Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6875—Nucleoproteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
  • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
  • G01N2333/16—HIV-1, HIV-2
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70514—CD4
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70517—CD8
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present inventors have focused on a means to elucidate better, more predictive information about a patient's immune system, by determining a patients' present state of immune system activation.
• Various markers for immune system activation namely CD 38, CD69, HLA-DR, CD4 counts and changes in plasma HIV viral loads were compared, and baseline Ki-67 expression in CD3, CD4 and CD8 T cell subsets was found to be more predictive than CD4 counts or baseline values of CD38 and other activation markers (i.e. CD69, HLA-DR).
• CD38 is a glycoprotein found on the surface of many immune system cells including CD4+, CD8+, B and natural killer cells. Masur et al. connected CD 38 with the appearance of multiple cases of opportunistic infections that occur in acquired immune deficiency patients.
• CD38 expression could be considered as a promising marker to evaluate progression of the disease with recommendations to use this marker as a diagnostic value (Coetzee LM, Cytometry B Clin Cytom. 2009 Nov;76(6):375-84.) in routine clinical testing.
• Ki-67 protein (also known as MKI67) is a cellular marker strictly associated with cell proliferation. During interphase (collectively, phases Gi, S and G 2 ), Ki-67 can be exclusively detected within the cell nucleus, and during mitosis most of the protein is located on the surface of the chromosomes. Ki-67 protein is present during all active phases of the cell cycle (Gl, S, G2, and mitosis), but is undetectable in resting cells (GO). Maximal virus replication occurs in Gl and S phases (Foli A. et al, J Infect Dis. 2007 Nov 1 ; 196(9): 1409-15 ), when Ki-67 is also up-regulated, so that the only phase when Ki-67 is present and maximal virus replication is not occurring is the relatively short, intermediate, G 2 phase.
• the inventors posited that the comparison of activation markers might yield better ways to evaluate the clinical status of HIV-infected patients and to monitor the effects of anti-HIV therapy.
• Ki-67 expression is dramatically up-regulated in HIV infected patients (IB) up to 2...10% of total T cells compared to 0.05....0.5% in non-infected individuals (1A) and decreases to 0.5...2%> after 24 weeks of VS411 AV- HALT treatment (1C).
• the Ki-67 decrease is statistically significance (p ⁇ 0.001) by paired Student's t-test.
• Patient's PBMCs were stained for surface T cell markers and for Ki-67 by intracellular staining and analyzed on BD LSR-II flow cytometer.
• One representative dot- plot out of 10 subjects in figure 1A and out of 32 subjects in figures IB and 1C is presented, respectively.
• Fig.2 Cohort 1 VS411 study.
• the expression of Ki-67 is strongly correlates with the reduction of HIV viral load.
• 28 subjects from Phase 2A VS-411 study were analyzed for Ki-67 expression on CD3+, CD3+CD4+ and CD3+CD8+ T cells and the Pearson's bi- variate correlation was applied between the baseline values of Ki-67 positive cells percentage and HIV viral load decrease from baseline to last day of anti-HIV treatment.
• Fig.3 Cohort 2 RIGHT702 study.
• Ki-67 baseline expression in CD3 cells strongly correlates with HIV viral load decrease for short-term and long-term therapy outcomes.
• PBMC peripheral blood mononuclear cells
• steps comprising: a. Isolation of peripheral blood mononuclear cells (PBMC) b. Staining of T-cells among the PBMC for surface markers c. Intracellular staining of T cells for Ki-67 d. Measurement of the percentage of Ki-67 positive cells by flow cytometry. Does this yield actively dividing immune system cells v. quiescent immune system cells? The diagnostic assay proof of principle was demonstrated in a chronic disease, namely HIV-1/AIDS.
• PBMC peripheral blood mononuclear cells
• the cells may be frozen PBMCs derived from HIV- infected patients and Ki-67 expression in different T cell subsets is detected by the intracellural staining with anti-Ki-67 antibodies and analyzed by multi-color flow cytometry, as demonstrated in our examples for two independent cohorts of VS411 Phase2A and RIGHT702 studies, or freshly isolated PBMCs, or sorted CD3 cells. Sorted cells could by lyzed and the Ki-67 expression could also be detected by ELISA or any other method. In the present application, Ki-67 was analyzed in CD3 , CD4 and CD8 T cells. For HIV-1, a dramatic increase in number of circulating Ki-67 positive T cells is a particular indicator for disease progression.
