Cross-Reference to Related Applications

[0001] This application claims benefit of priority under PCT Chapter I,

Article 8 of U.S. Provisional Patent Application No. 63/231,034 entitled, “COMPOSITIONS, KITS, AND METHODS FOR DETECTING PRECLINICAL ALZHEIMER’S DISEASE,” filed on August 9, 2021, which is incorporated herein by reference in its entirety.

Statement Regarding Federally Sponsored Research or Development

[0002] This invention was made with government support under

APP1048082 (2013-2015), APP1061419 (2014-2016), APP1120095 (2017-2019) awarded by the National Health and Medical Research Council (Australia). The Australian government may have certain rights in the invention. Further support was provided by the Bethlehem Griffiths Research Foundation.

Field of Invention

[0003] This invention relates to the detection and prognosis of Alzheimer’s

Disease, in some instances before clinically-significant symptoms emerge.

Background of the Invention

[0004] The causes of Alzheimer’s Disease (AD) are poorly understood, yet the disease correlates with a dramatic decrease in quality of life and high costs of care for patients suffering from its most advance stage. AD typically develops over years. Progressing from cognitively normal (CN), through mild cognitive impairment (MCI), to clinical AD dementia, a patient experiences diminishing short-term memory, attentiveness, and abstract thinking along with increasing apathy, social withdrawal, and isolation. Accumulation of amyloid beta (AP) protein in the form of oligomers and plaques has been detected in both familial and sporadic instances of AD.

[0005] Employing a novel hypothesis, Applicants have examined a suite of cell-surface leukocyte biomarkers using flow-cytometric techniques for their association with cerebral AP accumulation as potential biomarkers for AD. That study, on CN, MCI, and AD patient groups, have yielded surprising correlations allowing for the diagnosis and prognosis of AD. Applicants’ discoveries have yielded inventive compositions and methods disclosed herein.

Summary of the Invention

[0006] Certain embodiments of the present invention allow for the use of individual biomarkers not associated with AD before. Applicants have unexpectedly identified several biomarkers suitable for the methods, compositions, and kits of the present invention such as, for example, CD 163, CD91, and MerTK. Those biomarkers have been found to be diminished or increased (down-regulated or up-regulated) in AD patients and MCI patients, relative to CN patients, showing their usefulness in diagnosing and prognosing AD, alone or in combination with one or more other biomarkers. Specifically, CD 163 and MerTK are down-regulated in AD, while CD91 is up-regulated. See Table 1. Accordingly, some compositions of the present invention include a binding agent such as an antibody for a biomarker such as CD 163, CD91, and MerTK, and the antibody is coupled to a detectable moiety such as a fluorophore. In addition, such compositions may include the use of particular binding agents, such as, for example, mouse anti-human IgG monoclonal antibodies for those and other biomarkers. Compositions optionally include an anticoagulant and possibly whole human blood. Kits, such as comprising an antibody -fluorophore conjugate for a biomarker, also appear in embodiments of the present invention.

[0007] Further embodiments employ those antibody-fluorophore conjugates in useful methods. Thus, Applicants have invented the use of binding agents for certain biomarkers in methods for the diagnosis of AD in a human patient, for example. Certain embodiments relate to methods for diagnosing AD in a human patient, one such method comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with an antibody for a biomarker conjugated with a fluorophore to obtain bound biomarker; measuring the concentration of bound biomarker in the blood sample by fluorescence of the fluorophore; comparing the concentration with a concentration range of biomarker in healthy humans; observing a change in concentration of biomarker in the blood sample; thereby diagnosing AD in the human patient. [0008] Other aspects relate to methods for determining the efficacy of an AD therapeutic candidate in a human patient, one such method comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with an antibody for a biomarker conjugated with a fluorophore to obtain first bound biomarker; measuring a first concentration of first bound biomarker in the first blood sample by fluorescence of the fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with the antibody for the biomarker conjugated with the fluorophore to obtain second bound biomarker; measuring a second concentration of second bound biomarker in the second blood sample by fluorescence of the fluorophore;

(D) observing a change from the first concentration of first bound biomarker to the second concentration of second bound biomarker;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[0009] Further embodiments relate to panels of biomarkers for diagnosis of

AD and evaluation of AD drug efficacy. Accordingly, some additional embodiments include compositions and kits, such as, for example, a composition or kit comprising a first binding agent for a first biomarker conjugated with a first detectable moiety, and a second binding agent for a second biomarker conjugated with a second detectable moiety. Other embodiments include a third binding agent for a third biomarker conjugated with a third detectable moiety. Still other embodiments contain more binding agents each conjugated with a detectable moiety. Ideally, the emission spectra of the several fluorophores do not significantly overlap, or otherwise can be distinguished for quantitative assessment of the presence of the several biomarkers. Panels of antibody-fluorophore conjugates can appear in compositions or kits. A composition indicates one or more ingredients in a single mixture, while a kit includes one or more compositions. [0010] Suitable biomarkers for diagnosis of AD and evaluation of AD drug efficacy include CDl lc, CD59, and CD163, in one panel, for example. Another example includes antibody-fluorophore conjugates for CDl lb, CDl lc, and CD18. An additional panel includes antibody-fluorophore conjugates for CD91, CD59, and CD163, for example. A further panel comprises antibody-fluorophore conjugates for one or more scavenger receptors and their regulators. Another panel comprises antibody-fluorophore conjugates for one or more scavenger receptors and their regulators, and one or more antibody-fluorophore conjugates for one or more leukocyte CD markers. Scavenger receptors and their regulators include, but are not limited to, CD163, CD91, P2X7, MerTK, CD59, CDl lc, CDl lb, and CD18. Further scavenger receptors have been identified in the literature, for example, in M. PrabhuDas et al., “Standardizing Scavenger Receptor Nomenclature,” J. Immunol., 192(5), (2014) 1997-2006. Leukocyte CD markers include, but are not limited to, CD14, CD15, CD16, CD19, and CD3. Other panels include antibody-fluorophore conjugates for one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE, and one or more of CDl lb, CDl lc, CD18, CD163, and MerTK. Yet another panel includes antibody-fluorophore conjugates for one or more of CDl lc, CDl lb, CD18, CD59, and one or more of CD14, CD15, CD16, CD19, and CD3. Further panels include antibody-fluorophore conjugates for one or more of CD91, CD 163, P2X7, and MerTK, and one or more of CD14, CD15, CD16, CD19, and CD3. Still other panels include three groups, such as antibody-fluorophore conjugates for one or more of CD91, CD163, P2X7, and MerTK; one or more of CD11c, CD1 lb, CD18, CD59; and one or more of CD14, CD15, CD16, CD19, and CD3.

[0011] Still other instances of the present invention relate to methods of measuring relative expression of biomarkers in a human patient, one such method comprising: obtaining a sample of whole blood from the human patient; contacting the sample with monoclonal antibodies for the biomarkers conjugated with fluorophores to form bound biomarkers; measuring fluorescence of the fluorophores, thereby determining the relative expression of the biomarkers.

[0012] Yet additional embodiments relate to methods for diagnosing AD in a human patient, one such method comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with monoclonal antibodies for the biomarkers conjugated with fluorophores to form bound biomarkers; measuring a concentration of bound biomarkers in the blood sample by fluorescence of the fluorophores; observing changes in the concentration of each of the bound biomarkers relative to concentration ranges of biomarkers in healthy humans; thereby diagnosing AD in the human patient.

[0013] Still other aspects relate to methods for determining the efficacy of an

AD therapeutic candidate in a human patient, one such method comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a first aliquot of monoclonal antibodies for biomarkers conjugated with fluorophores to obtain first bound biomarkers; measuring a first concentration of first bound biomarkers;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a second aliquot of the monoclonal antibodies for biomarkers conjugated with fluorophores to obtain second bound biomarkers; measuring a second concentration of second bound biomarkers by fluorescence of the fluorophores;

(D) observing changes from the first concentration of first bound biomarkers to the second concentration of second bound biomarkers in the human patient;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[0014] Other examples of the present invention relate to methods for diagnosing AD in a human patient, another such method comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a monoclonal antibody for a scavenger receptor conjugated with a fluorophore to obtain bound scavenger receptor; measuring a concentration of bound scavenger receptor in the blood sample by fluorescence of the fluorophore; observing a decrease in the concentration of bound scavenger receptor relative to a concentration range of scavenger receptor in healthy humans; thereby diagnosing AD in the human patient.

[0015] Still further embodiments relate to methods for determining the efficacy of an AD therapeutic candidate in a human patient, one such method comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a first aliquot of a plurality of antibody-fluorophore conjugates, each antibody-fluorophore conjugate in the plurality comprising a monoclonal antibody for a scavenger receptor conjugated with a fluorophore; thereby forming first bound scavenger receptors; measuring first concentrations of the first bound scavenger receptors in the first blood sample by fluorescence of the fluorophores;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a second aliquot of the plurality of antibody-fluorophore conjugates; thereby forming second bound scavenger receptors; measuring second concentrations of the second bound scavenger receptors in the second blood sample by fluorescence of the fluorophores;

(D) observing an increase from the first concentration of first bound scavenger receptors to the second concentration of second bound scavenger receptors in the human patient;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient. [0016] Still further embodiments of the present invention relate to compositions, kits, and methods optimized for assessing AD risks in patients, efficacy of AD medicines, and other uses. In some cases, those embodiments include binding agents for certain biomarkers conjugated with detectable moieties. For example, certain of those embodiments relate to a composition comprising: a monoclonal antibody for CD 11c conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore.

[0017] For another example, others of those embodiments relate to a composition comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore.

[0018] Further instances of the present invention relate to a kit comprising: a monoclonal antibody for CD11c conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore.

[0019] Other instances relate to a kit comprising: a first binding agent for CD91 conjugated with a first detectable moiety; a second binding agent for CD59 conjugated with a second detectable moiety; and a third binding agent for CD 163 conjugated with a third detectable moiety.

