JP2008500832A - Methods for assessing treatment response to FCGR3A Gebotype (GEBOTYPE) and non-depleting antibodies - Google Patents

Abstract

  The present invention relates to methods and compositions for assessing or assessing a subject's response and / or side effects to a particular therapeutic treatment. More specifically, the invention neutralizes a target without depleting the target cell in a method for determining a subject's response and / or side effects, or treatment protocol for a subject treated with therapeutic antibodies. It provides a way to adapt to the situation where it is desired. The present invention is based on the determination of the subject's FCGR3A genotype. Preferably, the therapeutic antibody is an antibody or protein comprising an Fc portion of the G4 subclass.

Description

  The present invention relates to methods and compositions for assessing or assessing a subject's response and / or side effects to a particular therapeutic treatment. More specifically, the present invention provides a method for determining a subject's response and / or side effects or adapting a treatment protocol for a subject treated with a therapeutic antibody. The present invention can be used for patients treated with antibodies that are designed to target membrane antigens, but do not need to deplete target cells, or increase the therapeutic response to that therapy or Suitable for identifying subjects who show a decrease or for subjects who show an increased or decreased risk of side effects, particularly undesirable side effects such as cytokine release syndrome.

Introduction Various therapeutic strategies in humans are based on the use of therapeutic antibodies, which need not be depleting to the cells to which they are bound. Some of the strategies aim to use antibodies that antagonize or agonize with membrane receptors and / or target membrane antigens. Its purpose is the modulation of cell function. The strategies include a wide range of treatment modalities including, but not limited to, strategies that modulate immune cell function (eg, B cells, T cells, NK cells), inflammatory symptoms and / or autoimmune disorders. Strategies to treat, anti-tumor strategies (eg, anti-erbB1 and erbB2 antibodies such as trastuzumab, pertuzumab and ABX-EGF; anti-TRAIL-R antibody, anti-CD33 antibody Mylotarg®), and heart disease Or treatments related to antithrombosis (eg ReoPro®) are included. In almost all such applications, the antibody is thought to act by antagonizing key membrane receptors or induce intracellular signaling. Typically, the antibody is an IgG species, typically an IgG4 monoclonal antibody, most preferably a full length antibody so as to have a long in vivo half-life. They are often developed in the treatment of inflammatory disorders such as psoriasis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, but are used, for example, in cancer where Fc-mediated ADCC mechanisms are not required (See, eg, Mylotarg, ABX-EGF targeting tumor cells, or anti-NKp30 antibodies that activate NK cells). Preferential use of IgG4 prevents cell lysis, while maintaining a long in vivo half-life thanks to interaction with the FcRn receptor. Thus, this immunoglobulin subclass is commonly used when effector mobilization is not required. A specific example of such a therapeutic antibody is CDP571 (also referred to as Humicade ^™ ), which is an anti-TNFα IgG4 monoclonal antibody. Examples of other IgG4 antibodies include clenoliximab used for the treatment of rheumatoid arthritis or ch5D12 used for the treatment of multiple sclerosis, aselizumab (HuDreg55), an anti-selectin L antibody of the IgG4 type, Cedelizumab OKTcdr4a, humanized (anti-CD4) and rovelizumab Hu23F2G (humanized (anti-CD11a)) are included. Yet another example of an antibody that is not required to have the activity of depleting target cells is an antibody that modulates NK cells by targeting NK cell surface receptors, such as anti-NKG2D, anti-NKp30, anti-NK NKp46 or anti-NKp44 or anti-CD94 / NKG2A antibody. However, it is possible to obtain IgG1 antibodies that do not have the ability to induce cytolytic activity, for example, Mason U et al. (J Rheumatol. 2002; 29 (2): 220-9) have shown that IgG1-type anti-CD4 An example is provided in which the antibody is only effective if it does not substantially induce lysis. The document demonstrates that certain IgG1 antibodies can eliminate depletion activity, but with respect to the suggestion, IgG1-based products, particularly because lytic activity may depend on glycosylation status Emphasizes the dangers of developing.

  An antibody can be a recombinant antibody (chimeric, humanized, primatized, “fully” human, or an antibody that has been modified in any manner, modified to include functional domains of various species or origin or specificity. May include, but are not limited to, modifications that reduce binding to FcγRIIIa). Also contemplated are fusion proteins comprising an Fc portion.

  The non-depleting antibodies are a novel and target specific approach to human therapy, but do not always show strong efficacy and often show undesirable side effects. Thus, its use can be improved by assessing the nature of the subject's response. For example, some antibodies have been suggested to be depleting in some patients even though they do not require target cell depletion for efficacy (Isaacs JD, Wing MG, Greenwood JD, Hazleman BL, Hale G, Waldmann H. “A therapeutic human IgG4 monoclonal antibody that depletes target cells in humans.” Clin Exp Immunol. 1996 Dec; 106 (3): 427-33). This depleting effect will reduce the efficacy of the product in applications where depletion is not desirable; therefore, if the patient can be treated with a different product or according to a different therapy It is expected that the depletion effect may have important therapeutic significance.

  In another example, depleting antibodies are known to often cause cytokine release syndrome, including severe cytokine release syndrome. It would be therapeutically useful to predict the effects in patients who are not required to be target depleting or are treated with unintended antibodies. Similarly, it would also be very important to predict the effects in patients who are being treated with depleting antibodies. The purpose is to modify the treatment regime, for example, by giving the patient a premedication effective to reduce or prevent cytokine release syndrome. Examples of depleting antibodies suggested to induce cytokine release syndrome include CAMPATH 1-H monoclonal antibody (Wing et al., 1996, J Clin Invest 98, 2819-2826) and anti-CD20 antibody Mabthera (rituximab) In this case, symptoms presumed to be due to cytokine release syndrome include fever and chills, flushing, angiodema, nausea, urticaria / rash, hypotension and bronchospasm ( bronchiospasm) (Committee for Proprietary Med. Prod. Europ. Public Assessment Report, CPMP / 259/98, The Europ. Agency for the Evaluation of Med. Prod. (1998)).

  The availability of methods that allow assessment of patient response and / or side effects to treatment with antibodies greatly improves the therapeutic efficacy of the therapy. The reason is that the profit / loss ratio of the treatment can be well evaluated, the treatment can be adjusted, and finally a new therapeutic product can be developed.

SUMMARY OF THE INVENTION The present invention proposes novel methods and compositions for assessing (assessing) a subject's therapeutic response to a therapeutic composition, preferably a therapeutic antibody, comprising an Fc moiety. Preferably, the therapeutic antibody is an antibody that does not have or is not required to deplete target cells, and more preferably induces ADCC-mediated target cell depletion. An antibody that does not have the ability, does not induce, or is not required to have that ability. In a preferred example, the antibody is an IgG4 antibody. In another example, the antibody is an antibody other than IgG4 that does not have, or does not need, or is not required to induce ADCC- or ADCP-mediated target cell depletion. is there. The antibody may be an antibody other than IgG4 that does not substantially induce target depletion or preferably does not induce target depletion in an individual having phenylalanine at position 158 of the FcγRIIIa receptor. Preferably, said non-IgG4 antibody comprises one or more modifications in the Fc region that reduce binding to FcγRIIIa.

  Preferably, the subject's therapeutic response relates to efficacy in the treatment of disorders in which membrane antigen is the target of the antibody, but in particular through the ADCC- or ADCP mechanism where target cell depletion is not required. Preferred examples of the disorder to be treated include, but are not limited to, infectious diseases, immunodeficiencies such as inflammatory and autoimmune disorders, transplantation, cancer, and cardiovascular disorders. In general, a disorder will also include any disorder whose immune response can be enhanced or inhibited. For example, antibodies that have the ability to activate NK cells (eg, anti-NCR or anti-KIR) or T cells (eg, anti-CTLA4), but do not require lysis of target NK or T cells, and even try to avoid It can be used for the treatment or prevention of cancer or infectious diseases. In other examples, antibodies that target and antagonize the membrane antigens erbB1 et erbB2 (trastuzumab, pertuzumab, cetuximab, ABX-EGF) act via antiproliferative or anti-antiogenic activity Thus, no lysis of the target cells is required, especially lysis via the ADCC mechanism. In another example, anti-VEGFR antibodies that inhibit the VEGF receptor but do not induce target cell lysis may be sought. Its purpose is to avoid potential damage to any endothelium, avoid inflammation and avoid potential thrombosis. Preferably, the subject's therapeutic response relates to modulation of the physiological pathway, and more preferably relates to anti-inflammatory effects, anti-proliferative effects, inhibition or enhancement of the immune response.

