ES2360373T3 - CD86 AND CD80 RECEIVER COMPETITION TESTS. - Google Patents

Abstract

An in vivo or in vitro assay procedure for monitoring the efficiency of binding of L104EA29Ylg to a CD86 receptor in a patient, said method comprising the steps of: (a) providing a blood sample from a patient dosed with L104EA29Ylg, including the sample of blood leukocyte cells; (b) add CD14-FITC to the blood sample; (c) add labeled competitive and non-competitive anti-receptor antibodies to the blood sample; (d) lyse the blood sample and fix leukocytes; (e) isolate leukocytes from the lysed blood sample; (f) measure the expression of the total receptor and the available leukocyte cell receptor, in which the non-competitive antireceptor antibody is 2D4, an antibody that is obtainable from the Mus musculus hybridoma cell line, spleen cell: 2D4, deposited with Deposit Number ATCC PITA-7305.

Description

Field of the Invention

The present invention relates to methods for monitoring compounds used to treat diseases of the immune system such as graft rejection after organ transplantation. Specifically, the present invention relates to a competition based assay of CD86 receptor based on flow cytometry to measure the binding efficiency of L104EA29Ylg to CD86 receptors by comparing the binding of non-competitive anti-CD86 monoclonal antibodies to CD86 receptors (expression of Total CD86) and binding of competitive anti-CD86 antibodies to CD86 receptors not bound by L104EA29Ylg.

Background of the invention

The characteristic property of a vertebrate immune system is the ability to discriminate "the proper" from "the non-self" (strange). This property has led to the evolution of a system that requires multiple signals to achieve optimal immune activation (Janeway, Cold Spring Harbor Symp. Qant. Biol. 54: 1-14 (1989)). T-cell B interactions are essential for the immune response. The levels of many cohesive molecules found in T lymphocytes and B lymphocytes are increased during an immune response (Springer et al., A. Rev. Immunol. 5: 223-252 (1987); Shaw and Shimuzu, Current Opinion in Immunology, Eds Kindt and Long, 1: 92-97 (1988)); and Hemler Immunology Today 9: 109-113 (1988)). Increased levels of these molecules can help explain why activated B cells are more effective in stimulating proliferation of antigen-specific T cells than are resting B cells (Kaiuchi et al., J. Immunol. 131: 109- 114 (1983); Kreiger et al., J. Immunol. 135: 2937-2945 (1985); McKenzie, J. Immunol. 141: 2907-2911 (1988); and Hawrylowicz and Unanue, J. Immunol. 141: 4083 -4088 (1988)).

The generation of an immune response of T lymphocytes ("T cells") is a complex procedure that involves cell-cell interactions (Springer et al., A. Rev. Immunol. 5: 223-252 (1987)), particularly between cells T and accessory cells such as B cells and production of soluble immune mediators (cytokines or lymphokines) (Dinarello and Mier, New Engl. Jour. Med. 317: 940-945 (1987)). This response is regulated by several T cell surface receptors, including the T cell receptor complex (Weiss et al., Ann. Rev. Immunol. 4: 593-619 (1986)) and other "accessory" surface molecules ( Springer et al. (1987) supra). Many of these accessory molecules are cell surface differentiation (CD) antigens that occur in nature defined by the reactivity of monoclonal antibodies on the cell surface (McMichael. Ed, Leukocyte Typing III. Oxford Univ. Press, Oxford, NY (1987)).

In order to achieve effective T lymphocyte activation, two receptors on the cell surface must be joined by their respective ligands and must deliver a signal to the cell. First, the T cell receptor must recognize the antigen in the context of MHC on an antigen presenting cell. Second, a co-stimulator receptor must bind to the appropriate ligand, or co-receptor, in the antigen presenting cell. The most studied T-cell costimulator receptor is CD28, which binds to B7 molecules (CD80 and CD86) in antigen presenting cells. Green JL, Leytze GM, Emswiler J, Peach R, Bajorath J, Cosand W, Linsley PS. Covalent dimerization of CD28 / CTLA-4 and oligomerization of CD80 / CD86 regulate T cell costimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994. Inhibition of the CD28 / B7 pathway in vitro inhibits T cell proliferation, cytokine production and induces non-responsiveness of antigen specific T cells. Green JL, Leytze GM, Emswiler J, Peach R, Bajorath J, Cosand W, Linsley PS. Covalent dimerization of CD28 / CTLA-4 and oligomerization of CD80 / CD86 regulate T cell costimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994; and Kelly S, Linsley P, Warner G, Shyu WC and Paborji M. Investigator Brochure, BMS-188667, CTLA4lg. In animal models, this route has been shown to be important in T-cell dependent immune responses, including alloantigenic recognition and autoimmunity. Green JL, Leytze GM, Emswiler J, Peach R, Bajorath J, Cosand W, Linsley PS. Covalent dimerization of CD28 / CTLA- 4 and oligomerization of CD80 / CD86 regulate T cell costimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994; and Kelly S, Linsley P, Warner G, Shyu WC and Paborji M. Investigator Brochure, BMS-188667, CTLA4lg. Larsen, CP, Pearson, TC, Adams, AB, Tso, P, Shirasugi, N, Strobert, E, Anderson, D, Cowan, S, Price, K, Naemura, J, Emswiler, J, Greene, J, Turk, L, Bajorath, J, Townsend, R, Hagerty, D, Linsley, PS, and RJ Peach. 2005. Rational Development of LEA29Y, a High-Affinity Variant of CTLA4-lg with Potent Immunosuppressive Properties. American Journal of Transplantation. 5 (3): 443-53. Thus, the CD28 / B7 route represents a logical target, viable for an immunomodulatory therapeutic agent.

CTLA4lg (BMS-188667), a condensation protein comprising the extracellular domain of human CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and a fragment of the human IgGI Fc domain, blocks the CD28B7 pathway by binding to COSO and CD86 over the surface of the antigen presenting cells. This compound has been found to be clinically useful as an immunosuppressant. See United States Patent Application 10 / 419,008 (Publication No. 20040022787 A1) which describes and discusses CTLA4lg and L104EA29Ylg and the procedures for preparing and using them. United States Patent Documents Numbers: 5,844,095, 5,885,796, and 5,851,795 describe and discuss CTLA4lg.