[0020] Any suitable antibodies can be used in the various embodiments of the present invention, such as, for example, mouse anti-human IgG monoclonal antibodies.

[0021] Another aspect relates to a composition comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore.

[0022] An additional aspect relates to a composition comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore.

[0023] Further aspects relate to a kit comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore.

[0024] In certain additional aspects, a kit may comprise: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore.

[0025] Still further aspects relate to using compositions and kits comprising binding agent-detectable moiety conjugates such as antibody-fluorophore conjugates for CDl lc, CD14, CD59, and CD163 for (1) determining the relative expression of those biomarkers in a human patient; (2) diagnosing AD in a human patient; and (3) determining the efficacy of an AD therapeutic candidate in a human patient, as those methods are described herein.

[0026] While the disclosure provides certain specific embodiments, the invention is not limited to those embodiments. A person of ordinary skill will appreciate from the description herein that modifications can be made to the described embodiments and therefore that the specification is broader in scope than the described embodiments. All examples are therefore non-limiting.

Brief Description of the Drawings

[0027] Fig. 1 depicts a mechanism that may explain the usefulness of some embodiments of the present invention, without binding that invention to theory.

Detailed Description

[0028] As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. The figures are not necessarily to scale, and some features may be exaggerated to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event that there is a plurality of definitions for a term herein, those in this disclosure prevail unless stated otherwise.

[0030] Wherever the phrase “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly “an example,” “exemplary” and the like are understood to be nonlimiting.

[0031] The term “substantially” allows for deviations from the descriptor that don’t negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0032] The term “about” when used in connection with a numerical value refers to the actual given value, and to the approximation to such given value that would reasonably be inferred by one of ordinary skill in the art, including approximations due to the experimental and or measurement conditions for such given value.

[0033] The terms “comprising” and “including” and “having” and “involving”

(and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a device having components a, b, and c” means that the device includes at least components a, b and c. Similarly, the phrase: “a method involving steps a, b, and c” means that the method includes at least steps a, b, and c.

[0034] A biomarker is a specific chemical moiety expressed in the human body.

An antibody is an immunologic protein adapted to bind to a specific biomarker. Antibodies can be present in monomeric or polymeric forms. “Fluorophore” indicates a chemical species that absorbs light at an excitation wavelength and emits fluorescence at an emission wavelength for the purposes of detection and quantification. As known in the art, a fluorophore will exhibit a characteristic absorption spectrum and an emission spectrum. Antibody-fluorophore conjugates include an antibody and at least one fluorophore chemically connected together so that the antibody can bind to a biomarker and be detected or even quantified by the fluorescence intensity of the connected fluorophore(s).

[0035] A kit includes two or more compositions such as those described herein.

Optionally, a kit includes other useful items such as sample preparation reagents, collection tubes, control samples, instructions for use, or a combination of such items.

[0036] Certain abbreviations used herein and their definitions include [0037] Ap Amyloid P peptide

[0038] AD Alzheimer’ s Disease

[0039] AIBL Australian Imaging, Biomarkers and Lifestyle study

[0040] APC Allophycocyanin

[0041] APOE Human apolipoprotein E (symbol for gene is italicized; for protein is not).

[0042] APP Amyloid precursor protein

[0043] ATN Amyloid, tau, neurodegeneration

[0044] AUC Area under curve

[0045] CDR Clinical dementia rating

[0046] CN Cognitively normal

[0047] CSF Cerebrospinal fluid

[0048] FACS Fluorescence-activated cell sorting

[0049] FCIP Flow cytometry immunophenotyping

[0050] FITC Fluorescein isothiocyanate

[0051] GWAS Genome-wide association study

[0052] IDI Integrated discrimination improvement

[0053] mAb monoclonal antibodies

[0054] MAC Membrane attack complex

[0055] MCI Mild cognitive impairment

[0056] MFI Mean fluorescent intensity

[0057] MRI Magnetic resonance imaging

[0058] MMSE Mini Mental State Examination

[0059] PACC Preclinical Alzheimer’s Cognitive Composite

[0060] PE R-phycoerythrin

[0061] PerCP peridinin-chlorophyll-protein complex

[0062] PET Positron emission tomography

[0063] RAGE Receptor for advanced glycation end products

[0064] ROC Receiver operating characteristic

[0065] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. [0066] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0067] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

I. Individual Biomarkers

[0068] As stated above, Applicants have unexpectedly identified several biomarkers suitable in such methods and compositions, such as, for example, CD 163, CD91, and MerTK. Those biomarkers have been found to be changed in AD patients and MCI patients, relative to CN patients. Specifically, CD163 and MerTK are down-regulated in AD, while CD91 is up- regulated, with strong correlation with AD. See Table 1 below. They can be used individually or in combination with each other and/or other biomarkers for diagnosing AD and other uses. Table 1 includes results obtained in accordance with the protocols set forth in the Examples.

Table 1 Biological relevance and changes of potential biomarkers in AD.

•

Potential

GO class (direct) biomarkers Biological relevance to AD Changes in AD

Amyloid-beta binding & CD1 lb Integrin subunit alpha M; Complement Phase I, III Nil; Phase microglial cell activation component receptor 3 alpha; Phagocytosis; II J,

Integrins; C3b binding.

Integrin binding CD11c Integrin subunit alpha X; Complement Phase I, II, III J, component receptor 4 alpha; Regulation of actin cytoskeleton.

Amyloid-beta binding & CD18/ITGB2 Integrin subunit beta 2; Combines with Phase III J, microglial cell activation different alpha chains, e.g. CD1 lb and

CD11c, to form different integrin heterodimers also referred to as CR3 and

CR4.

Protein binding CD33 GW AS associated gene for AD [21]; Myeloid Phase II J, cell surface antigen; Immunoglobulin superfamily cell adhesion molecule.

Complement component CD35/CR1 GW AS associated gene for AD [22]; Phase III f

C3b/4b binding Phagocytosis; Integrins; Complement receptor 1.

Amyloid-beta binding & CD36 Scavenger receptor class B member 1; Phase III J, antigen processing and Phagocytosis; Receptor for oxidized low- presentation density lipoprotein (LDL) [32]; Receptor for

A[> [33].

Protein binding CD59 Complement inhibitory protein; Prevent Phase III f formation of the complement membrane attack complex (MAC) [20] .

Amyloid-beta binding & CD91 Low-density lipoprotein receptor-related Phase III f scavenger receptor activity protein 1 (LRP1);

Scavenger receptor activity CD163 Scavenger receptor. Phase III 1 Protein binding & protein MerTK Receptor protein-tyrosine kinase; Mediates Phase III J. phosphorylation phagocytosis in microglial cells [44] and retinal pigment epithelium [45].

Extracellularly ATP-gated P2X7/P2RX7 Innate phagocytosis [7]. Phase I, III Phase II cation channel activity Nil

Amyloid-beta binding & RAGE/ AGER Mediates Af> transport across the blood-brain Phase III f microglial cell activation barrier and accumulation in brain [30-31].

Amyloid-beta binding & SC ARA- Macrophage scavenger receptor 1; Phase III f scavenger receptor activity 1/MSR1 Phagocytosis; Mediates uptake of fibrillar amyloid [5].

GO: Gene ontology, up-regulation.

J,: down-regulation.

Nil: No change.

[0069] Embodiments of the present invention employ binding agents for biomarkers, such as, for example, from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof. The binding agents should exhibit adequate selectivity and affinity for the desired biomarkers. Any suitable antibodies can be used in the embodiments of the present invention. For example, antibodies can be monoclonal, and be derived from mice, horses, rabbits, or pigs, for example via hybridomas. In some cases, the antibody is a mouse anti-human IgG monoclonal antibody for the biomarker. Care should be taken to avoid the presence of antibody inhibitors, such as, for example, heparin.

[0070] Further embodiments employ detectable moieties, such as, for example, radioisotopes, stable isotopes, fluorophores, and combinations thereof. The detectable moieties should be selected to avoid an overlap in their signaling that would confuse the detecting technology. For example, the same radioisotope should not be conjugated to different binding agents in the same kit. Similarly, fluorophores should be selected to avoid overlapping emission spectra. Any suitable fluorophores can be used in the embodiments of the present invention. Suitable fluorophores include, but are not limited to, those sold under the Alexa Fluor® trademark, such as Alexa Fluor® 488, Alexa Fluor® 647; allophycocyanin; fluorescein compounds such as fluorescein isothiocyanate, fluorescein amidite, and 6-carboxyfluorescein; R-phycoerythrin; erythrosine; a peridinin-chlorophyll- protein complex such as that known as PerCP; rhodamine dyes such as carboxytetramethylrhodamine, tetramethylrhodamine, and sulforhodamine; coumarin; lanthanide phosphors; quantum dots; and other fluorophores known in the art. Care should be taken to avoid using two fluorophores with closely-overlapping emission spectra. For example, Alexa Fluor® 647 should not be used with allophycocyanin. Alexa Fluor® 488 should not be used with fluorescein isothiocyanate. Also, in some cases, to eliminate nonspecific binding, a blocking/bleaching reagent may be employed, such as is known in the art. [0071] Further examples of fluorophores include, but are not limited to, BD Horizon™ and BD Horizon Brilliant™ Ultraviolet family of dyes sold by Becton, Dickinson and Company (Franklin Lakes, NJ), such as, for example, those fluorophores known as BUV395; BUV563; BUV615; BUV661; BUV737; BUV805; BV421; BV480; BV510; BV605; BV650; BV711; BV750; and BV786. Further fluorophores include BF Horizon™ tandem conjugate PE-CF594; tandem conjugate PerCP with a cyanine dye known as PerCP- Cy™ 5.5 offered by Becton, Dickinson and Company (Franklin Lakes, NJ); R- phycoerythrin (PE); and BD Horizon™ Red 718; or a combination of two or more thereof. Additional examples include NovaFluor™ brand fluorescent labels offered by Phitonex, Inc. now part of Thermo Fisher Scientific Inc. (Waltham, MA), such as, for example, those fluorophores known as NovaFluor™ Blue 510, NovaFluor™ Blue 530, NovaFluor™ Blue 555, NovaFluor™ Blue 610-30S, NovaFluor™ Blue 610-70S, NovaFluor™ Blue 660- 120S, NovaFluor™ Blue 660-40S, NovaFluor™ Yellow 570, NovaFluor™ Yellow 610, NovaFluor™ Yellow 660, NovaFluor™ Yellow 690, NovaFluor™ Yellow 700, NovaFluor™ Yellow 720, NovaFluor™ Red 660, NovaFluor™ Red 685, NovaFluor™ Red 700, or a combination thereof.