  The present invention further proposes new methods and compositions for assessing a subject's side effects on therapeutic antibodies. The antibody should be non-depleting to the cells to which it binds, or more particularly avoid ADCC- or ADCP- lysis of cells covered with the antibody. Side effects can result from cytokine release or unwanted ADCC- or ADCP activity being induced. The present invention also proposes a method of selecting patients with the best response profile for treatment with therapeutic antibodies. The invention also relates to a method of treating a patient with a therapeutic antibody, comprising a prior step of assessing the patient's response. The invention also relates to compositions and kits suitable for practicing the invention. Similarly, the present invention may be used in clinical trials or experimental settings to assess or monitor a subject's response or to verify the mode of action of an antibody. Three classes of FcγRs (FcγRI, FcγRII and FcγRIII) and their subclasses are encoded by eight human genes, all of which are located on the long arm of chromosome 1. Some of these genes exhibit functional allelic polymorphisms, thus resulting in allotypes with various receptor properties. One of these genetic factors is gene dimorphism in FCGR3A, which encodes FcγRIIIa with phenylalanine (F) or valine (V) at amino acid position 158 of the mature protein (Koene et al., Blood. 1997; 90: 1109-1114; Wu J et al., J Clin Invest. 1997; 100: 1059-1070). Human IgG4 has been demonstrated to bind more strongly to homozygous FcγRIIIa-158V natural killer cells (NK) compared to homozygous FcγRIIIa-158F or heterozygous NK cells (Koene et al., 1997).

  The present invention intends to be substantially non-depleting by utilizing the correlation between a subject's genotype and its ability to respond to treatment with a composition, particularly an antibody, comprising an Fc portion. It is possible to predict the response and side effect sensitivity of a subject to treatment with the therapeutic composition. More specifically, the present invention provides an object for treatment with a composition comprising an Fc moiety, particularly an antibody, intended to be non-depleting utilizing the genotype of the FcγRIIIa receptor. It is possible to predict a person's response and susceptibility to side effects. As described further herein, by any suitable manner, such as by determining the nucleotide sequence present in the patient's DNA or by determining the amino acid present in the patient's FcγRIIIa receptor It is understood that the genotype of a patient can be determined. The latter follows a standard approach, for example to determine the phenotype or allotype of a patient's receptor.

  More specifically, the present invention provides that if valine is present at position 158 of the subject's FcγRIIIa receptor, it exhibits a reduced response to the treatment in terms of efficacy, and position 158 of the FcγRIIIa receptor. The presence of phenylalanine is indicative of an increased response to the treatment. In addition, the present invention provides that if valine is present at position 158 of the subject's FcγRIIIa receptor, it exhibits side effects or increased sensitivity to the treatment, and phenylalanine is present at position 158 of the FcγRIIIa receptor. That is, to show a side effect to the treatment or a decrease in sensitivity to the side effect.

  The identity of the amino acid residue present at position 158 of the FcγRIIIa receptor corresponds to a single nucleotide polymorphism of the nucleotide at position 559 of FCGR3A cDNA or position 4985 of FCGR3A genomic DNA. Thus, determination of a subject's genotype can be easily performed by determining the nucleotides present in the subject's DNA or by determining the amino acid residues present in the FcγRIIIa receptor.

  Side effects may be the result of ADCC- or ADCP-mediated induction of cytokine release or activation of FcγrIIIa-retaining cells, particularly by NK cells and / or macrophages. Examples of cytokines released are TNF-α, IFN-γ and IL-6. Symptoms of cytokine release often include, but are not limited to, fever and chills, flushing, angioedema, nausea, urticaria / rash, hypotension and bronchospasm.

  Accordingly, the present invention provides methods based on the association of FCGR3A genotypes and clinical and molecular responses to compositions comprising Fc moieties, particularly antibodies, intended to be non-depleting. Preferably, the composition is not intended to induce cytolytic activity of the relevant or bound cell, in particular ADCC or ADCP. The present invention thus provides a marker that can be used to monitor, evaluate or select a patient's response to said antibody. The present invention thus introduces a pharmacogenetic approach in the management of patients using treatment with therapeutic antibodies, more specifically treatment with substantially non-depleting antibodies, particularly IgG4 antibodies. In one aspect, the substantially non-depleting antibody is an antibody that binds to a receptor present on the surface of immune cells, more preferably B cells, T cells or NK cells. In another preferred aspect, the substantially non-depleting antibody is an antibody that binds to a receptor present on the surface of a tumor cell or endothelial cell; examples of receptors include, but are not limited to: , ErbB1 and erbB2 on tumor cells, and cellular VEGF-R on endothelial cells. Other examples or receptors are known in the art and / or are listed in Table 1 and further described herein.

  It will be appreciated that the methods of the invention can be practiced with a wide range of Fc moiety-containing compositions, particularly therapeutic antibodies. In general, the antibody or Fc moiety-containing composition is intended to be non-depleting, preferably does not substantially induce cytolytic activity, particularly ADCC or ADCP activity, on its associated or bound cells, or It is intended not to trigger. For example, the therapeutic antibody may be an IgG4 subtype antibody. Preferably, the therapeutic antibody specifically binds to a membrane antigen on the target cell, such as, but not limited to, tumor cells, endothelial cells, B lymphocytes, T lymphocytes or / and NK cells are included. In a preferred example, the therapeutic antibody binds to and activates a NK cell activatory receptor. In another example, the therapeutic antibody binds to an NK cell activated receptor and interferes with ligand binding thereto or inhibits its function. In another example, the therapeutic antibody binds to an NK cell inhibitory receptor and interferes with ligand binding thereto, or inhibits its function, thereby activating or activating NK cells. To strengthen. In another example, the therapeutic antibody binds to and activates an NK cell inhibitory receptor, thereby inhibiting NK cell activation. In another aspect, the composition comprising the Fc portion, particularly an antibody, specifically binds to a membrane proinflammatory cytokine or lymphocyte receptor.

  In a preferred embodiment, the subject to be treated according to the present invention has a disorder such as a tumor. In another example, the subject may have another proliferative disorder, such as a hyperproliferative symptom, such as hyperplasia, fibrosis (especially pulmonary fibrosis, as well as other types of fibrosis, such as renal fibrosis), Has angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in blood vessels such as stenosis or restenosis after angioplasty. In another example, the subject has an inflammatory or preferably autoimmune disorder, including but not limited to macrophages and neutrophilic granulocytes (polymorphonuclear leukocytes, PMN) and / or Or disorders mediated by phagocytic cells, including T cells. Examples include inflammatory skin diseases such as psoriasis; responses associated with inflammatory bowel diseases (eg Crohn's disease and ulcerative colitis); adult respiratory distress syndrome; dermatitis; CNS inflammatory disorders such as multiple sclerosis Uveitic disorders; allergic symptoms such as eczema and asthma and other symptoms involving T cell infiltration and chronic inflammatory reactions; skin hypersensitivity reactions such as poisonous poisons and poisons; self Immune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes, multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, Sjogren's syndrome, juvenile-onset diabetes, and typically tuberculosis, sarcoidosis, polymyositis, Immune responses associated with delayed-type hypersensitivity mediated by cytokines and T lymphocytes found in granulomatosis and vasculitis; Multi-organ injury syndrome secondary to sepsis or trauma; autoimmune hemolytic anemia; myethemia gravis; disease mediated by antigen-antibody complex; all types of transplant rejection, eg graft versus host Or host versus graft disease.