A related molecule, L104EA29Ylg (BMS-224818) (also known as LEA29Y), was found to be a

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particularly potent immunomodulatory therapeutic agent. This compound is a human CTLA4lg molecule that contains a two amino acid substitution that results in enhanced binding to CD80 and CD86 in relation to CTLA4lg. See Larsen, CP, Pearson, TC, Adams, AB, Tso, P., Shirasugi, N., Strobert, E., Anderson, D., Cowan, S., Price, K., NaemurFfia, J., Emswiler, J., Greene, J., Turk, L., Bajorath, J., Townsend, R., Hagerty, D., Linsley, PS, and RJ Peach. 2005. Rational Development of LEA29Y, a High-Affinity Variant of CTLA4-lg with Potent Immunosuppressive Properties. American Journal of Transplantation. 5 (3): 443-53. US Patent Application 09 / 865,321 (Publication No. 2002-0182211 Al) describes and discusses L104EA29Ylg. WO 02/094202 reports procedures for inhibiting rejection of islet cell transplantation, in particular for treating diabetes, by administering soluble CTLA4 mutant molecules such as L104EA29Ylg.

WO 98/19706 reports specific human CD80 antibodies capable of inhibiting the binding of CD80 to a CD28 receptor without inhibiting the binding of CD80 to a CTLA4 receptor.

CD80 and CD86 are discussed in Carreno, BM, and Collins, M., 2002 (The B7 Family of Ligants and Its Receptors: New Pathways for Costimulation and Inhibition of Immune Responses, Annu. Rev Immunol. 20: 29-53) and Salomon , B. and Bluestone, JA, 2001 (Complexities of CD28 / B7: CTLA-4 Costimulatory Pathways in Autoimmunity and Transplantation, Annu. Rev. Immunol. 19: 225-52). Since CTLA4lg and L104EA29Ylg bind to circulating leukocytes expressing CD80 and / or CD86 molecules, it would be informative to monitor the extent to which CD80 and / or CD86 is bound to the protein (s) of condensation, in addition to the amount of compound that circulates in the plasma during clinical use. In doing so, clinical workers would be able to correlate levels of compound exposure with levels of receptor saturation required for efficacy in order to monitor binding efficiency. Understanding the extent to which CD86 is saturated with L104EA29Ylg at various blood concentrations can be used to help justify different dosing schedules or regimens. For example, during the development phase, different formulations and routes of administration can be used (for example, monthly intravenous or weekly subcutaneous treatment). This test could be used to help establish the best route and course of administration that manifests maximum saturation for the longest period of time. The use of FACS-based competition assays to characterize monoclonal antibodies to bind OX40L epitopes in activated B cells and dendritic cells is described by Wang et al. (Tissue Antigens, Munksgaard, Copenhagen, DK, vol. 64, No. 5, pages 566-574, November 5, 2004).

Brief summary of the invention

The present invention relates to assays for monitoring and measuring the binding of L104EA29Ylg to a CD86 receptor. In this assay, peripheral mononuclear cells are isolated from a blood sample, and parts of the mononuclear cell sample are pre-incubated with increasing concentrations of L104EA29Ylg. After incubation, a labeled anti-CD86 antibody is added and binding of the labeled antibody is measured using flow cytometry. By comparing the binding of parts of the mononuclear cell sample with varying concentrations of L104EA29Ylg added to a part of the mononuclear cell sample without any added L104EA29Ylg, someone can determine that L104EA29Ylg is binding the receptor.

In yet another embodiment of the invention, an assay procedure is provided in which the binding efficiency of L104EA29Ylg to a receptor is measured using a competitive antibody and a non-competitive antibody concurrently. In such an assay, peripheral monocytes are isolated from a blood sample and parts of the monocyte sample are pre-incubated with L104EA29Ylg. After incubation, a labeled competitive anti-receptor antibody is added and then a non-competitive anti-receptor antibody is added. The binding of the two antibodies is measured using flow cytometry. Noncompetitive antibody binding represents the total receptor amount and competitive antibody binding represents the amount of available receptor, not bound by L104EA29Ylg. In this way, the binding efficiency of L104EA29Ylg can be determined using a single sample.

In yet another embodiment of the invention, an assay procedure is provided in which the binding efficiency of L104EA29Ylg to a receptor is measured using a competitive antibody and a non-competitive antibody in separate samples. In such an assay, whole blood is treated with L104EA29Ylg. PBS is used as an untreated control. Mouse IgG solution is added to all samples to block non-specific binding of detection reagents. To detect unbound levels of CD86, the fluorescently labeled competitive antireceptor antibody (eg, mAb HA5) is added to a group of samples. To detect total levels of CD86, a fluorescently labeled non-competitive antireceptor antibody (for example, 2D4 mAb) is added to another group of samples. To detect monocytes, anti-CD14-FITC is added to each sample. To assess the non-specific fluorescence associated with labeled anti-CD86 mAbs, excess unlabeled anti-human CD86 mAbs are added to a subset of relevant samples (for example, unlabeled HA5 is added to samples containing labeled HAS). Cells were lysed using lysis solution and antibody binding is measured using flow cytometry. Noncompetitive antibody binding represents the total receptor amount and competitive antibody binding represents the amount of available receptor, not bound by L104EA29Ylg. The specific binding (�MFI (medium fluorescence intensity)) is determined by the difference between the total binding (labeled anti-CD86 mAb alone) and the non-specific binding (labeled + anti-CD86 mAb not marked in excess). In this way, the binding efficiency of L104EA29Ylg can be determined using two separate samples.

In yet another embodiment of the invention, an assay procedure is provided to monitor the efficiency of

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union of L104EA29Ylg in a clinical setting. In such a trial, a patient is administered with L104EA29Ylg. A blood sample is obtained from the patient, and a mixture of mouse IgGs is added to aliquots of the blood sample to reduce non-specific binding mediated by Fc receptor. Anti-human CD14-FITC is added to identify monocytes. Then, labeled competitive anti-receptor antibody and labeled non-competitive anti-receptor antibody were added to the aliquots of the blood sample. As before, to evaluate the non-specific fluorescence associated with labeled anti-CD86 mAbs, excess unlabeled human anti-CD86 mAbs are added to a subset of the relevant samples (for example, unlabeled HA5 is added to samples containing labeled HAS ). A lysis / fixative solution is used to lyse red blood cells and fix leukocytes. The samples were centrifuged to remove the lysed blood cells and isolate the leukocytes. The total receptor and the available receptor (not bound by L104EA29Ylg) are measured by determining the binding of competitive and non-competitive antibodies (respectively). Specific binding (�MFI) is determined by the difference between total binding (labeled anti-CD86 mAb alone) and non-specific binding (labeled anti-CD86 mAb + anti-CD86 unbranded mAb).

In a preferred aspect of the above embodiments, the anti-receptor antibody is an anti-CD86 antibody. In particular, the competitive anti-CD86 antibody is FUN-1, IT2.2, or HA5 (clone of HA5.2B7). In another preferred aspect of the invention, the human anti-CD86 antibody is labeled with a fluorophore. In another preferred aspect of the invention, the fluorophore is phycoerythrin (PE).

In yet another preferred aspect of the above embodiments, the non-competitive anti-CD86 antibody is mAb 2D4.