[0072] Still other fluorophores may be chosen from fluorescent proteins such as GFP, YFP, RFP, eGFP, mCherry, tdtomato, FITC, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, Pacific Blue, Coumarin, BODIPY FL, Pacific Green, Oregon Green, Cy3, Cy5, Pacific Orange, TRITC, Texas Red, and combinations thereof.

[0073] Specific examples of antibody-fluorophore conjugates include mouse antihuman CD 163 antigen conjugated with fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR); mouse anti-human CD91 antigen conjugated with fluorescein isothiocyanate; and mouse anti-human MerTK antigen conjugated with fluorescent dye sold under the trademark BD Horizon Brilliant™ Blue 700 (Beckton, Dickinson and Company, Franklin Lakes, NJ).

[0074] Conjugating a binding agent to a detectable moiety, such as an antibody to a fluorophore, can be accomplished by any suitable method. For example, known chemistries are well-developed and commercially available. Fluorophore-conjugated antibodies are available, such as, for example, from Becton, Dickinson and Company. Radioisotopes such as ¹⁸ F and stable isotopes such as ² H can be incorporated into the binding agent, or into a molecule that stably attaches to the binding agent. Fluorodeoxy glucose containing ¹⁸ F, or a deuterated molecule can be used, for example. Quantum dots conjugated to a binding agent such as an antibody also may be mentioned.

[0075] Compositions useful in the present invention are not limited. Certain instances provide a composition comprising a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a fluorophore; and at least one anti-coagulant. Any suitable anti-coagulant can be used in the various embodiments of the present invention, such as, for example, ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing. Any suitable salt may be used, such as, for example, alkali metal salts such as monosodium, disodium, trisodium, or tetrasodium salts may be mention. Other instances relate to a composition comprising a mouse antihuman IgG monoclonal antibody for CD 163 conjugated with a fluorophore; ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing; and whole blood of a human patient. Any of those compositions can include additional useful ingredients, such as, for example, bovine serum albumin and sodium azide.

[0076] Various methods appear in embodiments of the present invention. Some methods relate to diagnosing AD in a human patient. Certain of those methods include obtaining a blood sample from the human patient; optionally storing the blood sample in ice, which can include refrigeration; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a monoclonal antibody for a biomarker such as CD 163, CD91, or MerTK conjugated with a fluorophore to obtain bound biomarker; measuring the concentration of bound biomarker in the blood sample by fluorescence of the fluorophore; comparing the concentration with a concentration range of the biomarker in healthy humans; observing a changed concentration of the biomarker in the blood sample; thereby diagnosing AD in the human patient.

[0077] In some cases, a blood sample is obtained from a human patient, and optionally stored on ice or in refrigeration. As used herein, “storing on ice” includes any sort of refrigeration, to include anything from a food-free refrigerator to an ice bath to dry ice, so long as the storage method does not harm the sample. The sample can be tested within any suitable time, such as within three hours of being withdrawn. The sample is tested by reacting the blood sample with an antibody for a biomarker conjugated with a fluorophore to obtain bound biomarker, and measuring the concentration of bound biomarker in the blood sample by fluorescence of the fluorophore. Fluorescence can be measured by any suitable technique, such as flow cytometry analysis.

[0078] Certain instances allow for the comparison of a concentration of the biomarker in the patient in question with a concentration range of the biomarker in healthy humans. This comparison can be achieved in any suitable manner. Data for healthy humans can be found in the literature, or can be developed by testing a number of healthy persons otherwise determined to be free of AD or MCI. Those healthy persons, for example, can exhibit any suitable indication of health, such as, for example, having <25CL as measured by amyloid PET imaging. The concentration can be compared at any suitable level. In some cases, the concentration is compared in whole blood. In other cases, the concentration is compared on leukocytes. In still other cases, the concentration is compared on subsets of leukocytes, such as, for example, wherein the leukocytes are chosen from monocytes, C14+ monocytes, CD14+CD16- monocytes, neutrophils, CD14- neutrophils, CD14-CD16+ neutrophils, lymphocytes, B lymphocytes, T lymphocytes, natural killer cells, and combinations thereof. The subsets of leukocytes can be identified according to any suitable method. Cell sorting can be performed, or the cells can be gated according to methods known in flow cytometry, for example.

[0079] The concentration of detectable moiety can be measured by any suitable method. Scintillation counting, mass spectroscopy, and fluorescence may be mentioned. Measuring the concentration of bound biomarker comprises measuring the mean fluorescence intensity of bound biomarker conjugated to a fluorophore, for example. In the case of CD 163, observing a decreased concentration of CD 163 in a blood sample can mean measuring a decrease in mean fluorescence intensity of at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, or at least about 19% compared to a mean fluorescence intensity of bound CD 163 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[0080] Observing an increased concentration of CD91 in a blood sample can mean measuring an increase in mean fluorescence intensity of at least about 44%, at least about 50%, at least about 55%, or at least about 59% compared to a mean fluorescence intensity of bound CD91 for a pool of human patients who have <25CL as measured by amyloid PET imaging.

[0081] Observing a decreased concentration of MerTK in the blood sample comprises measuring a decrease in mean fluorescence intensity of at least about 12% compared to a mean fluorescence intensity of bound MerTK for a pool of human patients who have <25CL as measured by amyloid PET imaging.

[0082] Other methods relate to the testing of AD therapeutic candidates to see if they would be efficacious as drugs to treat, prevent, or delay the onset of AD. Certain of those methods include

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; wherein the testing is performed by reacting the first blood sample with a first aliquot of a monoclonal antibody for a biomarker such as CD 163, CD91, or MerTK conjugated with a fluorophore to obtain first bound biomarker; measuring a first concentration of first bound biomarker in the first blood sample by fluorescence of the fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a second aliquot of the monoclonal antibody for the biomarker conjugated with the fluorophore to obtain second bound biomarker; measuring a second concentration of second bound biomarker in the second blood sample by fluorescence of the fluorophore;

(D) observing a change from the first concentration of first bound biomarker to the second concentration of second bound biomarker;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient. In the cases of CD 163 and MerTK, those biomarkers are down-regulated in AD, so an efficacious AD therapeutic candidate would up-regulate or increase the concentration of those biomarkers in a human patient. For CD91, this biomarker is up-regulated in AD, so an efficacious AD therapeutic candidate would down-regulate or decrease the concentration of that biomarker in a human patient.

[0083] Any suitable AD therapeutic candidate can be used. In some cases, the AD therapeutic candidate is a phagocytosis-promoting agent, a scavenger receptor agonist, or both. Thus, a candidate drug can act to promote phagocytosis; it can act as an agonist on a scavenger receptor; or both activities can appear. Of course, other mechanisms of action can be tested by this protocol as well. A suitable AD therapeutic candidate can have any regulatory approval status, from investigational new drugs to those approved therapeutics requiring post-market surveillance, for example, as the method is probing the candidate’s efficacy on the particular patient. The AD therapeutic candidate can be administered in any suitable amount, by any suitable route. In some cases, the amount is a therapeutically effective amount, or a suspected therapeutically effective amount. For example, an AD therapeutic candidate can be administered to the patient in an amount of less than 1 g/kg body weight of the patient, per day. For another example, an AD therapeutic candidate can be administered to the patient in an amount greater than about 1 ng/kg body weight of the patient, per day. In other cases, an AD therapeutic candidate can be administered to the patient in an amount of not more than about 0.1 pg/kg, not more than about 1 pg/kg, not more than about 10 pg/kg, or not more than about 100 pg/kg per day. Any suitable time period can be employed, such as, for example, 1 day, 5 days, 10 days, 30 days, 60 days, 90 days, 180 days, 365 days, or more than 365 days. Oral administration, injections via intravenous or intramuscular routes, transdermal patches, implants, and the like may be employed, for example.

II. Combinations of Biomarkers

[0084] Applicants have found that combinations of biomarkers hold predictive value in diagnosing and prognosing AD and drug efficacy evaluation. Those biomarkers can represent indicators of a single mechanism, such as, for example, phagocytosis-modulating molecules. Or those biomarkers can indicate more than one mechanism or aspect of the patient’s health, such as, for example, one or more phagocytosis-modulating molecules in combination with one or more leukocyte CD markers including CD 14 (monocyte), CD 16 (monocyte, NK cells, neutrophils), CD15 (neutrophils), CD19 (B lymphocytes) and CD3 (T lymphocytes).

[0085] Some embodiments of the present invention probe the expression of one or more scavenger receptors for diagnosing or prognosing AD. Table 1, set forth above, describes the expected activities of certain biomarkers including scavenger receptors. Particular panels, of scavenger receptors and other biomarkers include, for example, CDl lc, CD59, and CD163;

CDl lb, CDl lc, and CD18;

CD91, CD59, and CD 163; one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE, with one or more of CDl lb, CDl lc, CD18, CD163, and MerTK; one or more of CD163, CD91, P2X7, MerTK CD59, CDl lc, CDl lb, and CD18, with one or more of CD14, CD15, CD16, CD19, and CD3; one or more of CDl lc, CDl lb, CD18, and CD59, with one or more of CD14, CD15, CD16, CD 19, and CD3; one or more of CDl lc, CDl lb, CD18, and CD59, with one or more of CD14, CD15, CD16, CD 19, and CD3; one or more of CD91, CD 163, P2X7, and MerTK, with one or more of one or more of CD14, CD15, CD16, CD19, and CD3; one or more of CD91, CD163, P2X7, and MerTK, with one or more of CDl lc, CDl lb, CD18, and CD59, and with one or more of CD14, CD15, CD16, CD19, and CD3.