  It will be appreciated that any aspect of the invention relating to therapeutic antibodies may relate to whole antibodies or fragments or modified antibodies, so long as the antibody comprises an Fc portion. As discussed further herein, there are five human immunoglobulin Fc regions with various effector and pharmacokinetic properties: IgG, IgA, IgM, IgD, and IgE. IgG is the most abundant immunoglobulin in serum. IgG also has the longest serum half-life (23 days) of any immunoglobulin. Unlike other immunoglobulins, IgG is efficiently recycled after binding to a specific Fc receptor called FcRn. FcRn is structurally and genetically unrelated to FcγR. There are four IgG subclasses G1, G2, G3, and G4, each subclass having various effector functions. G1 and G3 bind to C1q and can activate complement through the classical pathway, while G2 and G4 cannot. G3 can bind C1q more efficiently than G1, but G1 is more effective in mediating complement-directed cell lysis. The C1q binding site in IgG is located in the carboxy terminal region of the CH2 domain. All IgG subclasses have the ability to bind to Fc receptors (CD16, CD32, CD64), and G1 and G3 are more effective than G2 and G4. The Fc receptor binding region of IgG is formed by residues located in both the hinge and carboxy terminal regions of the CH2 domain. IgA can exist in both a dimeric form organized by monomer and J chain. IgA is the second most abundant Ig in serum, but its half-life is only 6 days. IgA has three types of effector functions. It binds to IgA-specific receptors on macrophages and eosinophils that are associated with phagocytosis and degranulation, respectively, resulting in antibacterial activity and cytotoxicity, particularly against parasites, for example. It can also fix complement through alternative pathways. In a particularly preferred embodiment, the invention is according to the F4 region of the G4 subclass (also referred to herein as IgG4, also referred to as the γ4 isotype when referring to the complete set of constant regions on the heavy chain). Executed.

  Any protein comprising an Fc portion of an immunoglobulin, an analog of an Fc portion of an immunoglobulin, or a fragment of an Fc portion of an immunoglobulin can be used in the present invention. In particularly preferred embodiments, the protein comprises a protein of interest (eg, other than the Fc portion of Ig) fused to the Fc portion of an immunoglobulin. The protein of interest may be fused directly to the Fc portion or may be fused via a peptide linker. The Fc portion may be fused to the protein of interest at one or both ends thereof. These heterologous fusion proteins have biological activity and are known to have an increased half-life compared to the native protein of interest. An example is a TNF-alpha receptor protein fused to an Fc moiety, such as etanercept (ENBREL®, Amgen, USA). Another example is an alphacept (Amevive), or preferably a modified Fc (IgG1), which is a fusion protein comprising an Fc region of the IgG1 subtype fused to the first extracellular domain of human LFA-3 -LFA-3 or other Fc-LFA-3 fusion protein, which is substantially non-depleting or has reduced depleting activity. Preferably, as discussed with respect to therapeutic antibodies, the fusion protein is preferably intended to be non-depleting, preferably the cytolytic activity of the cell with which it is associated or bound, in particular ADCC or ADCP It is intended not to trigger. When referring to activity mediated by the fusion protein, ADCC or ADCP may also be interchangeably referred to as fusion protein dependent cell mediated cytotoxicity (FPDCC) and fusion protein dependent cell mediated phagocytosis (FPDCP). it can. Preferably, the Fc portion is a subtype of Fc portion that has relatively low binding to the FcγRIIIa receptor or is modified to reduce binding to the FcγRIIIa receptor.

  An additional subject of the present invention is a method for assessing a subject's response to treatment with a protein comprising the Fc portion of the IgG4 subtype, comprising determining the amino acid residue at position 158 of the FcγRIIIa receptor, The presence of valine at position 158 indicates a low response to the treatment, and the presence of phenylalanine at position 158 indicates a good response to the treatment.

  Another subject of the invention is a method of selecting patients for treatment with an Fc moiety-containing composition, preferably an antibody, which composition is intended to be non-depleting, preferably it is associated or bound Intended to elicit cytolytic activity of cells that do not specifically induce ADCC or ADCP, the method determines the amino acid residue at position 158 of the FcγRIIIa receptor and selects patients with phenylalanine at position 158 for the treatment Including doing. The method can also be carried out by determining the nucleotide present at nucleotide position 559 of the FCG3A cDNA or nucleotide position 4985 of the FCG3A gene. Preferably, the therapeutic antibody is IgG4.

  Another subject of the invention is a method for assessing a subject's side effects on treatment with a composition, preferably an antibody, comprising an Fc moiety, the composition being intended to be non-depleting, preferably Is not intended to elicit cytolytic activity of the cell to which it is associated or bound, particularly ADCC or ADCP, the method comprising determining the amino acid residue at position 158 of the FcγRIIIa receptor, wherein valine is present at position 158 For example, the side effect to the treatment is strong, and if phenylalanine is present at position 158, the side effect to the treatment is weak. The method can also be carried out by determining the nucleotide present at nucleotide position 559 of the FCG3A cDNA or nucleotide position 4985 of the FCG3A gene.

  The method of the present invention can be used particularly beneficially in methods of treating diseases. Preferably, the FcγRIIIa receptor genotype is indicative of the outcome of the treatment. In one example, the methods of the invention are used to determine the amount of therapeutic composition to be administered to a subject and the dosing schedule. In another example, the method of the invention is used to select a therapeutic composition to be administered to a subject. The composition is, for example, a therapeutic composition that is less likely to induce ADCC or ADCP in antibody-bound cells, or less likely to induce cytokine release by immune cells.

  If it is determined that the subject has valine at position 158 of the FcγRIIIa receptor, the subject is considered to exhibit increased sensitivity to side effects and / or decreased response to treatment (eg, less effective treatment) It is done. Such a subject may be treated more beneficially with a composition that is likely to provide high efficacy in the subject or according to a therapy that is likely. Examples of compositions that may be more suitable for such patients are low ADCC or ADCP activity, or low binding to FcγRIIIa receptors compared to another antibody that specifically binds to the same biological target protein. It is an antibody compound having In one aspect, antibody binding to the FcγRIIIa receptor using the method of Koene et al., Blood (1997) or using the BIAcore system (Okasaki A et al. J Mol Biol 2004, 336: 1239-1249). Can be assessed. Alternatively, subjects with valine at position 158 of the FcγRIIIa receptor can be treated with conjoint treatments (eg, cytokines) at various doses or regimens, preferably at lower doses than subjects with phenylalanine at position 158. The antibody is administered using premedication to treat or prevent the release syndrome. Preferably, the dosing regimen, dosing or compound selection is intended to result in decreased lysis of antibody-bound cells and / or decreased cytokine release. Antibodies with reduced ability to bind to the FcγRIIIa receptor are known and can be prepared according to methods. In certain embodiments, for example, certain residues within the Fc region known to reduce interaction with the Fc receptor are altered, or within the Fc region (or Ig that may reduce binding to the Fc receptor). The antibody contains amino acid modifications that reduce the interaction with the Fc receptor by using well-known methods that experimentally identify amino acid changes at other positions within). In the case of an Fc region derived from an IgG1 subtype in an antibody in which depletion is not desired, it is described in Shields et al. (J. Biol. Chem. 276: 6591-6604); PCT Publication (International Publication No. WO 2004/126750). Modifications can be performed as follows: The disclosure content of said document is hereby incorporated by reference.

The present invention also provides a method for monitoring or treating a subject,
Determining a subject's FCGR3A genotype, wherein the genotype correlates with or increases the likelihood of responding to treatment with an Fc moiety-containing composition, particularly an antibody, or treatment with the antibody Correlate with an increase or decrease in the likelihood of having side effects as a result of; and monitoring subjects for the occurrence of side effects on treatment or the efficacy of treatment with antibodies:
A method comprising:

The present invention is a method for treating a subject comprising:
Determining a subject's FCGR3A genotype, wherein the genotype correlates with an increased or decreased response to treatment with an Fc moiety-containing composition, or a side effect as a result of treatment with the antibody. Further comprising: correlating with an increase or decrease in the likelihood of exhibiting; and selecting or determining a treatment suitable for treating the subject. Preferably, the step of selecting or determining a treatment suitable for treatment of the subject comprises selecting a composition to be administered to the subject. In other aspects, determining or determining a treatment suitable for treatment of the subject comprises determining the dose of therapeutic composition administered to the subject, the frequency of administration, or the duration of treatment with that composition. Including selecting. Preferably, the method further comprises the step of (c) prescribing the treatment selected in step (b), preferably the composition to the subject. Preferably, the composition administered to the subject is an antibody composition having reduced binding to an FcγRIIIa polypeptide, preferably an FcγRIIIa polypeptide comprising valine at position 158.