In yet another embodiment of the invention, the monoclonal antibody mAb 2D4 and a hybridoma expressing this mAb are provided. The hybridoma used to produce this antibody was deposited with the ATCC on January 13, 2006, as deposit number PTA-7305 (hybridoma expressing mAb 2D4). These and other embodiments of the invention will be apparent in light of the detailed description below.

Brief description of the figures

Figure 1 illustrates the results of a CD86 competition in isolated peripheral blood mononuclear cells with competitive FUN-1 mAb.

Figure 2 illustrates (a) the results of competition in whole blood with competitive mAb FUN-1 (lug / ml) and (b) the results of competition in whole blood with a non-competitive mAb 2D4.

Figure 3 shows the nucleotide sequence (SEQ ID NO: 1) of the CTLA4lg molecule. The amino acid sequence (SEQ ID NO: 2) encoded by the nucleic acid is also shown. CTLA4lg molecules that can be produced from this nucleotide include molecules having the amino acid sequence of residues: (i) 26-383 of SEQ ID NO: 2, (ii) 26-382 of SEQ ID NO. : 2, (iii) 27-383 of SEQ ID NO: 2, or (iv) 26-382 of SEQ ID NO: 2, or optionally (v) 25-382 of SEQ ID NO: 2 , or (vi) 25-383 of SEQ ID NO: 2. The DNA and amino acid sequences comprise the following regions: (a) a signal sequence of Oncostatin M (amino acids 1-26 of SEQ ID NO: 2);

(b) an extracellular domain of human CTLA4 (amino acids 27-151 of SEQ ID NO: 2); (c) a modified part of the constant region of human IgG I (amino acids 152-383 of SEQ ID NO: 2), including a modified hinge region (amino acids 152-166 of SEQ ID NO: 2), a CH2 domain of modified human IgG1 (amino acids 167-276 of SEQ ID NO: 2) and a CH3 domain of modified human IgG 1 (amino acids 277-383 of SEQ ID NO: 2).

Figure 4 depicts a nucleotide sequence (SEQ ID NO: 3) and an amino acid sequence (SEQ ID NO: 4) of L104EA29Ylg (also known as "LEA29Y") comprising an Oncostatin M signal sequence; a mutated extracellular domain of CTLA4 starting at (as indicated in Figure 4) methionine at position +1 and ending at aspartic acid at position +124, or starting at alanine at position -1 and ending with aspartic acid at position +124; and an Ig region. SEQ ID NO: 3 and 4 indicate the first amino acid of the signal sequence of Oncostatin M (M, which is followed by G) as 1. SEQ ID NO: 3 and 4 represent a nucleotide and an amino acid sequence, respectively, of L104EA29Ylg comprising an Oncostatin M signal sequence; a mutated extracellular domain of CTLA4 that begins at methionine at the +27 position and ends with aspartic acid at the +150 position, or begins at the alanine at the +26 position and ends with aspartic acid at the +150 position; and an Ig region. L104EA29Ylg may have the amino acid sequence of residues: (i) 26-383 of SEQ ID NO: 4, (ii) 26-382 of SEQ ID NO: 4; (iii) 27-383 of SEQ ID NO: 4 or (iv) 27-382 of SEQ ID NO: 4, or optionally (v) 25-382 of SEQ ID NO: 4, or (vi ) 25-383 of SEQ ID NO: 4.

Figure 5 represents the results of a competition of CD86 receptor ex vivo in whole blood with mAb HA5 and mAb FUN1. (NHV = Normal Healthy Volunteer). This figure demonstrates that the concentration of L104EA29Ylg necessary to inhibit specific binding of HA5 by 50% is 0.13 ug / ml, and the concentration of L104EA29Ylg necessary to inhibit specific binding of FUN1 by 50% is 0.49 ug / ml.

Figure 6 depicts the competition characterization of CD86 with mAb FUN 1 after incubation of whole blood with L104EA29Ylg. (MFI = medium fluorescence intensity; MLR = mixed leukocyte reaction; NS = Not specific). This figure summarizes FUN-1 performance in the blood test collected from 6 different NHVs demonstrating the effect of inhibition of antibody binding L104EA29Ylg.

Figure 7 depicts the competition characterization of CD86 with mAb HA5 after blood incubation

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complete with L104EA29Ylg. This figure summarizes the performance of HA5 in the blood test collected from 6 different NHVs that demonstrates the effect of inhibition of L104EA29Ylg on antibody binding.

Figure 8 depicts a competition test of CD86 receptor in whole blood collected from NHV to which L104EA29Ylg has been administered subcutaneously. (SC = subcutaneous; squares indicate pre-dose, triangles indicate Day 5, circles indicate Day 14 and Diamonds indicate Day 42). Figure 8 demonstrates that the administration of L104EA29Ylg SC to normal healthy volunteers inhibits the binding of FUN1 to monocytes on day 5, but this effect is reversed on day 14.

Figure 9 represents the correlation between pharmacokinetic (PK) and pharmacodynamic (PD) properties of the results shown in Figure 8, which help to understand what serum drug concentrations are required to achieve a pharmacodynamic activity such as saturating the target. The open and closed triangles represent the two subjects who received L104EA29Ylg. This figure demonstrates that in the subjects to whom L104EA29Ylg SC has been administered, more inhibition of Fun-1 binding to monocytes in peripheral blood is observed with increasing serum concentrations of L104EA29Ylg.

Figure 10 represents a comparison of the specific binding of several anti-CD86 mAbs in NHV to monocytes in whole blood. The data shows that all three antibodies bind monocytes in a similar way.

Figure 11 depicts specific binding of 2D4 mAb in clinical samples (including blood from renal transplant patients treated with L104E29Ylg). In transplant patients who received L104EA29Ylg, the 2D4 junction is unchanged after administration of L104EA29Ylg and is not significantly different from that of NHVs.

Figure 12 depicts specific binding of mAb HA5 and mAb FUN 1 in clinical samples (including blood from transplanted patients treated with L104E29Ylg). These data demonstrate that unlike the 2D4 binding, the binding of FUN-1 and HA5 is significantly inhibited by administration of L104E29Ylg in transplant patients and is significantly reduced with respect to NHV.

Figure 13 represents simulated clinical PK and PD profiles. This figure represents a model suggesting how this assay could be used to monitor receptor saturation after administration of L104E29Ylg. This suggests that, shortly after the first dose, the saturation of the receptor is maximized and remains at the desired level despite changes in dosage regime such as dose frequency or dose strength.

Figure 14 represents the assessment of FUN1 PE anti-CD86, demonstrating the effects on the binding of L104EA29Ylg. These results were used to determine the concentration of antibodies for use in competition assays.

Figure 15 depicts the evaluation of PE HA5.2B7 anti-CD86, demonstrating the effects on the binding of L104EA29Ylg. These results were used to determine the concentration of antibodies for use in competition assays.