[0086] Those panels would be represented by the presence of a binding agent such as a monoclonal antibody for the particular biomarker conjugated with a suitable detectable moiety such as a fluorophore. In any combination of antibody-fluorophore conjugates, it can be appreciated, the fluorophores should be selected so that there is not significant or unresolvable overlap in their emission spectra, or one could not readily determine the relative intensities of the overlapping spectra. In some cases, care should be taken on the excitation parameters as well, so that qualitative information can be obtained. All fluorophores should be adequately illuminated under the intensities of excitation used, so that quantitative data emerges. Some embodiments of the present invention depend on the relative concentrations of biomarkers, or on the concentration of one or more biomarkers in a patient’s sample relative to that of healthy persons.

[0087] Certain instances relate to compositions and kits. For example, one instance relates to a composition or a kit comprising a monoclonal antibody for CDl lc conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore. Another instance relates to a composition or a kit comprising a mouse anti-human IgG monoclonal antibody for CDl lc conjugated with a first fluorophore; a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a second fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a third fluorophore. A further instance relates to a composition or a kit comprising a mouse anti-human IgG monoclonal antibody for CD91 conjugated with a first fluorophore; a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a second fluorophore; and a mouse antihuman IgG monoclonal antibody for CD 163 conjugated with a third fluorophore. Suitable fluorophores include those described above. In some cases, the first fluorophore is allophycocyanin or fluorescein isothiocyanate; the second fluorophore is R-phycoerythrin; and the third fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[0088] Optionally, the composition or kit includes at least one anticoagulant. Any suitable anticoagulant can be used as described above. Any suitable additional ingredients also may appear, such as, for example, bovine serum albumin and sodium azide.

[0089] Compositions according to the present invention may include whole blood from a human patient. Such a composition appears when a sample from the patient is being tested according to the present invention.

[0090] Various methods appear in several embodiments of the present invention. Some methods relate to measuring the relative expression of certain biomarkers in a patient. It has been found that for MCI and AD patients, certain biomarkers increase while other biomarkers decrease, relative to CN patients. For example, one such method relates to measuring relative expression of CDl lc, CD59, and CD163 in a human patient, the method comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a monoclonal antibody for CD11c conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore, to form bound CD11c, bound CD59, and bound CD163; measuring fluorescence of the first fluorophore, the second fluorophore, and the third fluorophore, thereby determining the relative expression of CDl lc, CD59, and CD163. As explained above, any suitable antibodies and fluorophores can be employed. With reference to Table 1, if CDl lc and CD163 appear down-regulated while CD59 appears up-regulated, the patient can be assessed for MCI or AD, and further treatment or prophylaxis measures may be taken.

[0091] Another such method relates to measuring relative expression of CD91, CD59, and CD163 in a human patient, the method comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore, to form bound CD91, bound CD59, and bound CD163; measuring fluorescence of the first fluorophore, the second fluorophore, and the third fluorophore, thereby determining the relative expression of CD91, CD59, and CD163. As explained above, any suitable antibodies and fluorophores can be employed. With reference to Table 1, if CD91 and CD59 appear up-regulated while CD 163 appears down-regulated, the patient can be assessed for MCI or AD, and further treatment or prophylaxis measures may be taken.

[0092] Methods for diagnosing AD in a human patient, which may include assessing whether the patient falls into a CN, MCI, or AD diagnosis, also can employ combinations of biomarkers. One such method includes reacting a blood sample from the patient with a plurality of antibody-fluorophore conjugates, each antibody-fluorophore conjugate in the plurality comprising a mouse anti-human IgG monoclonal antibody for a scavenger receptor conjugated with a fluorophore thereby forming bound scavenger receptors; measuring concentrations of the bound scavenger receptors in the blood sample by fluorescence of the fluorophores; and observing a decrease in the concentration of at least one bound scavenger receptor relative to a concentration range of a corresponding scavenger receptor in healthy humans; thereby diagnosing AD in the human patient. In some cases, the scavenger receptors are chosen from CD163, MerTK, CDl lb, CDl lc, CD18, and combinations thereof.

[0093] Measuring the concentration of bound biomarkers can proceed in any suitable manner. Whole blood, leukocytes, or subsets of leukocytes can be examined. One biomarker can be examined on one subset of leukocytes, for example, while another biomarker can be examined on a different subset of leukocytes. In some cases, for example, measuring the concentration of bound biomarker comprises measuring the mean fluorescence intensity of bound biomarker. Similarly observing a change (increase or decrease) in the concentration of bound biomarker may involve comparing a mean fluorescence intensity of bound biomarker for the human patient with a mean fluorescence intensity of bound biomarker for a pool of human participants who have <25CL as measured by amyloid PET imaging. For example, observing a decrease in the concentration of bound CDl lc may involve measuring a decrease in mean fluorescence intensity of at least about 15%, at least about 19%, at least about 22%, at least about 25%, at least about 28%, at least about 30%, or at least about 34% compared to a mean fluorescence intensity of bound CDl lc for the pool of human participants who have <25CL as measured by amyloid PET imaging. In another example, observing an increase in the concentration of bound CD59 comprises measuring an increase in mean fluorescence intensity of at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 78% compared to a mean fluorescence intensity of bound CD59 for the pool of human participants who have <25CL as measured by amyloid PET imaging. [0094] In certain instances, measuring the concentration of biomarker is correlated with specific cells. For example, the concentration of a biomarker may exhibit a significant difference from healthy people when measured on particular cells. Cell population subsets can be identified in any suitable manner, and correlated with other biomarkers. For example, leukocytes can be isolated from whole blood, and the concentration of CD 163 can be measured on leukocytes only. Or the leukocytes or subsets thereof can be gated according to known methods useful in flow cytometry. Natural killer (NK) cells, B lymphocytes, T lymphocytes, classical monocytes, intermediate monocytes, non-classical monocytes and mature neutrophils, for example, can be gated and probed for the expression of one or more biomarkers such as CD 163, CD91, and MerTK. Such gating strategy typically involves forward and side scatters of lymphocytes, monocytes and neutrophils, together with their surface expression of cell-specific CD markers, including, for example, monocytes (CD14+), neutrophils (CD 15+ or CD16+), B lymphocytes (CD19+), T lymphocytes (CD3+), natural killer cells (CD16+), and combinations thereof. As used herein, classical monocytes can be described as CD14+CD16-, non-classical monocytes are CD14dimCD16+, and intermediate monocytes are CD14+CD16+.

[0095] Certain further embodiments relate to determining efficacy of an AD therapeutic candidate in a human patient using a combination of biomarkers. One such method comprises

(A) obtaining a first blood sample from the human patient; reacting the first blood sample with a first aliquot of a plurality of antibody-fluorophore conjugates, each antibody-fluorophore conjugate in the plurality comprising a mouse antihuman IgG monoclonal antibody for a scavenger receptor conjugated with a fluorophore, thereby forming first bound scavenger receptors; measuring first concentrations of the first bound scavenger receptors in the first blood sample by fluorescence of the fluorophores;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; reacting the second blood sample with a second aliquot of the plurality of antibody- fluorophore conjugates, thereby forming second bound scavenger receptors; measuring second concentrations of the second bound scavenger receptors in the second blood sample by fluorescence of the fluorophores; (D) observing an increase from the first concentration of first bound scavenger receptors to the second concentration of second bound scavenger receptors in the human patient;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient. Any suitable AD therapeutic candidate can be tested, such as, for example, a phagocytosispromoting agent, a scavenger receptor agonist, or a candidate that exhibits both mechanisms.

III. Two Panels

[0096] Applicants have found two combinations of biomarkers useful in certain of the several compositions, kits, and methods of the present invention. One combination relates to biomarkers CD163, CD59, CDl lc, and CD14. The second combination relates to biomarkers CD163, CD59, CD91, and CD14. Those combinations can be represented by binding agents for the biomarkers, such as monoclonal antibodies, conjugated with distinctive detectable moieties, such as fluorophores. Accordingly, one embodiment relates to a composition comprising: a monoclonal antibody for CDl lc conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore. Another embodiment relates to a composition comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore. Those monoclonal antibodies can include any suitable antibodies, such as, for example, mouse anti-human IgG monoclonal antibodies. As stated above, monoclonal antibodies derived from mice, horses, rabbits, or pigs, for example via hybridomas can be used. At least one anticoagulant also can appear in those compositions, such as, for example, ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing. Suitable additional ingredients can be present, for example, bovine serum albumin and sodium azide, or for another example, phosphate buffered saline, gelatin, and sodium azide.

[0097] Detectable moieties include radioisotopes, stable isotopes, fluorophores, and combinations thereof. Any suitable fluorophores can be conjugated with those binding agents, taking care to avoid using two fluorophores with closely-overlapping emission spectra. In some cases, the first fluorophore is fluorescein isothiocyanate, allophycocyanin, or a combination thereof. In further cases, the second fluorophore is a peridinin- chlorophyll-protein complex. Still further cases provide the third fluorophore is R- phycoerythrin. Additional cases provide wherein the fourth fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR). In still further cases, the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR). The composition may include whole blood from a human patient, for example, when the patient is being assessed for AD.

[0098] Kits employing those combinations also appear in the present invention. Thus, another embodiment provides a kit comprising: a monoclonal antibody for CD11c conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore. And a further embodiment provides a kit comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD 14 conjugated with a second fluorophore; a monoclonal antibody for CD59 conjugated with a third fluorophore; and a monoclonal antibody for CD 163 conjugated with a fourth fluorophore.