  In another aspect, it is determined that the subject exhibits increased susceptibility to side effects (eg, cytokine release syndrome) that occur in response to treatment with an Fc moiety-containing composition, particularly an antibody, or When it is determined to show a reduced response (efficacy) to treatment with an Fc moiety-containing composition, particularly an antibody, to reduce the side effects or to improve the efficacy of the treatment, eg by NK and / or macrophages A treatment intended to inhibit mediated ADCC or ADCP can be prescribed to the patient.

Assessment and Clinical Testing of Test Compositions The methods of the present invention are also useful in clinical trials to assess a subject's therapeutic response to Fc moiety-containing compositions, particularly antibodies, or susceptibility to side effects induced by the treatment. Can be used. Such a method is expected to be useful, for example, in determining whether one, two or more treatment groups in a clinical trial are balanced with respect to the number of subjects exhibiting an increased or decreased response to treatment. Is done. The methods of the invention can also be used to select subjects for inclusion in clinical trials. The methods of the invention are expected to be particularly useful in clinical trials where the efficacy or side effects of the test composition are assessed.

  It is known to use the method of the invention in clinical trials that the test composition has the ability to induce antibody-mediated side effects, in particular NK cell or macrophage-mediated side effects such as cytokine release syndrome, Alternatively, it is particularly suitable when suspected or when the test composition is suspected to bind via its Fc region to an FcγRIIIa polypeptide, preferably an FcγRIIIa polypeptide comprising valine at position 158.

  For example, the methods of the invention can include determining a subject's FCGR3A genotype, wherein according to the genotype, the subject is a subgroup for treatment or analysis in clinical trials, or clinical Entered into the subgroup for inclusion in the exam.

In one aspect, the present invention provides a method for conducting a clinical test of a test composition, comprising the following steps.
(A) administering a test composition to a plurality of individuals; and (b) identifying one or more individuals having a first FCGR3A genotype and one or more individuals having a second FCGR3A genotype. Preferably in this case, the first FCGR3A genotype exhibits increased susceptibility to side effects or decreased response to treatment with an Fc moiety-containing composition, particularly an antibody,
The second FCGR3A genotype shows reduced susceptibility to side effects or reduced response to treatment with Fc moiety-containing compositions, particularly antibodies. The method optionally comprises (a) assessing the response of the individual (s) having the first FCGR3A genotype to the test composition; and / or (b) of the population having the second FCGR3A genotype. Assessing the response to the test composition. Preferably, the response to the test composition is assessed in both populations having the first and second FCGR3A genotypes. Preferably, the response is assessed separately in the first and second subpopulation populations. Assessing the response to the test composition preferably includes assessing the therapeutic efficacy of the test composition.

The present invention also relates to a method for conducting a clinical test of a test composition comprising the following steps.
-Identifying a first population having a first FCGR3A genotype and a second population having a second FCGR3A genotype;
-Administering the test composition to the individuals of the first and / or second population. In one embodiment, the test composition is administered to the first population of individuals and not to the second population of individuals. In one aspect, the test composition is administered to the second population of individuals and not to the first population of individuals. In another embodiment, a test composition is administered to both the first and second populations of individuals. Preferably, the test composition is an Fc moiety-containing composition, particularly an antibody.

  In the methods of monitoring or treating a subject and performing clinical trials described herein, the Fc moiety-containing composition is preferably intended to be non-depleting, preferably it is associated or bound. It is intended not to induce cytolytic activity of the cells that do, especially ADCC or ADCP. Most preferably, the Fc moiety-containing composition comprises a subclass G4 Fc region.

DETAILED DESCRIPTION The following terms used herein have the meanings ascribed to them unless specified otherwise.

The term “antibody” as used herein refers to polyclonal and monoclonal antibodies, and fragments and derivatives thereof. Antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM, depending on the type of constant domain of the heavy chain. Some of these are further divided into subclasses or isotypes such as IgG1, IgG2, IgG3, IgG4, etc. A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to the light and heavy chains, respectively. The heavy chain constant domains that correspond to the different classes of immunoglobulins are termed "alpha", "delta", "epsilon", "gamma", and "mu", respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and / or IgM are a preferred class of antibodies for use in the present invention, with IgG being particularly preferred because they are the most common antibodies in physiological situations, because they are laboratory This is because IgG can be prepared most easily by setting, and IgG is specifically recognized by the Fc gamma receptor. Preferably, the antibody of the present invention is a monoclonal antibody. Particularly preferred are antibodies that are suitable for humanized, chimeric, human, or other humans.

  “Therapeutic antibody” refers to any antibody, derivative of an antibody, or antibody fragment that can be used safely in humans, eg, with respect to the therapeutic methods described herein. Antibodies suitable for humans are elicited when all types of humanized, chimeric, or fully human antibodies, or at least part of the antibodies are derived from humans or when native non-human antibodies are used. Any antibody that has been otherwise modified to avoid an immune response is included.

  For purposes of the present invention, a “humanized” antibody refers to an antibody in which one or more human immunoglobulin constant and variable framework regions are fused to an animal immunoglobulin binding region, eg, a CDR. Such humanized antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding region is derived, but avoid immune responses to the non-human antibody.

  “Chimeric antibody” means (a) a completely different molecule in which the antigen binding site (variable region) confers new properties to another or altered class, effector function and / or species constant region or chimeric antibody; The constant region, or part thereof, has been altered, substituted or exchanged, eg, linked to an enzyme, toxin, hormone, growth factor, drug, etc .; or (b) another or altered antigen specificity. An antibody molecule in which a variable region, or part thereof, has been altered, substituted or exchanged with a variable region having. In a preferred embodiment of the invention, even a chimeric antibody retains the Fc region of an immunoglobulin, preferably a human Fc region, thereby allowing interaction with the human Fc receptor on the surface of the target cell.

  The term “specifically binds” means that the antibody can bind to a binding partner, preferably in a competitive binding assay, which binding partner is eg an NK receptor, eg NKp30, NKp44, NKp46, NKG2D or CD94. / NKG2A, membrane antigens such as CD2, CD3, CD4, CD25, CD28, CD40, CD11 / 18, ICAM, alpha-4-integrin or CTLA4, the binding properties of which are recombinant proteins, epitopes contained therein Or assessed using native protein present on the surface of isolated NK or related target cells. Competitive binding assays and other methods for measuring specific binding are further described below and are well known in the art.

  As used herein, “ADCC” or “antibody-dependent cell-mediated cytotoxicity” refers to nonspecific cytotoxic cells that express FcyR, in particular FcγRIIIa, recognize a bound antibody on a target cell, It then refers to a cell-mediated reaction that causes lysis of the target cell.

  As used herein, “ADCP” or “antibody-dependent cell-mediated phagocytosis” refers to nonspecific cytotoxic cells that express FcyR, in particular FcγRIIIa, recognize bound antibodies on target cells, It then refers to a cell-mediated reaction that causes phagocytosis of the target cell.

  As used herein, “amino acid modification” means an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. Preferred amino acid modifications herein are substitutions.

  The terms “isolated”, “purified” or “biologically pure” refer to material that is substantially or essentially free from the components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The most numerous species of proteins present in the preparation is substantially purified.

  The term “biological sample” as used herein includes, but is not limited to, biological fluids (eg, serum, lymph, blood), cell samples, or tissue samples (eg, bone marrow).

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers.

A “label” or “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³² P, fluorescent dyes, high electron density reagents, enzymes (eg, enzymes commonly used in ELISA), biotin, digoxigenin, or haptens and, for example, by incorporating radioactive labels within peptides Proteins that can be made detectable or that are used to detect antibodies that specifically react with peptides.

  The term “recombinant” when used with respect to, for example, a cell, or a nucleic acid, protein, or vector, is modified by introduction of a heterologous nucleic acid or protein, or modification of a native nucleic acid or protein. Or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in native (non-recombinant) cells, or are otherwise abnormally expressed, reduced in expression, or not expressed at all Express native gene.