Figure 16 depicts the evaluation of PE 2D4 anti-CD86, demonstrating the effects of L104EA29Ylg binding. L104EA29Ylg does not affect the binding of 2D4 mAb.

Figure 17 represents specific binding of a non-competitive 2D4 mAb in clinical samples of long-term and short-term subjects. In a transplant patient receiving L104EA29Ylg, the 2D4 junction is unchanged after administration of L104EA29Ylg and is not significantly different from that of control subjects treated with Cyclosporin A (CsA). The test is similar to the test described in Figure 11 except that subjects treated with CsA are used as controls and the results of subjects treated with L104EA29Ylg for a period of 6 months or less are also included.

Figure 18 depicts specific binding of a competitive monoclonal antibody HA5 in clinical samples of long-term and short-term subjects. These data demonstrate that HA5 binding is significantly inhibited by administration of L104EA29Ylg in transplant patients and is significantly reduced compared to control subjects treated with CsA. The test is similar to the test described in Figure 12 except that the subjects treated with Csa were used as controls and the results of subjects treated with L104EA29Ylg for a period of 6 months or less are also included.

Figure 19 depicts competition test of CD86 receptor in whole blood of normal healthy volunteers with mAb HA5. The data demonstrate that the concentration of CTLA4Ig necessary to inhibit specific binding of HA5 by 50% is 2.63 µg / ml, while the concentration of L104EA29Ylg required to inhibit specific binding of HA5 by 50% is 0.11 µg / ml. The trial is the same type of trial as shown in Figure 5, donor B, except that the results in Figure 19 represent the average response of 6 donors.

Figure 20 depicts competition test of CD80 receptor in whole blood of normal healthy volunteers with mAb L307.4. The data shows that the concentration of CTLA4lg required to inhibit specific binding of L307.4 by 50% is 0.01 µg / ml and that the concentration of L104EA29Ylg required to inhibit specific binding of L307.4 by 50% is 0, 01 µg / ml. The results represent the average response of 6 donors.

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Figure 21 depicts the results of CD80 and CD86 receptor saturation and inhibition of allo-response assay for comparison. The IC50 of CTLA4lg are represented in a mixed leukocyte reaction, in a competition test of the CD86 receptor using HA5 mAb and in a competition test of CD80 receptor using mAb L307.4. The IC50s of L104EA29Ylg are also represented in a mixed leukocyte reaction, in a competition test of the CD86 receptor and in a competition test of the CD80 receptor.

Detailed description of the invention

The present invention can be more readily understood by reference to the following detailed description of the preferred embodiments of the invention and to the Examples included herein. This invention describes the development of a "CD86 Receiver Competition Test" based on flow cytometry for L104EA29Ylg. Preliminary attempts to develop a competition trial of CD86 receptor based on CTLA4Ig failed. The relatively high affinity of L104EA29Ylg for CD86 (in relation to CTLA4lg or mAb) allows L104EA29Ylg to compete for union with the anti-CD86 mAbs tested.

Flow cytometry has been used with increasing regularity in clinical laboratories to perform leukocyte antigen immunophenotypes. The advantages of flow cytometry include speed, sensitivity, accuracy and objectivity. The components and operation of flow cytometers are well known to those skilled in the art and will not be described in detail herein. For purposes of a description of such things, applicants refer to US Pat. No. 5,567,627, issued October 22, 1996. It is sufficient to indicate that the components and flow cytometry methodology can be used to provide specific information on a number of parameters of a sample. For example it is possible to provide information on components of different sizes within a sample, while simultaneously providing information on the signals of different wavelengths received from different components received from the sample. Thus, when a sample includes components of various sizes and also includes components with markers that emit different wavelengths of light, the flow cytometry data obtained can provide multidimensional information to the user. The present invention utilizes this technology by providing different types of labeled antibodies and labeled and unlabeled cells that express known antigens. By exposing a sample to such things and thereafter analyzing such things within a flow cytometer it is possible to obtain substantial amounts of information regarding blood in a quick and efficient manner.

In this cytometric flow assay, total CD86 expression levels in peripheral monocytes were detected by a really non-competitive mAb (for example 2D4) and the level of CD86 molecules not bound by L104EA29Ylg was detected by a competitive anti-CD86 mAb (for example FUN-1 and IT2.2).

As used herein, a competitive anti-receptor antibody is an antibody that measurably prevents binding to a receptor by means of a given molecule, such as L104EA29Ylg. A non-competitive anti-receptor antibody is an antibody that does not measurably prevent the binding of a given molecule, such as L104EA29Ylg, to the receptor.

As used herein, "CTLA4lg" or "CTLA4-Ig" refers to a protein molecule having the amino acid sequence of residues: (i) 26-383 of SEQ ID NO: 2, (ii ) 26-382 of SEQ ID NO: 2; (iii) 27-383 of SEQ ID NO: 2, or (iv) 27-382 of SEQ ID NO: 2, or optionally (v) 25-382 of SEQ ID NO: 2, or ( vi) 25-383 of SEQ ID NO: 2. The expression of CTLA4lg in mammalian cells may result in the production of N- and C-terminal variants. CTLA4lg also refers to multimeric forms of the polypeptide, such as dimers, tetramers and hexamers. Dimeric combinations may include, for example: (i) and (i); (i) e (ii); (i) e (iii); (i) e (iv); (i) and (v); (i) and (vi); (ii) e (ii); (ii) e (iii); (ii) e (iv); (ii) e (v); (ii) and (vi); (iii) e (iii); (iii) e (iv); (iii) and (v); (iii) and (vi); (iv) e (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and (vi) and (vi). These different dimeric combinations can also be associated with each other to form tetramer CTLA4lg molecules. (The DNA encoding CTLA4lg as shown in SEQ ID NO: 2 was deposited on May 31, 1991 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209 as per stipulate the specifications of the Budapest Treaty and having agreed access number of ATCC 68629; a Chinese Hamster Ovary (CHO) cell line that expresses CTLA4lg as shown in SEQ ID NO: 2 was deposited on May 31, 1991 with ATCC identification number CRL-10762).

L104EA29Ylg (also known as "LEA29Y" or "L104EA29Y") is a condensation protein modified by genetic engineering similar in structure to CTAL4lg. Two amino acid modifications were made for CTLA4lg, leucine to glutamic acid at position 104 (L104E), which is position 130 of SEQ ID NO: 2, and tyrosine alanine at position 29 (A29Y), which is position 55 of SEQ ID NO: 2, to generate L104EA29Ylg.

