist is selected from the group consisting of an NCAM L1 antisense oligonucleotide, an anti-NCAM L1 antibody, a soluble NCAM L1, or a derivative or fragment thereof, and The method wherein the factor reduces or inhibits NCAM L1 production and / or costimulatory function. The method according to claim 12, wherein the anti-NCAM L1 antibody is a monoclonal antibody. 14. The method of claim 13, wherein said anti-NCAM L1 monoclonal antibody is mAb 5G3. 11. The method of claim 10, wherein the NCAM L1 antagonist reduces or inhibits L1-L1 homophilic interactions. 16. The method of claim 15, wherein the antagonist of NCAM Ll reduces or inhibits Ll-Ll ligation between antigen presenting cells and T cells. 17. The method of claim 16, wherein the antagonist of NCAM Ll reduces or inhibits Ll-Ll ligation without simultaneously causing NCAM Ll clustering and signaling. A method for reducing or inhibiting T cell activation, said method comprising administering to a mammal in need thereof an effective amount of an antagonist of NCAM L1, wherein said NCAM L1 The method wherein the antagonist is a small molecule antagonist that reduces or inhibits the interaction of NCAM L1 with integrins involved in T cell activation. A method for reducing or inhibiting T cell activation, said method comprising administering to a mammal in need thereof an effective amount of an antagonist of NCAM L1, wherein said NCAM L1 The method wherein the antagonist is a small molecule antagonist that reduces or inhibits the trans- or cis-trans action of NCAM L1 with integrin α5β1 or integrin αvβ3. 11. The method of claim 10, wherein the antagonist of NCAM L1 reduces or inhibits the interaction of NCAM L1 with a ligand involved in costimulation.
18. The method according to 18 or 19. A method for reducing or inhibiting T cell activation, said method comprising administering to a mammal in need thereof an effective amount of an antagonist of NCAM L1, wherein said NCAM L1 The method wherein the antagonist is a small molecule antagonist that reduces or inhibits the interaction of NCAM L1 with CD9 and / or CD24. 22. The method of claim 10, 18, 19, or 21, wherein the mammal is a human. 22. The method of claim 10, 18, 19, or 21, wherein activation of CD4 ⁺ cells, CD8 ⁺ cells or both is reduced or inhibited. 22. The method of claim 10, 18, 19, or 21, wherein the mammal has a disease or disorder selected from the group consisting of autoimmunity, graft rejection, and neuroimmunological disorders. A formulation, wherein the formulation comprises:
a) Multimerizing nerve cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding the L1 or a functional derivative or fragment thereof, or a function of producing and / or co-stimulating L1. An effective amount of an enhancing agent; and b) an effective amount of another costimulatory molecule, or an agonist thereof,
A formulation comprising: 26. The formulation of claim 25, which is in the form of a pharmaceutical composition. 26. The formulation of claim 25, wherein said costimulatory molecule is selected from the group consisting of CD28, OX40, 4-1BB and ICOS. 26. The formulation of claim 25, wherein the costimulatory molecule is derived from an antigen presenting cell (APC). 29. The formulation of claim 28, wherein the APC-derived co-stimulatory molecule is selected from the group consisting of LFA-1, LFA-3, ICAM-1, ICAM-2, ICAM-3, CD40 and B7. A method for enhancing T cell activation, said method comprising multimerizing neural cell adhesion molecule L1 (NCAM L1), or a functional derivative or fragment thereof, or a nucleic acid encoding said L1 or a functional derivative thereof. Administering to a mammal an effective amount of a derivative or fragment, or an agent that enhances said L1 production and / or costimulatory function, and an effective amount of another costimulatory molecule, wherein the T cell activity is Is preferred, thereby enhancing T cell activation in said mammal. A formulation, wherein the formulation comprises:
a) an effective amount of an antagonist of NCAM L1; and b) an effective amount of another costimulatory molecule;
A formulation comprising: 32. The formulation of claim 31, which is in the form of a pharmaceutical composition. 33. The formulation of claim 32, wherein the costimulation inhibitory molecule is T lymphocyte associated antigen 4 (CTLA-4) or ethanol. A method for reducing or inhibiting T cell activation, said method comprising administering to a mammal an effective amount of an antagonist of NCAM L1 and an effective amount of another costimulatory molecule. , Wherein the reduction or inhibition of T cells is preferred, thereby reducing or inhibiting T cell activation in said mammal.

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