[0099] Those kits can include the binding agents such as antibodies, detectable moieties such as fluorophores, anticoagulants, and additional ingredients as mentioned above. In some instances, the compositions and kits may include the use of particular antibodies, such as, for example, mouse anti-human IgG monoclonal antibodies for those and other biomarkers. Compositions optionally include an anticoagulant and possibly whole human blood. Suitable anticoagulants include, but are not limited to, ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing. The compositions and kits may further include any suitable further ingredients, such as, for example, bovine serum albumin and sodium azide, and phosphate buffered saline, gelatin, and sodium azide as another example.

[00100] Suitable fluorophores include those described above. In some cases, the first fluorophore is fluorescein isothiocyanate, allophycocyanin, or a combination thereof. In further cases, the second fluorophore is a peridinin-chlorophyll-protein complex. Additional cases provide R-phycoerythrin as the third fluorophore. Still other cases relate to the fourth fluorophore being fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00101] Methods of using those combinations of biomarkers also appear.

Methods for determining the relative expression, diagnosing AD in a human patient, and determining the efficacy of an AD therapeutic candidate as described herein can be employed.

Detailed Description of the Drawings

[00102] Further embodiments of the present invention can be understood by reference to the accompanying drawings.

[00103] Fig. 1 shows a proposed mechanism. Without wishing to be bound by theory, the proposed mechanism may explain the usefulness of certain embodiments of the present invention. Further elucidation appears in the Discussion below.

Examples

Testing Biomarkers as Indicative of AD

[00104] Applicants examined a suite of common cell-surface leukocyte biomarkers using flow-cytometric techniques for their association with cerebral amyloid-P (AP) accumulation as potential biomarkers for AD in a total of 418 human patients. Those patients included 241 cognitively normal (CN), 106 mild cognitive impairment (MCI) and 71 clinically AD dementia patients.

[00105] 2. Methods:

[00106] 2.1. Subjects and ethical approval

[00107] Participants in Phases I, II, and III were recruited from the Australian

Imaging, Biomarkers and Lifestyle study (AIBL). This study integrates data from neuroimaging, biomarkers, lifestyle, clinical and neuropsychological analyses. 99% of participants collected by AIBL study are genetic northern European Caucasians who are fluent in English. Classification of the AIBL participants is based on both clinical and neuroimaging/CSF evidence of AP accumulation. This study was approved by Human Research Ethics Committee, Research Governance Unit, St Vincent’s Hospital, Melbourne, Australia (Ref: HREC-A 028/06). All participants and patient caregivers completed written informed consent before participation. All clinical and demographic information of AIBL participants was masked until the collection of all biological measurements. For Phase IV, the sample was randomly recruited from subjects participating in the Anti-Amyloid Treatment in Asymptomatic Alzheimer's Disease (A4) Study. In this phase, whole blood was obtained from 112 participants who were blindly and randomly selected from the cohort of subjects seen at US sites as part of the screening process for the Anti-Amyloid in Asymptomatic AD (A4) clinical trial (e.g. A4 Biobank Addendum, PI Rissman) [52].

[00108] 2.2. Ap -PET imaging and the centiloid (CL) scale [00109] Participants underwent AP-PET imaging as previously described [17, 18]. A 3D T1 -weighted MRI was obtained for screening and co-registration with the AP-PET images. Researchers involved in blood assessments were blinded to all AP-PET and clinical data until reporting of the statistical analyses. AP-PET status is based on centiloids (CL): negative <25CL (including borderline >15CL and <25CL) and positive > 25CL. The thresholds are selected as per their association with risk of disease progression.

[00110] 2.3. Episodic memory and Preclinical Alzheimer’s Cognitive Composite

(PACC) score

[00111] The methods used to develop the episodic memory composite and PACC scores are published elsewhere [19, 20].

[00112] 2.4. Blood collection

[00113] For Phases I-III, peripheral whole blood was collected via venepuncture between 8:00am and 10:30am after overnight fasting. Whole blood was kept in EDTA anticoagulant Vacutainer® tube (Becton Dickinson Biosciences) and was kept on ice during transportation. Processing of whole blood was completed within three hours after collection. For Phase IV, whole blood via venepuncture was collected from overnight fasted participants. Blood collected was shipped overnight on ambient temperature gel packs and processed the following morning for analyses.

[00114] 2.5. Cell flow cytometry staining

[00115] Cell surface staining procedures were carried out using the BD standard protocol: aliquots of 100 pL fresh blood were added into fluorescence-activated cell sorting (FACS®) tubes with pre-mixed antibody cocktails. Mouse anti-human IgG monoclonal antibodies (mAbs) conjugated with fluorophores, which were excited at FITC, PE, PerCP and APC channels, were used to stain leukocytes (Tables A. Is below). An autofluorescence tube containing only whole blood and an IgG isotype control (BD Australia) tube were prepared for each AIBL sample. Optimal concentration for antibodies was determined by titration tests. For intracellular staining, cells were fixed and permeabilized before mixing with antibody cocktail according to the BD standard protocol. Immunophenotyping was performed by flow cytometry using FACSCalibre (BD Biosciences). All flow data were stored in digital form.

[00116] 2.6. Definition of Phase I, II, III, and IV

[00117] Phase I was a triple-color cytometric panel that consisted of 16 surface and 2 intracellular markers. Phase II was a quadruple-color cytometric panel that comprised 18 surface markers. Phase I, II covered some major leukocyte CD markers. Phase III had 19 surface markers and was also a quadruple-color cytometric panel; but was enriched to reflect the emerging evidence that pathways of innate immunity may play a key role in AD pathogenesis. In total, 34 leukocyte markers were examined. Antibody-fluorophore conjugate panels, together with catalogue numbers for the products used, appear in Tables A. Is.

[00118] Table A.1.1. Antibody panel for Phase I.

Phase I FL1 FL2 FL4

Surface 1 Autofluorescence

Surface 2 IgGi (BD#550616) IgGi (BD#554680) IgGi (BD#555751)

Surface 3 CD8 (BD#555366) CD4 (BD#555347) CD3 (BD#555342)

Surface 4 CD19 (Dako#F0768) CD4 (BD#555347) CDl lc (BD#559877)

Surface s CD19 (Dako#F0768) CDl lb (BD#555388) CD4 (BD#555349)

Surface 6 CD40 (BD#555588) CDl lb (BD#555388) CD14 (BD#340436)

Surface 7 CD16 (BD#555406) CDl lb (BD#555388) CD14 (BD#340436)

Surface s CD15 (BD#555401) CDl lb (BD#555388) CD14 (BD#340436)

Surface 9 CD8 (BD#555366) TCRrd (BD#555717) CD3 (BD#555342)

Surface 10 CD19 (Dako#F0768) CD4 (BD#555347) CD34 (BD#555824)

Surface 11 Linl (BD#340546) HLA-DR (BD#347367) CDl lc (BD#559877)

Surface 12 CD14 (BD#347493) CD16 (Dako#R7012) 2X7 (in house)

Intra 1 Autofluorescence

Intra 2 IgGi (BD#550616) IgGi (BD#554680) IgGi (BD#555751)

Intra 3 IFN-y (BD#340449) CD4 (BD#555347) CD3 (BD#555342)

Intra 4 _ CD25 (BD#555432) CD4 (BD#555349)

*FoxP3 (BD#560047)

Intra is intracellular staining.

[00119] A.1.2. Antibody panel for Phase II.

Phase

Surface 1 Autofluorescence

Surface 6 CD16 (BD#555406) CD4 (BD#555347) CD14 (BD#340585) (j _n house)

„ - _ Linl HLA-DR CDl lc

Surface 7 D69 (BD#340548)

[00120] A.1.3. Antibody panel for Phase III.

Phase III FL1 FL2 FL3 FL4 Surface 1 Autofluorescence

IgGl IgGl IgGl IgGl

[BD#550616) (BD#554680) (BD#550672) )BD#555751)

CD 16 SCARA1 CD 14 iBD#555406) (RD#FAB2708P) (BD#340436)

CD68 CD 11 CD14 MerTK (RD#

(BD#562117) (Dako#R7012) (BD#340585) FAB8912A)

CD16 GDI IB CD14 CD11C

(BD#555406) (BD#555388) jBD#340585) (BD#559877)

CD 16 CD 14 CD35 (BD#

Surface 6 (Dako#R7012) (BD#340585) 565329)

CD 16 CD 14 ^A P2X7 (in

Surface 7 (Dako#R7012) (BD#340585) house)

CD 16 CD59 CD 14 ^A CD163

Surface 8 (BD#555406) (BD#555764) (BD#340585) )BD#562669) CD36 (BD# CD91 (BD# CD14 CD16

Surface 9 555454) 550497) (BD#340585) )BD#561304)

CD 16 CD44 CD4 CD 14

Surface 10 iBD#555406) (BD#555479) (BD#347324) (BD#340436)

CD18 CD16 CD 14 RAGE (LS-

Surface 11 (BD#555923) (Dako#R7012) (BD#340585) C212626)

CD16 CX3CR1 (BD# CD14 ^A CCR2 (BD# (BD#555406) 565796) (BD#340585) 558406)

In Tables A. Is, FL1, FL2, FL3 and FL4 are fluorescence detection channels of BD FACSCalibur. The fluorochromes for FL1, FL2, FL3, and FL4 are FITC, PE, PerCP and APC, or equivalent.

*FoxP3 is labelled with Alexa Fluor® 488.

^A represents antibody is labelled with Alexa Fluor® 647.

BD is Becton Dickinson Biosciences.

Dako is Agilent Dako.

RD is R&D Systems.

LS is LifeSpan Biosciences.

[00121] A.1.4 Antibody Panel for Phase IV tubes

Surface 1 Autofluorescence

CDl lc CD59 CD14 ^A CD163

Surface . <BD#561355) (BD#555764) (BD#340585) (BD#562669)

CD91 CD59 CD 14 ^A CD163

Surface 4 < _BD#550496) (BD#555764) (BD#340585) (BD#562669) FL1, FL2, FL3 and FL4 are fluorescence detection channels of BD FACSCalibur. The fluorochromes for FL1, FL2, FL3, and FL4 are FITC, PE, PerCP and APC, or equivalent.