• This assay relies on detection of Ki-67 in patients PBMC before the anti-HIV treatment is started.
• HIV infection a chronic infection that represents a world pandemic emergency.
• An advantage of this assay is that it removes a major barrier to drug development for chronic infections.
• the development of effective therapy has been hampered for the past 25 years due to the absence of a rapid test that can correlate immune system markers with disease progression in a case where the anti-HIV therapy is applied.
• a test that yields a quantitative determination of a specific marker in T cells that can predict the therapy outcome in HIV infection.
• Another advantage of the invention is that it modifies the standard flow cytometry assays, such as CD4 enumeration and analysis of CD38 expression, which did not yield a good predictive potential (CD4counts) for the therapy outcome and good correlation (CD38) with HIV-1 viral load changes
• Ki-67+ T-cells detected with this test can be used as predictive tool to determine the immunological status of chronically-infected patients and to predict the therapy outcome.
• a certain range of baseline number of Ki-67+CD3+ cells can be correlated with a certain range of decrease of viral load ("VL").
• VL decrease of viral load
• the Ki-67-based assay provides an immune monitoring tool for patients suffering from HIV/ AIDS to (1) determine their specific immunological status; (2) to predict their individual disease progression; (3) for individual patient management (for example for determining when to start therapy or how intensely to monitor a patient); and (4) for the design, testing and screening of therapies based on anti-viral drugs, immune modulation, targeted immune amplification, and prophylactic and/or therapeutic vaccines.
• Ki-67 + T-cells play a critical role as a reservoir of HIV infection in humans and SIV in the macaque model, the precise immune correlates of HIV progression and responsiveness to the therapy remain to be identified.
• the flow cytometry is currently widely used to identify HlV-specific effects on T cells in humans.
• Current approaches include CD4 enumeration and monitoring of activation markers, like CD38 and HLA-DR, which have a predictive potential in terms of disease progression but not in the evaluation of possible anti-HIV therapy outcome.
• the present invention quantifies the Ki-67 positive T cells more likely representing the HIV-related pool of T cells.
• peripheral blood mononuclear cells are first collected from donors or derived from criopreserved blood samples and stained for surface T cell markers and for Ki-67 followed by the flow cytometry analysis of the Ki-67 expression level.
• the percentage of Ki-67+ T cells quantified by flow cytometry was compared with other activation markers such as CD38, CD69, PD-1 and HLA-DR to discern which parameter correlates with the viral load decrease in chronically HIV-infected patients receiving the anti-HIV therapy.
• Ki-67 marker in HIV-infected individuals for the therapy outcome evaluation The lower percentage of Ki-67+ T-cells is associated with higher decrease in plasma viremia and better therapy outcome.
• the Ki-67 assay will be useful to evaluate the status of the immune system and the recovery of its function in patients treated with antiretroviral drugs as well as to assess the efficacy of prophylactic and therapeutic vaccines.
• PD-1 and HLA-DR expressions were down -regulated from 15,3 ⁇ 7,53% to 11,67 ⁇ 6,59% and from 11,94 ⁇ 5,54% to 9,56 ⁇ 4,45%, respectively, with p values ⁇ 0.01.
• PD-1, CD38 and HLA-DR expressions were down-regulated from 16,75 ⁇ 8,11% to 11,93 ⁇ 6,57%, from 59,13 ⁇ 15,5% to 50,1 ⁇ 15,01% and from 36,03 ⁇ 12,69% to 30,02 ⁇ 10,68, respectively, with p values ⁇ 0.005.

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• 2011
  • 2011-02-03 EP EP11740332.9A patent/EP2531855A4/de not_active Withdrawn
  • 2011-02-03 WO PCT/US2011/023547 patent/WO2011097345A1/en not_active Ceased

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