As used herein, "L104EA29Ylg" refers to a protein molecule having the sequence of residues: (i) 26-383 of SEQ ID NO: 4, (ii) 26-382 of SEQ ID N .º: 4; (iii) 27-383 of SEQ ID NO: 4, or (iv) 27-382 of SEQ ID NO: 4, or optionally (v) 25-382 of SEQ ID NO: 4, or ( vi) 25-383 of SEQ ID NO: 4. The expression of L104EA29Ylg in mammalian cells can result in the production of N- and C-terminal variants. L104EA29Ylg also refers to multimeric forms of the polypeptide, such as dimers, tetramers and hexamers. Dimeric combinations may include, for example: (i) and (i); (i) e (ii); (i) e (iii); (i) e (iv); (i) and (v); (i) and (vi); (ii) e (ii);

(ii) e (iii); (ii) e (iv); (ii) and (v); (ii) and (vi); (iii) e (iii); (iii) e (iv); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and (vi) and (vi). These different dimeric combinations can also be associated with each other to form molecules.

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of L104EA29Ylg tetramers. (The DNA encoding L104EA29Ylg was deposited on June 20, 2000, with the American Type Culture Collection (ATCC) 1994. The ATCC access number PTA-2104 has been agreed. L104EA29Ylg is further described in the State patent applications United in process together with this document of Serial No. 09 / 579,927, 60 / 287,576 and 60 / 214,065, and in documents US20020182211A1 and WO / 01/923337 A2.)

Examples Example 1. In Vitro Tests

Initial experiments were carried out measuring the binding of L104EA29Ylg to CD86. Various antibodies were used individually or in combination developing a test procedure that allows the measurement of L104EA29Ylg binding in relation to the availability of the CD86 receptor.

materials

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Dulbecco PBS without Ca ++ and Mg ++ (Mediatech Inc., Herndon VA, Cat. No. 21-031-CM), or equivalent

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BMS-224818-01 (L104EA29Ylg), 100 mg, was resuspended and stored frozen as a 10 mg / ml stock solution in PBS

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Lymphocyte Separation Medium (Mediatech Inc., Herndon VA, Cat. No. 25-072-CV)

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Sodium azide (Sigma-Aldrich, Milwaukee, Wl, Cat. No. S2002), or equivalent

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Trypan Blue, 0.4% (Invitrogen, Grand island NY, Cat. No. 15250-061) or equivalent

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LAC Solution 10X FACS (BD Biosciences, San José CA, Cat. No. 349202)

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Bovine Fetal Serum (Mediatech Inc., Herndon VA, Cat. No. 35-011-CV), or equivalent

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Human IgGk, human plasma (Sigma-Aldrich, St. Louis, MO, Cat. No. 15154), or equivalent

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Human serum IgG (Sigma-Aldrich, San Luis, MO, Cat. No. I4506), or equivalent

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Murine serum IgG (Sigma-Aldrich, St. Louis, MO, Cat. No. I5381), or equivalent

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anti-CD14-FITC conjugate mAb (Becton Dickinson, San José, CA Cat. No. 555397)

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mAb anti-CD86-PE conjugate, clone FL1N-1 (Becton Dickinson, San José, CA Cat. No. 555658)

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mAb anti-CD86-PE conjugate, clone IT2.2 (Becton Dickinson, San José, CA Catalog No. 555665)

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mAb 2D4-PE anti-CD86-PE conjugate. (BMS patented mAb, conjugated to PE, Lot Control No. 14640, by Caltag Laboratories).

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Anti-CD80-PE conjugate mAb (1420-PE) (BMS patented mAb, PE conjugate, Lot Control No. 17312, by Caltag Laboratories.)

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CPT Vacutainer ™ with sodium citrate, 8 ml. (Becton Dickinson, Cat. No. 362761)

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CPT Vacutainer ™, with sodium heparin, 8 ml, (Becton Dickinson, Cat. No. 362753)

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mAb L307.4 anti-CD80 conjugate (Becton Dickinson, Cat. No. 340294)

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mAb 104 anti-CD80 conjugate (Beckman Coulter, Cat. No. IM1976)

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Anticoagulant Acid Citrate Dextrose Solution Formula (ACD-A) (2 mg / ml dextrose, 1.8 mg / ml sodium citrate, 0.6 mg / ml citric acid; Gambro No. 777967000) Heparin (Sigma H3149, 30 U / ml)

TO.

Detection of CD86 in Human PBMC and competition with L104EA29Ylg

Since monocytes represent the circulating leukocyte population that expresses the most abundant expression of CD86, CD86 expression in CD 14+ monocytes was evaluated in a two-color direct immunofluorescence assay (Figure 1). As expected, peripheral monocytes in PBMC (peripheral blood monocytes) expressed moderate levels of CD86 as detected by binding of anti-CD86 FUN-1 mAb (or by IT2.2 data not shown). When PBMC were pre-incubated with increasing concentrations of L104EA29Ylg (0.1 to 100 µg / test), the detectable anti-CD86 binding was inhibited in a concentration dependent manner. These data suggest that L104EA29Ylg may compete with selective anti-CD86 mAbs (for example FUN-1e IT2.2) for binding of CD86 in peripheral blood monocytes. Thus the reagents and the potential to generate a CD86 receptor competition assay to measure costimulation blockade by L104EA29Ylg exist and thus are only due to the unique high affinity that L104EA29Ylg has for CD86.

7

Without wishing to commit to any specific procedure, Applicants provide the following example procedure to demonstrate how the test can be performed in an in vitro setting:

PBMC procedure:

1. 40 ml of blood is withdrawn in a syringe containing heparin (30 U / ml), EDTA (5.4 mM) or ACD-A 5 (2 mg / ml dextrose, 1.8 mg sodium citrate / ml, citric acid at 0.6 mg / ml) as an anticoagulant.

2.

Phase of 20 ml of blood on 15 ml of Lymphocyte Separation Medium.

3.

Centrifuge at 1800 rpm for 25 minutes at room temperature.

Four.

Remove the PBMC phase and transfer to a 50 ml tube. Add 30 ml of PBS.

5.

Centrifuge at 1800 rpm for 10 minutes at room temperature.

10 6. Resuspend the pellet in 50 ml of PBS.

7.

Centrifuge at 1200 rpm for 10 minutes at room temperature.

8.

Resuspend the sediment in PBS and determine the number of cells using trypan blue staining.

9.

Resuspend the cells at a final concentration of 107 cells per ml in 0.5% FBS / PBS / 0.1% sodium azide.

15 10. Aliquot 106 cells (100 μl) into a 12 x 75 mm polystyrene tube on ice.

eleven.

Add L104EA29Ylg at the desired concentrations (for example, between 0 and 200 µg / ml) to the tube and incubate for 15 minutes on ice.

12.

Add 20 µg of either human lgG1 (200 µg / ml, final concentration) or mixed human IgG

(final concentration of 200 µg / ml) blocking potential FcR binding of detection antibodies. 20 Incubate 10 minutes on ice.