*FoxP3 is labelled with Alexa Fluor® 488; represents antibody is labelled with Alexa Fluor® 647.

BD is Becton Dickinson Biosciences; Dako is Agilent Dako; RD is R&D Systems; LS is LifeSpan Biosciences.

[00122] Flow data was analysed using FlowJo software (V10, FlowJo, LLC). Applicants used forward scatter, side scatter to gate lymphocytes, monocytes and neutrophils. Subpopulations were then gated based on fluorescent intensity of specific markers. In Phase III, Applicants used fluorescent intensity of anti-CD14 and anti-CD16 mAbs to gate natural killer (NK) cells, B and T lymphocytes, classical monocytes, intermediate monocytes, non-classical monocytes, mature neutrophils, and immature neutrophils. Cell count and frequency of subpopulations were calculated. The mean fluorescent intensity (MFI) of different markers on different cell groups was also measured.

[00123] 2.7. Statistical analysis

[00124] Statistical analysis was performed in GraphPad Prism for Windows (Version 8.4.2, San Diego, California USA) and IBM SPSS® Statistics Software (Version 25.0 Armonk, NY: IBM Corp). Fluorescent intensity of mAbs was compared between CN, MCI and AD groups using One-way ANOVA. Pearson product-moment correlational analysis was performed to examine the strength of relationship between fluorescent intensity of mAbs and PET Ap centiloid, episodic memory and PACC score. For the predictive analyses, Receiver Operating Characteristic (ROC) analyses was used to define values of sensitivity, specificity and accuracy. Thresholds were chosen via Youden’s Index. Models were calculated for individual biomarkers, individual biomarkers adjusted for covariates and combinatorial sets of biomarkers to predict outcome post Logistic Regression. Figures were created in GraphPad Prism for Windows (Version 8.4.2, San Diego, California USA) and statistical analyses were conducted using R (version 4.0).

[00125] 2.8 Biomarker assessment

[00126] Here Applicants try to discover whether adding new biomarkers can better predict disease risk. Applicants assessed biomarker performance in a logistic regression model created with the rms R package and assessed with the rap R package. R version 4.0.2 was used. The added value of biomarkers to a baseline model were assessed using the change in area under the receiver operator characteristic curve (AUC), the integrated discrimination improvement (IDI) metrics, and the Brier skill score. The change in AUC reflects the change in ranking of the probabilities of the baseline model and the new model. The IDI are presented separately for those with and without the event of interest. They represent the net mean increase in risk for those with the event (IDI event) and mean decrease in risk for those without the event (IDI non-event). The Brier improvement is the relative improvement of the Brier score from the baseline model. The Brier score is the mean squared error of the model and the Brier improvement gives the percent improvement in accuracy with the addition of the biomarker.

[00127] The process of model creation was, in brief, to construct an artificial baseline logistic regression model to predict subjects’ brain AP-PET status. First, variables were chosen by a variable-reduction technique, principal component regression, which reduced the number of variables by shrinking neuropsychological diagnoses together with demographics (i.e., age, sex and year of education). Second, Applicants added each biomarker one by one to the baseline model to assess if the new biomarker improved the diagnostic performance of the baseline model. These are the “new models.” Finally, Applicants combined the best performing biomarkers into a panel of biomarkers to achieve the optimal performance. Here Applicants defined disease state simply by centiloids >25CE for Phase III Model 1. Phase III Model 2 sought to explore whether certain biomarkers had physiological value that helps either with differentiation of disease early stage (i.e., borderline). However, the AIBE borderline range, >15CE & <25CE, was too narrow to include enough cases. Therefore, Applicants arbitrarily defined early stage of AD by centiloids >25CL & <100CE; and the individuals with centiloids >100CE were removed. Model 3 further considered patients’ clinical status alongside their Ap status to stratify study cohort into healthy controls (CN & <25CE), preclinical (CN & >25CL), or prodromal (MCI & Not considering CE) and AD (Not considering CL) stages. Then, Applicants arbitrarily defined disease status for model 3 by combining preclinical and prodromal stages (AD stage was removed). Data skewness was tested by Kolmogorov-Smirnova and Shapiro-Wilk methods.

[00128] 3. RESULTS:

[00129] 3.1. Demographic s

[00130] This cross-sectional study comprised four phases, in which Phase I, II served as discovery data sets and Phase III, IV as validation data sets. Applicants recruited 76, 142 and 200 participants from the AIBL study in Phases I, II, and III, and 112 participants from the A4 study in Phase IV. All data sets included a balanced number of individuals clinically classified as CN, MCI and AD dementia. All samples had corresponding AP-PET and cognitive data. Study demographics were simply stratified by high or low Ap burden, except for Phase III models 2 and 3 (see Methods 2.8).

[00131] 3.2. Candidate biomarkers in Phases I and II [00132] Phase I identified CDl lc and P2X7 as candidate biomarkers (Table A.2.1 below). The MFI of CDl lc decreased by 19-34% (P < 0.05) and P2X7 decreased by 20- 42% (P<0.01) in MCI and AD groups compared to that of CN group. They also showed a negative correlation with Ap CL such that the higher Ap load subjects had lower expression levels of CDl lc and P2X7. Phase II identified CDl lc, CDl lb and CD33; but not P2X7 as candidate biomarkers (Table A.2.2 below). The MFI of CDl lc, CDl lb and CD33 decreased in MCI and AD groups by 14-23% (P < 0.05), 14-21% (P < 0.05) and 16-18% (P < 0.05), individually, compared to CN group. However, only CDl lc on NK cells correlated with AP- PET data.

[00133] 3.3 Candidate biomarkers in Phase III

[00134] In Phase III, Applicants identified ten candidate markers related to the MFI of CDl lc, CD59, CD91 and CD163 that were different between CN, MCI and AD groups (top 10 in Table A.2.3 below) and correlated with the Ap burden. The MFI of CD11c and CD163 decreased by 15-19% (P < 0.01) and 11-16% (P < 0.01), respectively, in MCI and AD groups compared to that of CN group, while the MFI of CD59 and CD91 increased by 27- 78% (P < 0.0001) and 44-59% (P < 0.001), respectively, in MCI and AD groups. The bottom four in Table A.2.3 related to MerTK, CD18 and RAGE were excluded because of small sample sizes. Nevertheless, the MFI of CD 18 and MerTK decreased by 20-26% (P < 0.05) and 12% (P < 0.05), respectively and the MFI of RAGE increased on monocytes by 31% (P < 0.05) in MCI and AD groups compared to CN group. Changes of all potential biomarkers and their relevance to AD are listed in Table 1 above.

[00135] The absolute percentage of CD14- lymphocytes in whole blood was lowered by 11% (P < 0.01) in MCI and AD groups compared to CN group (Table A.2.4 below) and was correlated with CL, episodic memory and PACC scores. The differences in the percentage of monocytes in whole blood between CN, MCI and AD groups were trivial, so Applicants divided the whole monocyte population into CD 14+ CD16+, CD 14+ CD 16- and CD 14- CD 16+ subpopulations and evaluated their relative percentage from the whole monocyte population. The relative percentage of CD14+ CD16- monocytes was increased by 13% (P < 0.01) in AD and MCI groups compared to CN group (Table A.2.4). Interestingly, the relative percentage of CD14+ CD16+ monocytes decreased by 30% (P = 0.02) in MCI groups compared to CN group, but AD and CN did not show any group differences. A lower relative percentage of CD14- CD16+ monocytes in MCI group (-29% P = 0.02) was also observed in another tube with smaller sample size. Thereafter, the percentages of CD 14- lymphocytes and CD 14+ CD 16- monocytes were added to the ten, making twelve biomarkers for further assessment. Table A.2.1. Phase I differences of mean fluorescent intensity and standard deviation of

CD11c and P2X7 in CN, MCI and AD.

Change of MCI & AD Markers CN MCI AD MCI&AD (%)

CDUconlym 8.61 ± 2.48 7.17 ±1.51 6.66 ±1.31 6.98 ±1.43 -19

CD11conmono 73.67 ± 37.28 47.99 ± 15.14 50.31 ± 7.94 48.87 ±12.77 -34

CD11c on neutro 21.43 ±8.71 15.26 ±4.95 16.04 ±4.52 15.56 ±4.72 -27

P2X7 on lym 17.03 ±4.72 13.02 ± 1.99 13.25 ±2.13 13.11 ±2.01 -23

P2X7 on NK 21.34 ±7.7 14.58 ±2.44 15.36 ±4.11 14.88 ±3.13 -30

P2X7 on T&B 16.21 ±4.34 12.82 ± 1.88 13.06 ±1.95 12.91 ±1.88 -20

P2X7 on CD14-CD16+ mono 40.58 ±20.37 24.96 ± 6.03 27.08 ± 12.69 25.8 ± 9.07 -36

P2X7 on CD14+CD16- mono 70.7 ±40.01 40.02 ± 10.53 42.48 ± 20.76 40.99 ±15.06 -42

P2X7 on neutro 32.67 ± 14.37 23.31 ± 2.45 23.78 ± 4.46 23.48 ± 3.24 -28

P2X7 on CD14-CD16+ neutro 31.08 ± 12.08 22.82 ± 2.51 23.31 ± 4.33 23 ± 3.21 -26

Lym: lymphocytes.

NK: natural killer cells.

T&B: T cells and B cells.

Mono: monocytes.

Neutro: neutrophils.

Table A.2.2. Phase II differences of mean fluorescent intensity and standard deviation of CD 11c, CD 11b and CD33 in CN, MCI and AD.