  In one example, a composition comprising an Fc portion, preferably a therapeutic antibody or Fc fusion protein, blocks or neutralizes important cytokine or receptor-ligand interactions of the immune system. The therapeutic activity of the antibody does not require or is based on NK cell cytolytic activity and ADCC induction. The therapeutic antibody may be polyclonal or, preferably, monoclonal. They may be produced by hybridomas or recombinant cells that have been engineered to express the desired variable and constant domains. Methods for producing antibodies in hybridomas are known, for example, in US Pat. No. 6,677,138 and PCT Publication (International Publication) WO 00/58499, the disclosure of which is hereby incorporated by reference. Incorporated into the book. The antibody may be a single chain antibody or other antibody derivative that retains antigen specificity and low hinge region or variants thereof. These may be multifunctional antibodies, recombinant antibodies, ScFv, humanized antibodies, or variants thereof. The therapeutic antibody is specific for a surface antigen, such as a membrane antigen or a free antigen. In preferred aspects, the therapeutic antibody may be specific for a membrane-bound cytokine, such as TNFα, or a cell surface molecule, such as a T lymphocyte or B lymphocyte cell surface molecule, or an NK receptor. Examples of membrane-bound molecules include NKp30, NKp44, NKp46, NKG2D, FcγRIIIa or CD94 / NKG2A, CD2, CD3, CD4, CD25, CD28, CD40, CD11 / 18, ICAM, alpha-4-integrin or T lymphocyte Related antigen-4 (also known as CTLA-4 and CD152 antigen) is included. In another preferred embodiment, an antibody is used in the treatment of transplant rejection, eg, graft versus host or host versus graft disease, such as non-depleting IgG1 anti-CD25 antibodies (eg, basiliximab and daclizumab) )), An IgG4-type anti-CD25 antibody, an antibody that inhibits T cell costimulatory receptors, and an anti-CD28 antibody. In this case, T cell depletion and / or APC-mediated proliferation is a target to be avoided. The therapeutic antibody is preferably an IgG4 type or IgG1 type antibody and includes an amino acid modification or is made to exhibit reduced binding to FcγRIIIa.

  Typical examples of therapeutic antibodies used in accordance with the present invention that do not need to be depleting to target cells are listed in Table 1.

  Within the context of the present invention, a subject or patient includes any mammalian subject or patient, more preferably a human subject or patient.

Antibodies that Modulate NK Cells In a preferred embodiment, a therapeutic antibody that is not intended or required to be depleting to a target cell can bind to membrane-bound molecules on NK cells by binding to NK cells. It is an antibody that regulates function. The activity of NK cells is regulated by a complex mechanism that involves both stimulatory and inhibitory signals. Thus, effective NK cell-mediated therapy, including inducing NK cell activation or proliferation, can be achieved by both stimulating these cells or neutralizing inhibitory signals. . Such an approach is useful, for example, in the treatment of tumors or infections where NK cells mediate lysis of target cells, but the NK cell receptor to which the therapeutic antibody binds does not contribute to NK cell lysis. Other therapies target a decrease in NK cell activity. These therapies may involve activation of inhibitory NK cell receptors or blocking of activated NK cell receptors and may be useful in the treatment or transplantation of immune deficiencies such as autoimmune disorders.

  As used herein, “NK” cells refer to a subpopulation of lymphocytes involved in immunity that is not normal immunity. NK cells can be identified based on specific characteristics and biological properties. Its characteristics and properties include, for example, expression of special surface antigens including FcγRIIIa, CD56 and / or CD57, absence of cell surface alpha / beta or gamma / delta TCR complexes, activation of special cytolytic enzymes The ability to bind to and kill cells that do not express "self" MHC / HLA antigens, to kill tumor cells or other pathological cells that express ligands for NK activating receptors, and immune responses The ability to release protein molecules called cytokines that stimulate or inhibit. Any of these features and activities can be used to identify NK cells using methods well known in the art.

  Within the context of the present invention, an “active” or “activated” NK cell refers to a biologically active NK cell, more specifically an NK cell that has the ability to lyse target cells. For example, “active” NK cells can kill NK activated receptor-ligand cells that do not express “self” MHC / HLA antigens (KIR-incompatible cells). Examples of target cells suitable for use in a redirected killing assay are P815 and K562 cells, but any number of cell types can be used and are well known in the art (See, eg, Sivori et al. (1997) J. Exp. Med. 186: 1129-1136; Vitale et al. (1998) J. Exp. Med. 187: 2065-2072; Pessino et al. (1998) J. Exp. Med. 188: 953 -960; Neri et al. (2001) Clin. Diag. Lab. Immun. 8: 1131-1135). An “active” or “activated” cell can also be any other property or activity associated with NK activity known in the art, such as cytokines (eg, IFN-gamma and TNF-alpha production, free intracellular calcium levels). Increase).

  NK cells are negatively regulated by major histocompatibility complex (MHC) class I specific inhibitory receptors (Karre et al., 1986; Ohlen et al., 1989; the disclosure of which is incorporated herein by reference) . These specific receptors bind to major histocompatibility complex (MHC) class I molecules or polymorphic determinants of HLA and inhibit natural killer (NK) cell lysis. In humans, a family of receptors called killer Ig-like receptors (KIRs) recognize a group of HLA class I alleles.

  In the present invention, the term “an antibody that blocks NK cell inhibitory receptors” is specific to NK cell inhibitory receptors, ie, KIR or NKG2A / C of NK cells, and suppresses KIR or NKG2A / C. Represents compounds that neutralize sex signals, preferably antibodies or fragments thereof. Preferably, said compound, preferably an antibody or fragment thereof, can block the interaction between HLA and NK cell inhibitory receptors. Said antibody may be polyclonal or, preferably, monoclonal. They may be produced by hybridomas or recombinant cells that have been engineered to express the desired variable and constant domains. The antibodies may be single chain antibodies or other antibody derivatives that retain antigen specificity and low hinge regions or variants thereof, such as Fab fragments, Fab'2 fragments, CDRs and ScFvs. These may be multifunctional antibodies, recombinant antibodies, humanized antibodies, or variants thereof.

  Preferably, the antibody blocking the inhibitory receptor of NK cells has an inhibitory signal of at least one inhibitory receptor selected from the group consisting of KIR2DL2, KIR2DL3, KIR2DL1, KIR3DL1, KIR3DL2, NKG2A and NKG2C. The antibody or fragment thereof to be summed. Optionally, GL183 (available from KIR2DL2, L3, Immunotech, France and Beckton Dickinson, USA); EB6 (available from KIR2DL1, Immunotech, France and Beckton Dickinson, USA); AZ138 (KIR3DL1, Moretta et al., Univ. Genova, Available from Italy); Q66 (KIR3DL2, available from Immunotech, France); Z270 (available from NKG2A, Immunotech, France); P25 (available from NKG2A / C, Moretta et al., Univ. Genova, Italy); and Antibodies can be selected from the group consisting of DX9, Z27 (available from KIR3DL1, Immunotech, France and Beckton Dickinson, USA).

  The term “activated NK receptor” as used herein, when stimulated, is any property or activity associated with NK activity known in the art, such as cytokines (eg, IFN-gamma and Surfaces of NK cells that produce a measurable increase in TNF-alpha production, an increase in intracellular free calcium levels, or the ability to target cells in, for example, the redirect killing assay described elsewhere herein. Represents any molecule of Examples of such receptors include the natural cytotoxicity receptor (NCR) known as NKp30, NKp44, and NKp46. Another particularly preferred example is the NKG2D receptor, which is also present on CD4 + and CD8 + T cells and gamma-delta T cells. Hereinafter, a method for determining whether NK cells are active will be described in more detail.

  Several other NK-specific receptors have been identified that play an important role in NK cell-mediated recognition and killing of HLA class I deficient target cells. These receptors termed NKp30, NKp46 and NKp44 are members of the Ig superfamily. Their cross-linking induced by specific mAbs results in strong NK cell activation resulting in increased intracellular Ca ++ levels, induction of cytotoxicity, and lymphokine release. Importantly, mAb-mediated activation of NKp30, NKp46, and / or NKp44 activates NK cytotoxicity against many types of target cells. These findings provide evidence for the central role of these receptors in natural cytotoxicity.