13.

Add indicated amount (for example 1 µg) of human anti-CD86 PE antibody (PE-labeled antibody, ie phycoerythrin) (for example clone FUN1 of BD), incubate 30 minutes on ice.

14.

Add 1 ml FACS Lysis Solution, incubate 30 minutes on ice.

fifteen.

Centrifuge at 1500 rpm for 5 minutes at 4 ° C. Resuspend sediment in 250 µl of Lysis Solution 25 of FACS.

16. Read on flow cytometer. For acquisition, fix the monocyte population, as identified by direct and lateral dispersion properties (G1). Acquisition stops after accumulation of 5000 G1 events.

17.

For analysis, the average fluorescence of G1 events was determined in an event histogram of 30 FL2 (CD86 PE) and used to determine the relative level of CD86 present on the monocyte surface.

B. Detection of CD86 in Samples of Human Complete Blood and competition with L104EA29Ylg

To be useful as a clinical trial, the competition of CD86 observed in PBMC must also be detectable in whole blood samples. To demonstrate this potential, whole blood was taken from a normal healthy volunteer in vacutainers containing either ACD or EDTA. The blood was then pre-incubated with L104EA29Ylg at 0, 5, 120 and 35 250 µg / ml at 37 ° C for 1 hour. After incubation, CD86 not occupied by L104EA29Ylg was measured by incubating blood samples at 4 ° C with FUN-1 (and / or IT2.2). The results of the flow cytometry measurements suggest that the expected levels of CD86 expression in CD14 + monocytes were detected; however, the use of different anticoagulants resulted in different levels of detectable CD86 in the absence of L104EA29Ylg. Increasing concentrations of L104EA29Ylg inhibited binding of FUN-1 in a manner dependent on

40 concentration (Figure 2a) and there appeared to be no effect of anticoagulant choice in this inhibition.

Without wishing to commit to any specific procedure, Applicants provide the following example procedure to demonstrate how the test should be done:

Whole blood procedure

1. Draw blood inside ACD-A anticoagulant

45 2. Dispense L104EA29Ylg in a 12 x 75 mm polypropylene tube for the desired final concentration (for example, between 0 and 200 µg / ml).

8

3.

Add whole blood to L104EA29Ylg. Incubate in a rotator in the incubator under a CO2 atmosphere at 37 ° C for 1 hour.

Four.

Aliquot 200 ul of blood sample into a 12 x 75 mm polystyrene tube on ice.

5.

To each tube, add 25 ug of mouse IgG mixed blocking binding to potential FcR detection antibody. Incubate for 15 minutes on ice.

6.

To each tube, add 20 ul of human anti-CD14 FITC (5 µg / ml) y and indicated amount (for example 1 lug) of PE-labeled human anti-CD86 (for example clone FUN1 BD PE). Incubate on ice for 30 minutes.

7.

Add 2 ml of FACS Lysis Solution (BD) to each tube. Incubate on ice for 30 minutes.

8.

Spin the tubes for 5 minutes at 1500 rpm, at 4 ° C.

9.

Resuspend in 200 µl of FACS Lysis Solution.

10.

Read on flow cytometer, adjust compensation settings as necessary. For acquisition, fix the monocyte population, as identified by direct and lateral dispersion properties (G1). Fix CD14 + monocytes using a direct scattering immunoblot chart against CD 14 (G2). The additive events of G1 and G2 (called G3) are observed in a histogram looking on the FL2 channel (CD86 PE). Acquisition stops after accumulation of 3000 G3 events.

eleven.

For analysis, the average fluorescence of G3 events is determined in a FL2 event histogram (CD86 PE) and is used by determining the relative level of CD86 present on the surface of CD14 + monocytes.

C. Detection of CD86 in Human Complete Blood Cells with Concurrent Use of Competitive and Non-Competitive Anti-CD86 Antibodies.

Additionally, the binding of 2D4, an anti-CD86 mAb that does not compete with CTLA-4, is not affected by preincubation with a high concentration (200 µg / ml) of L104EA29Ylg (Figure 2b). This suggests that 2D4 does not compete with L104EA29Ylg to bind to CD86 and can be used to measure the total surface expression of CD86 even in the presence of L104EA29Ylg.

 D. Summary

The data for these experiments are shown in Figures 1, 2a and 2b. These data demonstrate that selected anti-CD86 mAbs (for example FUN-1 and IT2.2), which do not compete with CTLA4lg, will compete with L104EA29Ylg for binding to CD86 on the surface of antigen presenting cells. The data also demonstrates that non-competitive anti-CD86 mAbs (for example 2D4) can truly be used by measuring total surface CD86 expression even in the presence of high concentrations of L104EA29Ylg. These observations are unique to L104EA29Ylg due to enhanced affinity for CD86 as compared to CTLA4lg and other CD86 ligands. These observations are the basis for the novel application of these reagents for the development of a clinical trial to measure costimulation blockade by L104EA29Ylg.

Example 2. Clinical Ex Vivo and In Vivo Studies

To be useful as a clinical trial, the competition of CD86 observed in PBMC must also be detectable in whole blood samples. To demonstrate this potential, whole blood is taken from a normal healthy volunteer in vacutainers that contain either ACD or EDTA. The blood was then pre-incubated with varying concentrations of L104EA29Ylg. Test concentrations were determined by titration of FUN-1 PE, HA5 PE and 2D4 PE. (See Figures 14-16, and Tables 1 and 2). After incubation, CD86 not occupied by L104EA29Ylg was measured by incubating blood samples at 4 ° C with HA5.2B7. The concentration of L104EA29Ylg necessary to inhibit antibody binding by 50% varies, depending on the antibody. For example, 0.13 µg / ml of L1104EA29Ylg is required to inhibit binding of HA5 by 50%, while 0.49 �g / ml of L104EA29Ylg is required to inhibit binding of FUN-1 by 50%. (See Figure 5). The performance of FUN-1 and HA5 in the blood test collected from 6 different normal healthy volunteers (NHV) demonstrating the effect of inhibition of antibody binding L104EA29Ylg is shown in Figures 6 and 7. In general, HA5 appears to be more sensitive since it detects greater receptor saturation at similar concentrations of L104EA29Ylg and lower IC50. All three monoclonal antibodies (FUN-1, HA5, and 2D4) bind to CD86 (in monocytes) at similar levels. (See Figure 10).