Change of MCI & AD

Markers CN MCI AD MCI&AD (%)

CDllc on NK 24.83 ± 11.46 18.81 ±7.16 19.48 + 7.36 19.11 ±7.21 -23

129.39 ±39.77 102.28 + 27.75 105.05 ±31.84 -19

CD14+ CD16+ mono 34.81

CDllc on neutro 22.01 ±5.69 19.02 ±5.41 18.81 ±6.16 18.92 ±5.73 -14

^C ^° ^nCD14 ’ 27.22 ±6.55 23.33 ±6.22 22.41 ±6.75 22.91 ±6.44 -16

CD16+ neutro

CDllc on CD14-CD16- _2094±779 I _8.52 ±6.66 15.88 ±4.87 17.34 ±6.04 -17 neutro

CDllb on CD15- T&B 17.39 ±6.34 15.22 ±4.92 14.63 ±4.43 14.95 ±4.68 -14

CDllb on CD15+ _{39969 ± 12} 4.42 ³ ^'^ ^± 307.63 ±79.39 314.12 ±76.47 -21 mono* 74.43 294.47 ±

CDllb on CD15- mono 345.5 + 118.92 292.72 + 76.1 293.67 + 75.64 -15

76.26

CDllb on CD15- 321.79 ±

378.83 ± 136.7 318.72 + 85.98 320.39 + 85.65 -15 CD14+ CD16- mono 86.51

CDllb on CD15+ 432.55 ±

516.45 ± 124.25 419.03 + 99.88 426.37 + 93.23 -17 CD14- neutro 88.13

190.62 ±

CD33 on mono 232.55 ± 93.31 188.46 + 100.44 189.65 + 90.99 -18

83.82

CD33 on neutro 34.66 ± 11.76 29.87 ± 10.59 28 ± 11.16 29.03 ± 10.82 -16

Lym: lymphocytes.

NK: natural killer cells.

T&B: T cells and B cells.

Mono: monocytes.

Neutro: neutrophils.

*Only 10-15% of subjects were found to weakly express CD15 on their monocytes.

Table A.2.3. Phase III differences of mean fluorescent intensity and standard deviation of CDl lc, CD59, CD163, CD91, MerTK, CD18 and RAGE in CN, MCI and AD.

Markers CN MCI AD AD&MCI %

CDllc on NK 14.58 + 4.7 12.01 + 4.63 11.7 + 5.34 11.87 + 4.92 -19

CDllc on CD14-CD16+ mono 57.35 + 22.02 47.29 + 18.78 47.22 + 28.09 47.26 + 23.22 -18

CDllc on CD14- neutro 11.81 + 3.71 9.79 + 3.21 10.33 + 3.54 10.03 + 3.34 -15

CD59 on T&B 7.9 + 2.14 13.22 + 6.12 15.11 + 6.52 14.06 + 6.33 78

CD59 on CD14-CD16+ mono 10.28 + 3.05 14.74 + 4.74 14.61 + 4.81 14.68 + 4.74 43

CD59 on CD14-CD16+ neutro 16.64 + 3.35 20.56 + 6.41 21.73 + 6.72 21.08 + 6.53 27

CD163 on CD14+CD16- mono 165.03 + 43.75 139.16 + 48.79 139.76 + 35.52 139.43 + 43.11 -16

CD163 on CD14- neutro 13.75 + 2.71 12.18 + 2.69 12.31 + 1.58 12.24 + 2.25 -11

CD91 on NK 4.03 + 1.63 5.51 + 2.84 6.1 + 3.9 5.78 + 3.36 44

CD91 on CD14+CD16+ mono 175.2 + 59.23 255.02 + 114.66 304.57 + 121.16 277.89 + 119.4 59 MerTK on CD14-CD16- neutro 6.85 ± 2.04 5.99 + 0.96 6.1 + 1.04 6.04 + 0.99 -12

CD18 on NK 232.13 + 57.55 189.42 + 40 179.42 + 47.42 185 + 43.19 -20

CD18 on T&B 96.64 + 28.1 68.62 + 20.98 75.17 + 34.45 71.51 ± 27.58 -26

RAGE on CD14-CD16+ mono 6.64 ± 1.7 8.26 ± 2.6 9.21 ± 1.99 8.68 ± 2.38 31

Lym: lymphocytes.

NK: natural killer cells.

T&B: T cells and B cells.

Mono: monocytes.

Neutro: neutrophils.

[00136] Table A.2.4. Phase III differences of blood cell percentage and standard deviation in CN, MCI and AD.

Markers CN MCI AD AD&MCI %

Absolute %lym 0.28 ± 0.07 0.25 ± 0.07 0.24 ± 0.09 0.25 ± 0.08 -11

Relative

%CD14+CD16- mono 0.57 ± 0.12 0.64 ± 0.11 0.66 ± 0.1 0.65 ± 0.11 13

[00137] 3.5. Biomarker assessment

[00138] In Tables 3s, the twelve selected biomarkers in Phase III were named as Biomarker 1 (BM1), Biomarker 2 (BM2), ... and Biomarkerl2 (BM12). Distribution tests found that Biomarker7 and Biomarker 8 nearly followed normal distributions (Tables A.6s). Others were transformed by using both natural logarithm (InBM) and square root (sqrtBM). Collinearity test was used to choose only independent biomarkers when combining them into a panel (Tables A.7s). [00139] Table 3.1 Phase III: Model 1 biomarker performance.

Rrier.

Biomarkers Descnntives Division of Goodness of Goodness of AUC.orig AUC.corrected _T IDI.events IDI.nonevents biomarkers tit (original) nt (corrected) (95% CI) (95% CI) men?' ^{(95% CI) (95% CI)}

Demo-graphics

Baselinel (Age, Sex, Ed, Reference 0.39 0.35 ^t ° 0.81 (0.76 to 0.88) NA NA NA Psy)

Biomarkerl CDllc on NK 0.047 (0.03 to -0.006 (-0.022 to

CDllc 0.47 cells 0.40 °' ⁸ Q 9° ₁₎ ^{8 t} ° 0.83 (0.77 to 0.9) 4 0.063) 0.011)

InBMl CDllc 0.47 0.045 (0.029 to -0.007 (-0.022 to

0.40 °' ⁸ Q 9° ₁ ^{8 t} ° 0.83 (0.79 to 0.9) 3.7 0.061) 0.008) sqrtBMl CDllc 0.47 0.046 (0.03 to -0.006 (-0.022 to

0.40 °' ⁸ Q 9° ₁ ^{8 t} ° 0.84 (0.77 to 0.91) 4 0.062) 0.01)

CDllc on non-

Biomarker2 0.047 (0.029 to -0.006 (-0.02 to classical CDllc 0.47 0.40 °' ⁸ Q 9° ₁ ^{8 t} ° 0.83 (0.78 to 0.9) 4.6 0.064) monocytes 0.009) lnBM2 CDllc 0.47 0.048 (0.03 to -0.004 (-0.02 to

0.41 °’ ⁸ Q 9° ₁ ^{8 t} ° 0.84 (0.79 to 0.91) 4.6 0.067) 0.012) sqrtBM2 CDllc 0.47 0.048 (0.03 to -0.005 (-0.02 to

0.42 °' ⁸ Q ^ ^{8 TO} 0.84 (0.78 to 0.9) 4.2 0.066) 0.01)

Biomarker3 CDllc on CDllc 0.055 (0.035 to 0.001 (-0.02 to neutrophils 0.49 0.43 °’ ⁸ 0 92) ^{8 t} ° 0.84 (0.79 to 0.91) 6.2 0.075) 0.022)

0.054 (0.033 to 0.001 (-0.02 to lnBM3 CDllc 0.49 0.43 °' ⁸ Q ^TO 0.84 (0- ^{78 to} 0- ⁹ ) 6.6 0.075) 0.021)

0.054 (0.034 to 0.001 (-0.02 to sqrtBM3 CDllc 0.49 0.43 °’ ⁸ 0 92) ^{8 t} ° 0.84 (0.78 to 0.91) 6.4 0.075) 0.022)

Biomarker4 CD59 on T&B 0.037 (0.014 to 0.017 (0.003 to lymphocytes CD59 0.49 0.43 °’ ⁸⁷ o ⁽ 93 ^{82 t} ° 0- ⁸⁵ (0- ^{78 to} 0-92) 7.7 0.059) 0.031) lnBM4 0.032 (0.013 to CD59 0.49 0.42 ° ' ⁸⁶ ₀ ⁽ 9 ₂ ^{81 tO} 0.84 (0.79 to 0.91) 7.4 3.012 (0 to 0.025) 0.051) sqrtBM4 0.034 (0.014 to CD59 0.015 (0.001 to

0.49 0.42 ° ' ⁸⁷ ₀ ⁽ 9 ₃ ^{81 tO} 0.84 (0.8 to 0.92) 7.3 0.055) 0.028)

ZD59 on non-

Biomarker5 0.032 (0.015 to 0.012 (-0.002 to classical CD59 0.48 0.40 ° ' ⁸⁶ ₀ ⁽ 9 ₂ ^{81 tO} 0.84 (0.79 to 0.91) 5.5 es 0.048 0.027) nonocyt ) lnBM5 CD59 0.031 (0.015 to

0.48 0.012 (-0.003 to

0.41 °’ ⁸ 0 92) ^{8 t} ° 0.84 (0.79 to 0.91) 7.5 0.048) 0.026) sqrtBM5 CD59 0.032 (0.015 to 0.012 (-0.002 to

0.48 0.42 ° ' ⁸⁶ ₀ ⁽ 9 ₂ ^{81 tO} 0.84 (0.79 to 0.92) 6.9 0.049) 0.027)

Biomarker6 ^^59 on CD59 0.031 (0.014 to 0.011 (-0.002 to leutrophils 0.48 0.43 ° ' ⁸⁷ ₀ ⁽ 9 ₂ ^{81 tO} 0.85 (0.78 to 0.91) 6.5 0.048) 0.024)

0.027 (0.013 to 0.008 (-0.003 to lnBM6 CD59 0.48 0.41 °’ ⁸ 0 92) ^{8 tO} 0.84 (0.78 to 0.91) 0.041) 0.019)

0.029 (0.013 to 0.01 (-0.002 to sqrtBM6 CD59 0.48 0.40 ^tO 0.84 (0.79 to 0.91) 0.045) 0.022)