  Antibodies against NK cell receptors may be generated by any technique known in the art. Typically, they are made by immunizing a non-human animal, preferably a mouse, with an immunogen comprising an activating receptor present on the surface of NK cells. Activating receptors include whole NK cells or cell membranes, receptors such as NKp30 (see eg PCT WO01 / 36630, the disclosure of which is incorporated herein by reference in its entirety), NKp44 (eg Vitale et al. (1998) J. Exp. Med. 187: 2065-2072, the disclosure of which is incorporated herein by reference in its entirety, or NKp46 (eg, Sivori et al. (1997) J. Exp. Med). 186: 1129-1136; see Pessino et al. (1998) J. Exp. Med. 188: 953-960; the disclosures of which are incorporated herein by reference in their entirety. Or a fragment or derivative thereof, typically an immunogenic fragment, ie the surface of a cell expressing any of the above receptors or any other receptor that, when stimulated, induces activation of NK cells so It would include a portion of a polypeptide comprising an epitope which is out. The fragment typically contains at least 7 contiguous amino acids of the mature polypeptide sequence, even more preferably at least 10 contiguous amino acids. They are essentially derived from the extracellular domain of the receptor. Any present on the surface of an NK cell in response to stimulation that induces cellular activation as measured by cytotoxicity, increased intracellular free calcium levels, cytokine production, or any other method known in the art It will be appreciated that any of the receptors can be used to generate antibodies. In a preferred embodiment, the activated NK cell receptor used to generate the antibody is a human receptor.

  In one aspect, the immunogen comprises wild type human NKp30, NKp44, or NKp46 polypeptide in a lipid membrane, typically on the surface of a cell. In a particular aspect, the immunogen comprises intact NK cells, particularly intact human NK cells, which are optionally treated or lysed. Examples of preferred isolated antibodies of the present invention are directed against at least one isolated amino acid compound of the present invention and induced by NK cells placed in a 1: 1 ratio in the presence of target cells. Isolated antibodies that are capable of inducing an increase of at least about 4 fold, preferably at least about 5 fold, more preferably at least about 6 fold, for natural cytotoxicity are included. In certain embodiments, the antibody binds to an epitope that is essentially the same as any of the following monoclonal antibodies: AZ20, A76, Z25, Z231, or BAB281. In the present specification, such antibodies are referred to as “AZ20-like antibodies”, “A76-like antibodies”, and the like. The term “binding to substantially the same epitope or determinant” as monoclonal antibody x means that the antibody can “compete” with x, where x is AZ20, A76, etc. Identification of one or more antibodies that bind to substantially the same epitope as the monoclonal antibody in question is readily determined using any one of a variety of immunological screening assays that can assess antibody competition. can do. All the above assays are conventional in the art (see, eg, US Pat. No. 5,660,827 issued Aug. 26, 1997, which is specifically incorporated herein by reference). . It will be appreciated that in order to identify antibodies that bind to the same or substantially the same epitope as the monoclonal antibody in question, it is not required in any way to actually determine the epitope to which the antibody binds.

  In other aspects, the antibody may bind to a molecule other than the natural cytotoxic receptor (NCR) or inhibitor receptor on NK cells. For example, the antibody may bind to any other NK surface molecule, such as an activating molecule, costimulatory receptor or adhesion molecule, including but not limited to NKp80, 2B4 (CD244), NKRP-1A (CD161), BY55 (CD160), PEN5 (CD162R), L-selectin (CD62L) and CD31 are included.

Methods for Classifying Individuals In the present invention, the term FCGR3A gene refers to any nucleic acid molecule that encodes a subject's FcγRIIIa polypeptide. The term includes in particular any synthetic nucleic acid comprising genomic DNA, cDNA, RNA (pre-rRNA, messenger RNA, etc.), etc., or all or part of its sequence. Synthetic nucleic acids include cDNA, which is prepared from RNA and contains at least a portion of the sequence of FCGR3A genomic DNA, such as one or more introns or one containing one or more mutations. Most preferably, the term FCGR3A gene represents genomic DNA, cDNA or mRNA, typically genomic DNA or mRNA. The FCGR3A gene is preferably a human FCGR3A gene or nucleic acid, ie, includes a nucleic acid sequence encoding all or part of an FcγRIIIa polypeptide having the sequence of a human FcγRIIIa polypeptide. Such nucleic acids can be isolated or prepared according to known techniques. For example, they may be isolated from gene libraries or banks by hybridization techniques. They can also be synthesized genetically or chemically. The gene structure of the human FCGR3A gene is as shown in FIG. The amino acid sequence of human FcγRIIIa is as shown in FIG. Amino acid position 158 is numbered from residue 1 of the mature protein and corresponds to residue 176 of the preprotein with the signal peptide. The sequence of the wild type FCGR3A gene is as shown in FIG. 3 (see also Genbank accession number AL590385 or NM_000569 for partial sequence).

  Within the context of the present invention, a portion or part means at least 3 nucleotides (eg codons), preferably at least 9 nucleotides, even more preferably at least 15 nucleotides, and as much as 1000 nucleotides. It can contain a large number of nucleotides. Such moieties can be obtained by any technique known in the art, such as enzymatic cleavage and / or chemical cleavage, chemical synthesis or combinations thereof. For purposes of clarity, the sequence of the portion of the FCGR3A gene that encodes amino acid position 158:

を示す。

Indicates.

  As shown throughout the present disclosure, the present invention includes a method comprising determining a subject's FCGR3A 158 genotype in vitro. In any aspect of the invention relating to the determination of FCGR3A genotype, determining its phenotype, ie determining the identity of the amino acid residue present (or encoded) at position 158 of the FcγRIIIa polypeptide. It is understood that the genotype can be easily determined. Thus, the determination of the FCGR3A genotype can comprise or consist of determining the identity of the amino acid residue present (or encoded) at position 158 of the FcγRIIIa polypeptide.

  Preferably, valine homozygous at position 158 of the FcγRIIIa receptor indicates a reduced response to the treatment in terms of potency, and phenylalanine (heterozygous or homozygous at position 158 of the FcγRIIIa receptor. (If present) indicates an increased response to the treatment. Regarding side effects derived from treatment with antibodies, preferably if position 158 of the FcγRIIIa receptor is homozygous for valine, it indicates an increased sensitivity to side effects, and if phenylalanine is present at position 158 of the FcγRIIIa receptor, Shows a reduction in side effects or susceptibility to side effects to treatment. Thus, the effect of the FcγRIIIa receptor position 158 genotype on therapeutic response or side effects is generally characterized more strongly than usual, and the effect is treated if the subject is a homozygote for position 158 valine. Decreased response to and / or increased side effects. However, subjects who are homozygous or heterozygous for phenylalanine at position 158 have a similar response to treatment and compared to observations in patients who are homozygous for position 158. The response of both these groups is increased. Also, subjects who are homozygous or heterozygous for phenylalanine at position 158 are similar in terms of side effects resulting from antibody treatment and compared to observations in patients who are homozygous for position 158. Thus, the susceptibility to side effects of both these groups is reduced.

  Determination of the genotype of the subject's FCGR3A gene or corresponding polypeptide may be accomplished by a variety of techniques including analysis of the encoding nucleic acid molecule or encoded polypeptide. Analysis may include sequencing, sequencing, migration, electrophoresis, immunological techniques, amplification, specific digestion or hybridization, and the like.

  In certain aspects, the determination of the amino acid residue at position 158 of the FcγRIIIa receptor comprises sequencing the FCGR3A receptor gene or RNA or those portions comprising nucleotides encoding amino acid residue 158.

  In other particular embodiments, determining the amino acid residue at position 158 of the FcγRIIIa receptor comprises amplifying the portion of the FCGR3A receptor gene or RNA or those portions that comprise nucleotides encoding amino acid residue 158. For example, amplification may be performed by polymerase chain reaction (PCR) such as simple PCR, RT-PCR or nested PCR using conventional methods and primers. For determining the amino acid residue at position 158 of the FcγRIIIa receptor, a preferred genotyping method, including the disclosure of nucleic acid primers, is Dall'Ozzo S, Andres C, Bardos P, Watier H, and Thibault G, J Immunol Methods. 2003 Jun 1; 277 (1-2): 185-92. The disclosure is hereby incorporated by reference in its entirety, including, but not limited to, the specific nucleotide sequences disclosed in the literature.