In clinical studies, specific binding to FUN-1, as measured by change in medium fluorescence intensity (MFI), is substantially inhibited on Day 5 after dosing with L104EA29Ylg. (See Figure 8). L104EA29Ylg inhibits the binding of FUN-1, and, when administered subcutaneously, as the concentration of L104EA29Ylg increases, so does the inhibition of binding of FUN-1. (See Figure 9). In transplant patients who continuously receive L104EA29Ylg, the 2D4 junction is unchanged following an individual administration of L104EA29Ylg and is not significantly different from NHV. (See Figure 11). The term "depression" refers to the time point just before the next administration of the drug, when the drug concentration is at its

9 5

10

fifteen

twenty

25

30

35

lower blood level in a patient. Unlike the binding of 2D4, the binding of Fun-1 and HA5 is significantly inhibited by administration of L104EA29Ylg in transplant patients and is significantly reduced compared to NHV. (See Figure 12; 2D4 data not shown.) Figure 13 is a model that suggests how this assay could be used by monitoring receptor saturation after administration of L104EA29Ylg. This suggests that, shortly after the first dose, the saturation of the receptor is maximized and remains at the desired level despite changes in the dosage regimen such as dose frequency or dose strength. Without wishing to commit to any specific procedure, Applicants provide the following example procedure to demonstrate how the test can be performed:

materials

•

L104EA29Ylg, BMS Syracuse, lot 4E82288 / MSF521A; 100 mg vial reconstituted with 10 ml of PBS, at a final concentration of 10 mg / ml. 50 aliquots stored at -20 ° C

•

Lot 15 of CD86 PE (clone HA5.2B7) from Beckman Coulter No. IM2729 (conc. 6.25 µg / ml)

•

APC CD86 2D4 clone; 0.09 mg / ml; BMS generated mAb, APC conjugated by Calbiochem

•

CD14 BD FITC No. 555397

•

Purified anti-CD86 (clone HA5.2B7) from Beckman Coulter No. IM2728, 200 µg lyophilized; resuspended at 200 µg / ml in dH2O / 0.1% azide

•

2.29 mg / ml of purified human anti-CD86 (2D4 clone)

•

Mouse IgG, reagent grade, Sigma No. 15381; 10 mg lyophilized; Resuspend in 5 ml of PBS at a final concentration of 2 mg / ml, store at 4 ° C

•

Lisis Solution FACS, 10x, BD No. 349202

In Vitro Complete Blood Procedure

one.

Draw blood inside ACD-A vacutainer or inside syringe with ACD-A as anticoagulant.

2.

Dilute L104EA29Ylg to 10 mg / ml in PBS at a concentration that is 10 times above the desired final concentration. See dilutions in Table 1 below.

Table 1. Concentration of L104EA29Ylg used in Tests

3.

Dispense 150 µl of L104EA29Ylg diluted in a 12 x 75 mm polypropylene tube. Dispense 150 µl of PBS to additional tube for untreated control.

Four.

Add 1.35 ml of whole blood to L104EA29Ylg or PBS. Incubate in a rotator in the incubator with a CO2 atmosphere at 37 ° C for 1 hour.

5.

On ice, dispense 10 µl of mouse IgG solution to 12 x 75 mm polystyrene sample tubes.

6.

Dispense 100 µl of blood for each sample in 12 12 x 75 polystyrene tubes.

7.

Incubate in icc 10 minutes.

8.

To three tubes, add 3.5 µl purified, mAb HA5 unlabeled (purified). To three additional tubes, add 4.4 μl of purified 2D4 mAb, not labeled. (See table below). Incubate 15 minutes on ice.

9.

To all tubes, add 10 μl of CD14 FITC.

Final conc. L104EA29Ylg

from He PBS He of L104EA29Ylg at 10 mg / ml Correlation with clinical values

250 µg / ml

150 fifty peak

125 µg / ml

175 25 intermediate

5 µg / ml

100 First dilute 1: 100, then add 100 depression

2 µg / ml

160 First dilute 1: 100, then add 40 depression

10 5

10

fifteen

twenty

25

30

10.

Add 20 µl of human anti-CD86 antibody PE (clone HA5.2B7) to tubes as indicated in the table below. Add 10 μl of 2D4-APC to tubes as indicated in Table 2 below.

Table 2. Addition of unlabeled / labeled antibody reagents.

eleven.

Incubate 30 minutes on ice.

12.

Dilute Lysis Solution FACS 10x 1:10 with dH2O. Add 1 ml of FACS Lysis Solution to each tube. Stir and incubate 15 minutes at RT.

13.

Spin the tubes for 5 minutes at 1500 rpm, at 4 ° C.

14.

Resuspend in 200 µl of FACS Lysis Solution.

fifteen.

Read on flow cytometer, adjust compensation settings as necessary. For acquisition, fix the monocyte population, as identified by direct and lateral dispersion properties (G1). Fix CD14 + monocytes using a direct scattering immunoblot chart against CD14 (G2). The additive events of G1 and G2 (called G3) are observed in a histogram looking on the FL2 channel (CD86 PE, FUN1) or on the FL4 channel (CD86 APC, 2D4). Acquisition stops after accumulation of 3000 G3 events.

16.

For analysis, the average fluorescence of G3 events is determined in a histogram of FL2 events (CD86 PE, HA5) or FL4 events (CD86 APC, 2D4) and is used by determining the relative level of CD86 present on the CD14 + monocyte surface. .

TUBE

HA5 PE, He HA5 not marked, he 2D4-APC, He 2D4 not marked, he CD14-FITC, He

one

twenty - - - 10

2

twenty - - - 10

3

twenty - - - 10

4

twenty 3.5 10

5

twenty 3.5 - - 10

6

twenty 3.5 - - 10

7

- - 10 - 10

8

10 10

9

- - 10 - 10

10

- -  10 4.4

10

eleven

- - 10 4.4 10

12

- - 10 4.4 10

Example 3. Clinical Trial

A. CD86 Clinical Trial Procedure

Performing such an assay, blood samples obtained from patients dosed with L104EA29Ylg can be aliquoted into 200 µl samples within a 12 x 75 mm polystyrene tube on ice (4 ° C). To each tube, 25 µg of mouse mixed IgG can be added by blocking potential FcR binding of detection antibodies (for example FUN-1, 2D4, 1420, etc.) and incubated for 15 minutes at 4 ° C. To each tube, 20 µl of human anti-CD14 FITC and the indicated amount (for example 1 µg) of human anti-CD86 (for example FUN1, HA5 or 2D4) fluorescently labeled can be added and incubated at 4 ° C for 30 minutes By evaluating the non-specific fluorescence associated with labeled anti-CD86 mAbs, excess unlabeled human anti-CD86 mAbs are added to a subset of relevant samples (for example, unlabeled HA5 is added to samples containing HA5). 2 ml of FACS Lysis Solution (BD) can be added to each tube by lysing the red blood cells and fixing the leukocytes. Blood can be subsequently incubated at 4 ° C for 30 minutes. To isolate the leukocytes and eliminate lysed RBCs and excess reagents, samples can be centrifuged at 1500 rpm for 5 minutes at 4 ° C and resuspended in 200 µl of FACS Lysis Solution for analysis in the flow cytometer as It is described in the procedures. The total CD86 expressed on the surface of the APC can be determined using 2D4 mAb while

eleven

that the available CD86 can be measured using mAb FUN-1 or HA5 as examples. Specific binding (�MFI) is determined by the difference between total binding (labeled anti-CD86 mAb alone) and non-specific binding (labeled anti-CD86 mAb + anti-CD86 unbranded mAb). With this data someone could calculate the proportion of CD86 not bound to total CD86 and determine the extent of receptor saturation by L104EA29Ylg in a given blood sample. Alternatively, someone could also perform the same procedure on the patient's blood obtained before administration of L104EA29Ylg by determining total CD86 levels and repeating the analysis after administration of the compound by measuring the decrease in competitive antibody binding as compared with pre treatment. In this way, someone would be able to determine the extent of receptor saturation after compound administration.