  In this regard, amplification primers used in the present invention more preferably contain less than about 50 nucleotides, even more preferably contain less than 30 nucleotides, typically less than about 25 or 20 nucleotides. Of nucleotides. Also, preferred primers usually contain at least 5 nucleotides, preferably at least 8 nucleotides, to ensure specificity. The sequence of the primer can be prepared based on the sequence of the FCGR3A gene, and preferably can be allowed to be completely complementary thereto. The probe may be labeled using any known technique, such as radioactivity, fluorescence, enzyme, chemical techniques, and the like. In this labeling, for example, phosphor 32, biotin (16-dUTP), digoxygenin (11-dUTP) can be used. It will be understood that the present invention is not bound or limited by any particular detection or labeling technique. The primer may further comprise a restriction site for introducing an allele-specific restriction site into the amplified nucleic acid, as disclosed below.

  Specific examples of such amplification primers are, for example, SEQ ID NOs: 1-4.

  One skilled in the art can design other primers for use in the amplification process, such as any fragment of the FCGR3A gene and in particular a primer pair comprising a forward sequence and a reverse sequence, wherein the primer of the pair is It will be appreciated that it hybridizes with a region of the FCGR3A gene and allows amplification of at least a portion of the FCGR3A gene containing codon 158. In a preferred embodiment, each primer pair contains at least one primer that is complementary to and overlaps codon 158, allowing 158V (gtt) and 158F (ttt) to be distinguished. Those skilled in the art may also adjust amplification conditions based on common general knowledge and guidance contained herein.

  Thus, in a particular embodiment, the method of the invention PCR amplifies a portion of FCGR3A mRNA or gDNA using a specific oligonucleotide primer in a cell or biological sample, wherein the portion comprises codon 158; And, for example, directly or indirectly analyzing PCR products by electrophoresis, particularly denaturing gel gradient electrophoresis (DGGE).

  In another specific embodiment, the determination of the amino acid residue at position 158 of the FcγRIIIa receptor comprises an allele-specific restriction enzyme digestion step. This step can be performed using a restriction enzyme that cleaves the coding sequence of a particular allele (eg, 158V allele) but does not cleave other alleles (eg, 158F allele, or vice versa). If such allele-specific restriction enzyme sites do not occur naturally in the sequence, they are sequenced by amplifying the nucleic acid using an allele-specific amplification primer containing the site in the sequence. May be artificially introduced. After amplification, determination of the presence of the allele may be performed by analyzing the digestion product, eg, by electrophoresis. This technique also makes it possible to identify subjects who are homozygous or heterozygous for the selected allele.

  An example of an allele-specific amplification primer includes SEQ ID NO: 3, for example. SEQ ID NO: 3 introduces the first 3 nucleotides of the NlaIII site (5'-CATG-3 '). Cutting occurs behind G. This primer, except for nucleotide 31 (A), which creates a restriction site, 11 bases that do not hybridize to FCGR3A (this extends the primer for the purpose of facilitating analysis of the amplified product by electrophoresis), and Contains 21 bases that hybridize to FCGR3A.

  In another specific embodiment, determination of the amino acid residue at position 158 of the FcγRIIIa receptor is accomplished by converting the portion containing the FCGR3A receptor gene or RNA or nucleotide encoding amino acid residue 158 to genotype valine or phenylalanine. Hybridizing with a specific nucleic acid probe and determining the presence or absence of the hybrid.

  It will be appreciated that the above methods can be used alone or in various combinations. Furthermore, other techniques known to those skilled in the art may be used as well to determine the FCGR3A-158 genotype. Other techniques include, for example, any method using amplification (eg PCR), specific primers, specific probes, migration, etc., typically quantitative RT-PCR, LCR (ligase chain reaction), TMA (transcription Mediated amplification), PCE (enzyme amplified immunoassay) and bDNA (branched DNA signal amplification) assays.

In a preferred embodiment of the invention, the determination of the amino acid residue at position 158 of the FcγRIIIa receptor is
-Obtaining genomic DNA from a biological sample;
Amplifying the FcγRIIIa receptor gene or a portion thereof comprising a nucleotide encoding amino acid residue 158; and determining the amino acid residue at position 158 of the FcγRIIIa receptor gene:
including.

Amplification can be accomplished using any specific technique such as PCR using specific primers as described above, eg, nested PCR. In the most preferred embodiment, the determination of the amino acid residue at position 158 is performed by allele-specific restriction enzyme digestion. In that case, the method
-Obtaining genomic DNA from a biological sample;
Amplifying the FcγRIIIa receptor gene or part thereof comprising nucleotides encoding amino acid residue 158;
-Introducing allele-specific restriction sites;
Digesting the nucleic acid with an enzyme specific for the restriction site; and-analyzing the digestion product, ie by electrophoresis, the presence of the allele if the digestion product is present:
including.

  In other specific embodiments, the genotype is extracted in vitro or ex vivo from total (or messenger) RNA from cells or biological samples or fluids, optionally using cDNA synthesis, FCGR3A specific oligonucleotide primers (PCR) Amplification, and analysis of PCR products.

  The methods of the invention also include FcγRIIIa receptor polypeptides, or portions thereof containing amino acid residue 158, by direct sequencing, or using reagents specific for each allele of FcγRIIIa polypeptide. Determining the amino acid residue at position 158 of the receptor may be included. This can be determined by any suitable technique known to those skilled in the art, including immunoassays (ELISA, EIA, RIA, etc.). It can be performed using any affinity reagent specific for the FcγRIIIa158 polypeptide, more preferably any antibody or fragment or derivative thereof. In a particular embodiment, the FcγRIIIa158 polypeptide is detected using an anti-FcγRIIIa158 antibody (or fragment thereof), more preferably a monoclonal antibody, that distinguishes between FcγRIIIa158V and FcγRIIIa158F. The antibody (or affinity reagent) may be labeled by any suitable mode (radioactivity, fluorescence, enzyme, chemical mode, etc.). Alternatively, for example, a labeled secondary reagent (eg, an antibody) that binds to the anti-FcγRIIIa158 antibody may be used to reveal (and / or quantify) the presence of the FcγRIIIa158 antibody immune complex.

  The above method is based on the determination of the genotype of FCGR3A158 in a subject's biological sample. The biological sample may be any sample containing the FCGR3A gene or the corresponding polypeptide, in particular blood, bone marrow, lymph node or fluid containing the FCGR3A158 gene or polypeptide, in particular blood or urine. Furthermore, since the FCGR3A 158 gene is generally present in cells, tissues or fluids, the method of the present invention has been processed to make the gene or polypeptide available for detection or analysis. Samples are usually used. Processing may include any conventional immobilization technique, cell lysis (mechanical or chemical or physical), or any other conventional method used, for example, in immunohistology or biology.

  The method is particularly suitable for determining a subject's response to treatment with a therapeutic antibody. In this regard, in certain embodiments, the subject has a disorder, and treatment with a therapeutic antibody aims to ameliorate the disorder. In this case, the antibody need not be depleting to the cells to which it binds. The membrane target of the antibody is as discussed above.

  In the following examples, other aspects and advantages of the present invention are disclosed. This example should be considered exemplary and should not be considered as limiting the scope of this application.

Example 1
FCGR3A-158V / F polymorphism genotyping materials and methods FCGR3A-158V / F genotyping All samples are analyzed in the same laboratory and DNA is extracted using standard procedures. The procedure includes precautions to avoid cross-contamination. DNA can be isolated from peripheral blood, bone marrow or lymph nodes. The genotyping of the FCGR3A-158V / F polymorphism is performed by nested PCR followed by allele-specific restriction enzyme digestion as described by Koene et al. (Koene et al., Blood. 1997; 90: 1109-1114). To implement. Briefly, two FCGR3A specific primers

（Eurobio, Les Ulis, France）を使用して、多型部位を含有する１．２kb断片を増幅する。

(Eurobio, Les Ulis, France) is used to amplify a 1.2 kb fragment containing the polymorphic site.

  The PCR assay uses 1.25 μg genomic DNA, 200 ng of each primer, 200 μmol / L of each dNTP (MBI Fermentas, Vilnius, Lithuania) and 1 U Taq DNA polymerase (Promega, Charbonniere, France) according to the manufacturer's recommendations. To implement. This first PCR consists of 95 minutes at 95 ° C, then 35 cycles (each cycle consists of 3 steps of 95 ° C for 1 minute, 57 ° C for 1.5 minutes, 72 ° C for 1.5 minutes) and full extension For 8 minutes at 72 ° C. Primer

（Eurobio）を使用する第二のＰＣＲでは、９４bp断片を増幅し、ＦＣＧＲ３Ａ−１５８Ｖ対立遺伝子においてのみ、ＮｌａＩＩＩ制限部位を創出する。

In a second PCR using (Eurobio), a 94 bp fragment is amplified and a NlaIII restriction site is created only in the FCGR3A-158V allele.

  This nested PCR is performed using 1 μL of amplified DNA, 150 ng of each primer, 200 μmol / L of each dNTP, and 1 U of Taq DNA polymerase. The first cycle is at 95 ° C for 5 minutes, followed by 35 cycles (each cycle consisting of 3 steps of 95 ° C for 1 minute, 64 ° C for 1 minute, 72 ° C for 1 minute) and at 72 ° C Complete stretching in 9.5 minutes. The amplified DNA (10 μL) is then digested with 10 U NlaIII (New England Biolabs, Hitchin, England) for 12 hours at 37 ° C. and separated by electrophoresis on an 8% polyacrylamide gel. After staining with ethidium bromide, the DNA band is visualized with ultraviolet light. In homozygous FCGR3A-158F patients, only one undigested band (94 bp) is seen. Three bands (94 bp, 61 bp and 33 bp) are observed in heterozygous individuals, while only two digested bands (61 bp and 33 bp) are obtained in homozygous FCGR3A-158V patients.

FIG. 1 shows the gene structure of the human FCGR3A gene. FIG. 2 is the amino acid sequence of human FcγRIIIa-158F (SEQ ID NO: 7). FIG. 3 shows the nucleic acid sequence of human FCGR3A-158F (SEQ ID NO: 8).

Claims (30)

  A method for assessing a subject's response to treatment with a composition comprising an Fc portion, comprising determining the amino acid residue at position 158 of the FcγRIIIa receptor, wherein the subject has phenylalanine at position 158 A determination indicates an increase in response to the treatment, and a determination to have valine at position 158 indicates a decrease in response to the treatment, where the treatment requires depletion of cells to which the composition is associated or bound And no way.   A method for assessing a subject's response to treatment with a composition comprising an Fc portion, comprising determining the amino acid residue at position 158 of the FcγRIIIa receptor, wherein the subject has phenylalanine at position 158 The determination indicates an increase in response to the treatment, and the determination of having valine at position 158 indicates a decrease in response to the treatment, wherein the Fc portion is an Fc portion of the G4 subclass.   A method for selecting a subject for treatment with a composition comprising an Fc portion, for monitoring a subject for treatment, or for identifying a sub-population of subjects for treatment, said method comprising FcγRIIIa Determining the amino acid residue at position 158 of the receptor, wherein determining that the subject has phenylalanine at position 158 indicates an increased response to the treatment, determining that valine is at position 158, A method showing a decrease in response to the treatment, wherein the treatment does not require depletion of the cells with which the composition is associated or bound.   A method for assessing side effects to treatment with a composition comprising an Fc moiety, comprising determining the amino acid residue at position 158 of the FcγRIIIa receptor, and determining that the subject has phenylalanine at position 158, Showing an increase in response to the treatment and determining having valine at position 158 indicates a decrease in response to the treatment, wherein the treatment does not require depletion of the cells with which the composition is associated or bound, Method.   The method according to any one of claims 1 to 4, wherein the composition comprises an antibody.   5. The method of any one of claims 1-4, wherein the composition comprises a fusion protein comprising an Fc moiety.   7. The method of any one of claims 1-6, wherein the Fc portion is a G4 subclass Fc portion.   8. The method of any one of claims 1-7, wherein the treatment is for enhancing or decreasing the subject's immune response.   8. The method of any one of claims 1-7, wherein the treatment is effective to treat the subject's tumor.   8. The method of any one of claims 1-7, wherein the composition specifically binds to an NK cell surface receptor.   8. The method of any one of claims 1-7, wherein the composition specifically binds to a T cell surface receptor.   11. The method of claim 10, wherein the NK cell surface receptor is selected from the group consisting of inhibitory and activated cell surface receptors.   13. The method of claim 12, wherein the NK cell surface receptor is an inhibitory cell surface receptor.   13. The method of claim 12, wherein the NK cell surface receptor is an activated cell surface receptor.   15. The method of claim 14, wherein the activated cell surface receptor is selected from the group consisting of NKp30, NKp44, NKp46 and NKG2D.   14. The method of claim 13, wherein the inhibitory cell surface receptor is selected from the group consisting of KIR and CD94 / NKG2A.   17. The determination of the amino acid residue at position 158 of the FcγRIIIa receptor comprises sequencing the FcγRIIIa receptor gene or RNA or a portion thereof comprising a nucleotide encoding amino acid residue 158. The method according to claim 1.   18. Determination of the amino acid residue at position 158 of the FcγRIIIa receptor comprises amplifying the FcγRIIIa receptor gene or RNA or a portion thereof comprising a nucleotide encoding amino acid residue 158. The method according to claim 1.   19. The method of claim 18, wherein amplification is performed by polymerase chain reaction (PCR) such as PCR, RT-PCR and nested PCR.   The method according to any one of claims 1 to 19, wherein the determination of the amino acid residue at position 158 of the FcγRIIIa receptor comprises an allele-specific restriction enzyme digestion step.   Determining the amino acid residue at position 158 of the FcγRIIIa receptor can be performed by combining the FcγRIIIa receptor gene or RNA or those portions containing nucleotides encoding amino acid residue 158 with nucleic acid probes specific for genotype valine or phenylalanine. 21. The method according to any one of claims 1 to 20, comprising a step of hybridization. Determination of the amino acid residue at position 158 of the FcγRIIIa receptor
-Obtaining genomic DNA from a biological sample;
Amplifying the FcγRIIIa receptor gene or a portion thereof comprising a nucleotide encoding amino acid residue 158; and determining the amino acid residue at position 158 of the FcγRIIIa receptor gene;
23. A method according to any one of claims 1 to 22, comprising: Determination of the amino acid residue at position 158 of the FcγRIIIa receptor
-Obtaining genomic DNA from a biological sample;
Amplifying the FcγRIIIa receptor gene or part thereof comprising nucleotides encoding amino acid residue 158;
-Introducing allele-specific restriction sites;
Digesting the nucleic acid with an enzyme specific for the restriction site; and-analyzing the digested product, ie, the presence of the digested product by electrophoresis indicates the presence of the allele;
23. A method according to any one of claims 1 to 22, comprising:   Determination of the amino acid residue at position 158 of the FcγRIIIa receptor extracts total (or messenger) RNA from cells or biological samples or fluids in vitro or ex vivo, optionally using cDNA synthesis, specific FCGRIIIa oligonucleotide primers 23. A method according to any one of claims 1-22, comprising (PCR) amplification and analysis of PCR products.   6. The method of any one of claims 1-5, wherein determining the amino acid residue at position 158 of the FcγRIIIa receptor comprises sequencing the FcγRIIIa receptor polypeptide or portion thereof comprising amino acid residue 158. .   26. The method according to any one of claims 1 to 25, wherein the subject is a human subject.   27. The method of claim 26, wherein the subject has a tumor.   27. The method of claim 26, wherein the subject has an inflammatory disorder.   Subject has inflammatory skin disease including psoriasis; inflammatory bowel disease (eg Crohn's disease and ulcerative colitis); adult respiratory distress syndrome; dermatitis; CNS inflammatory disorder such as multiple sclerosis; uveitis Disorders; allergic symptoms such as eczema and asthma and other symptoms involving T cell infiltration and chronic inflammatory reactions; skin hypersensitivity reactions (including poisonous poisons and poisons); autoimmune diseases such as rheumatoid arthritis, Systemic lupus erythematosus (SLE), diabetes, multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, Sjogren's syndrome, juvenile-onset diabetes, and typically tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis Immune response associated with delayed hypersensitivity mediated by recognized cytokines and T lymphocytes; pernicious anemia; following sepsis or trauma Group consisting of: autoimmune hemolytic anemia; myasthenia gravis; disease mediated by antigen-antibody complex; and all types of transplant rejection including graft-versus-host or host-versus-graft disease 27. The method of claim 26, having a disorder selected from.   30. The method of any one of claims 1-29, wherein the therapeutic antibody binds to a lymphocyte membrane antigen.

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