B. CD80 Clinical Trial Procedure

Although the expression of CD80 in monocytes is very low or nothing is given at all, other cell types or activated monocytes express higher levels of CD80. Total and available CD80 expression can be measured in a similar manner in these cell types using non-competitive CD80 mAbs 1420 by measuring expressed total CD80 and competitive L307.4 mAbs or MAB104 mAbs by measuring CD80 not bound by L104EA29Ylg. With this data someone could calculate the proportion of unbound CD80 versus total CD80 and determine the extent of receptor saturation by L104EA29Ylg in a given blood sample.

Alternatively, the same procedure could also be performed on the blood of the patient obtained before administration of L104EA29Ylg by determining the levels of CD80 and repeating the analysis after administration of the compound by measuring the decrease in binding of the competitive antibody compared to pre-treatment. In this way, someone would be able to determine the extent of receptor saturation after compound administration.

As shown in Figure 20, whole blood was stimulated with LPS at 1 µg / ml for 4 hours at 37 ° C inducing CD80 expression. The level of unbound CD80 was determined by the level of competitive antibody binding L307.4. The results shown in Figure 20 represent the average response of 6 donors.

C. Alorresponse test

To measure the responses, mixed leukocyte (MLR) reactions were carried out. For MLR proliferation assays that measure CTLA-4lg or L104EA29Ylg titres, T lymphocytes were grown at 1 X 105 / well in quadruplicate wells together with 1 X 103, 2 X 103 or 0.4 X 103 MoDC allogeneic as presenting cells of antigens (APC) in 96-well round bottom plates in a total volume of 200 µl of 10% FCS -RPMI. CTLA-4Ig or L104EA29Ylg was added to wells at a final starting concentration of 30 µg / ml, followed by semilogarithmic dilutions to a final concentration of 1 ng / ml. On day 5 after the initiation of the MLR, the cultures were pulsed with a �Ci of 3 [H] -thymidine (PerkinElmer, Boston, MA) for 6 hours, harvested in a Packard cell collector ( PerkinElmer), and were counted by liquid scintillation in a + Packard TopCount NXT (PerkinElmer). The saturation results of the CD80 receptor and the CD86 receptor and inhibition of the alhorresponse assay are shown in Figure 21.

Example 4. Procedures for Producing Antibodies

Antibody production is discussed in detail in US Patent Application. No. 10/375157 (Publication No. 2003-0224458). The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

The 2D4 mAb and the 1420 mAB were labeled with fluorochromes typically used in studies in flow cytometry (FITC, PE, etc.). The assay is not dependent on the type of fluorochrome used, different brands can be used in this assay and can be mixed and matched to suit the purpose of the researchers.

Sequence Listing

<110> BRILTOL-MYERS SQUIBB COMPANY

TOWNSEND, ROBERT M.

PRIETO, FRANCISCO L.

FLEENER, CATHERINE A.

<120> CD86 AND CD80 RECEIVER COMPETITION TESTS

<130> 10733 PCT

<150> 60 / 761,624

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<151> 24-7-2006

<150> 60 / 832,012

<151> 20-7-2006

<160> 4 5 <170> PatentIn version 3.3

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<212> DNA

<213> CTLA4Ig 10 <400> 1

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fifteen

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16 17

image 1

Claims (8)

1. An in vivo or in vitro test procedure for monitoring the efficiency of binding of L104EA29Ylg to a CD86 receptor in a patient, said procedure comprising the steps of:

(to)

provide a blood sample from a patient dosed with L104EA29Ylg, including the blood sample leukocyte cells;

(b)

add CD14-FITC to the blood sample;

(C)

add labeled competitive and non-competitive anti-receptor antibodies to the blood sample;

(d)

lyse the blood sample and fix leukocytes;

(and)

isolate leukocytes from the sample of lysed blood;

(F)

measure the expression of the total receptor and the available leukocyte cell receptor,

wherein the non-competitive antireceptor antibody is 2D4, an antibody that is obtainable from the Mus musculus hybridoma cell line, spleen cell: 2D4, deposited with ATCC Deposit Number PITA-7305.

2.

The assay of Claim 1, further comprising detecting the presence of antibodies using flow cytometry.

3.

The test of claim 1, subsequent to step (a) and before step (b) further comprising step (a ') of blocking non-specific binding mediated by Fc receptor.

Four.

The assay of Claim 3, wherein the non-specific binding mediated by Fc receptor is blocked by the addition of mixed mouse IgG to the blood sample.

5.

The assay of Claim 1, wherein the binding efficiency of L104EA29Ylg is measured by comparing bound and unbound receptors to determine the percentage of receptor saturation by L104EA29Ylg.

6.

The test of Claim 1, comprising the steps of

(to)

 provide a blood sample from a patient dosed with L104EA29Ylg; (a ') add a mixture of mouse IgG to aliquots of the blood sample;

(b)

 add human anti-CD14-FITC to aliquots;

(C)

 add competitive and non-competitive receptor antibodies to separate aliquots from the blood sample;

(c ') adding excess unlabeled human CD86 monoclonal antibody to a subset of relevant samples;

(d)

 lyse the blood sample aliquots and fix leukocytes;

(and)

 isolate leukocytes from aliquots of lysed blood sample;

(F)

 measure the expression of total receptor and available receptor of leukocyte cells,

in which the non-competitive anti-receptor antibody is 2D4, an antibody that is obtainable from the Mus musculus hybridoma cell line, spleen cell: 2D4, deposited with the Deposit Number in ATCC PTA-7305.

7.

A 2D4 antibody, obtainable from Mus musculus hybridoma cell line, spleen cell: 2D4, deposited with the Deposit Number in ATCC PTA-7305.

8.

A hybridoma cell line of Mus musculus, spleen cell: 2D4, deposited with Deposit Number in ATCC PTA-7305, which produces the 2D4 antibody.