ZD163 on 0.027 (0.014 to 0.008 (-0.003 to

Biomarker7 classical CD163 0.47 0.41 ⁰ ’ ⁸ 0 92) ^{8 t} ° 0.84 (0.78 to 0.91) 0.041) 0.019) monocytes

0.026 (0.013 to 0.007 (-0.003 to lnBM7 CD163 0.47 0.40 0.84 (0.78 to 0.91) 0.039) 0.017)

0.027 (0.013 to 0.008 (-0.003 to sqrtBM7 CD163 0.47 0.41 0.84 (0.77 to 0.91) 0.04) 0.018)

ZD163 on 0.057 (0.022 to 0.039 (0.013 to

Biomarker8 CD163 0.55 0.48 0.86 (0.81 to 0.93) leutrophils 0.091) 0.064) 0.06 (0.024 to 0.041 (0.015 to

InBMS CD163 0.56 0.49 °' ⁸⁷ < ⁰ ' ^{83 TO} °' ⁹⁴⁾ 0.096) 0.067)

0.058 (0.023 to 0.04 (0.014 to sqrtBM8 CD163 0.55 0.49 °- ^{87 (0} ' ^{82 TO} °' ⁹³⁾ 0.093) 0.066) i'DO I on NK 0.046 (0.021 to 0.005 (-0.021 to

Biomarker9 CD91 0.47 0.38 ⁰ - ⁸³ (0

:ells - ^{77 to 0} - ⁹¹ ) 0.07) 0.032)

0.048 (0.023 to 0.008 (-0.021 to lnBM9 CD91 0.48 0.40 °- ^{83 (0} - ^{78 to} °- ⁹²⁾ 0.074) 0.036) 0.047 (0.022 to 0.007 (-0.021 to sqrtBM9 CD91 0.48 0.39 ⁰ - ⁸³ (0- ^{77 to 0} - ⁹¹ ) 0.072) 0.034)

ZD91 on 0.047 (0.021 to 0.013 (-0.014 to

Biomarker 10 intermediate CD91 0.48 0.39 ° ⁸ ^93) ^{9 tO} °- ^{83 (0} - ^{77 to} °’ ⁹³⁾ 0.073) 0.039) monocytes

0.048 (0.022 to 0.014 (-0.014 to

InBMIO CD91 0.48 0.40 0.074) 0.041)

0.048 (0.022 to 0.013 (-0.014 to sqrtBMIO CD91 0.48 0.40 ^tO 0.83 (0.76 to 0.91) 0.073) 0.04)

% of

Biomarkerll lymphocytes in %Ly _m ph _o-C y _te Q .48 0.03 (0.013 to 0.011 (-0.003 to

0.42 ° whole ’ ⁸⁷ ₀ ⁽ 9 ₂ ⁸ ₎ ^{1 tO} 0.84 (0.79 to 0.92) 0.047) 0.024) leukocytes

0.03 (0.013 to 0.011 (-0.003 to

InBMll %Lympho-cyte 0.48 0.41 ^tO 0.84 (0.79 to 0.91) 3.6 0.047) 0.024)

0.03 (0.013 to 0.011 (-0.003 to sqrtBMll %Lympho-cyte 0.48 0.42 ^tO 0.84 (0.79 to 0.91) 5.8 0.047) 0.024)

% of classical Biomarker 12 monocytes in %Monocyte 0.48 0.86 (0.79 to _{n oQ zn T7 t} 0.048 (0.02 to 0.013 (-0.015 to

0.40 0 92) 0.83 (0.77 to 0.91) total monocytes 0.075) 0.041) lnBM12 0.047 (0.02 to %Monocyte 0.48 0.012 (-0.015 to

0.40 ^tO 0.83 (0.78 to 0.9 1) 0.074) 0.04) sqrtBM12 0.047 (0.02 to %Monocyte 0.48 0.013 (-0.015 to

0.40 0.075) 0.041)

Age, Sex, Ed, Reference, Newl 0.085 (0.049 to 0.05 (0.019 to Psy, lnBM8, CD163, CD59 0.61 0.54 0.122) 0.081) lnBM5, lnBM3 & CDllc

Psy is Neuropsych. SimpleClassification. ^a . A Hosmer-Lemeshow goodness of fit was used to test calibration of the model. Ln is natural logarithm of the biomarker and sqrt is square root of the biomarker. Underscores are best performance biomarkers.

Baseline 1 is the baseline of Phase III: Model 1.

Newl is a proposed panel of biomarkers for Phase III: Model 1.

[00140] Table 3.2 Phase III: Model 2 biomarker performance.

, .x - Goodness of Goodness of AUC.orig AUC.corrected _T Brier IDLevents IDLnonevents

Biomarkers Descnptives _{fit (original)a fit (corrected) (95% C} I) (95% CI) ^Im P^ ^e ' (95% CI) (95% CI)

Demographics

Baseline2 (Age, Sex, Ed, 0.32 0.26 n qi/ 0.78 (0.71 to 0.87) NA NA NA

Psy) ^U - ⁹¹⁾

Biomarkerl CDllc on NK _{Q 46 0 39} 0.83 (0.74 to 0.91) 9.8 3.067 (0.038 to 0.097) 0.011 (-0.007 to 0.029) cells

InBMl 0.45 0.38 0.83 (0.76 to 0.91) 9.8 3.065 (0.036 to 0.094) 0.01 (-0.007 to 0.027) sqrtBMl 0.46 0.37 0.83 (0.76 to 0.91) 10.1 3.066 (0.037 to 0.096) 0.01 (-0.007 to 0.028)

CD11c on non- „ „„ „

Biomarker2 classical 0.48 0.40 9 94) 0.84 (0.77 to 0.92) 13.8 3.082 (0.042 to 0.121) 0.018 (-0.002 to 0.038) monocytes ' ^J lnBM2 0.48 0.40 ^tO 0.84 (0.77 to 0.92) 13.1 3.082 (0.044 to 0.121) 0.018 (-0.003 to 0.04) sqrtBM2 0.48 0.40 °' ⁸⁷ ^ ^{8 tO} 0.84 (0.77 to 0.92) 14.2 3.083 (0.044 to 0.123) 0.019 (-0.002 to 0.039)

CDllc on 0.86 (0.79 to

Biomarker3 0.84 (0.76 to 0.93) 14.7 3.082 (0.047 to 0.117) 0.018 (-0.003 to 0.04) neutrophils 0.93)

0.86 (0.79 to lnBM3 0.47 0.83 (0.76 to 0.92) 15.2 3.079 (0.044 to 0.115) 0.017 (-0.003 to 0.037) 0.93)

0.86 (0.79 to sqrtBM3 0.48 0.83 (0.75 to 0.91) 14.4 3.081 (0.045 to 0.116) 0.018 (-0.003 to 0.039) 0.93)

CD59 on T&B 0.86 (0.79 to

Biomarker4 0.82 (0.76 to 0.92) 4.4 3.046 (0.023 to 0.068) 0.014 (0.001 to 0.027) lymphocytes 0.93)

0.85 (0.78 to lnBM4 0.45 0.92) 0.82 (0.76 to 0.91) 5.4 3.045 (0.024 to 0.066) 0.014 (0.001 to 0.026)

0.86 (0.79 to sqrtBM4 0.45 0.82 (0.76 t 5.2 3.045 (0.024 to 0.067) 0.014 (0.001 to 0.026) 0.92) o 0.91)

CD59 on non-

0.87 (0.8 to

Biomarker5 classical 0.47 0.83 (0.77 to 0.93) 9.5 0.056 (0.03 to 0.082) 0.019 (0.003 to 0.035) monocytes 0.93)

0.86 (0.79 to lnBM5 0.46 0.83 (0.76 to 0.91) 8 3.052 (0.028 to 0.077) 0.017 (0.002 to 0.032) 0.93)

0.86 (0.8 to sqrtBM5 0.46 0.83 (0.76 to 0.92) 7.1 0.054 (0.029 to 0.08) 0.018 (0.002 to 0.034) 0.93)

, CD59 on 0.85 (0.78 to

Biomarker6 neu _t . .. trophils 0.45 0.92) 0.82 (0.75 to 0.9) 6.3 3.045 (0.024 to 0.066) 0.014 (0.001 to 0.026)

0.85 (0.78 to lnBM6 0.45 0.92) 0.82 (0.76 to 0.92) 6.8 3.047 (0.025 to 0.069) 0.015 (0.001 to 0.028)

0.85 (0.78 to sqrtBM6 0.45 0.82 (0.75 to 0.92) 5.2 3.046 (0.025 to 0.067) 0.014 (0.001 to 0.027) 0.92)

CD163 on

0.85 (0.78 to

Biomarker7 classical 0.45

0.92) 0.82 (0.76 to 0.91) 6.9 3.046 (0.023 to 0.069) 0.014 (0.001 to 0.027) monocytes

0.85 (0.78 to lnBM7 0.45 0.36 0.92) 0.82 (0.76 to 0.92) 6.2 3.045 (0.024 to 0.066) 0.014 (0.001 to 0.026)

0.85 (0.78 to sqrtBM7 0.45 0.36 0.82 (0.76 to 0.92) 6 3.046 (0.024 to 0.068) 0.014 (0.001 to 0.027) 0.92)

CD163 on 0.9 (0.84 to

Biomarker8 neutrophils 0.46 0.86 (0.81 to 0.95) 19.7 3.101 (0.047 to 0.156) 0.049 (0.022 to 0.075) 0.95)

0.9 (0.84 to lnBM8 New2 0.56 0.48 0.87 (0.82 to 0.94) 20 0.106 (0.05 to 0.162) 0.051 (0.024 to 0.077) 0.96)

0.9 (0.84 to sqrtBM8 0.56 0.47 0.87 (0.81 to 0.94) 19.4 3.104 (0.048 to 0.159) 0.05 (0.023 to 0.076) 0.96)

„ CD91 on NK

Biomarker9 „ 0.46 0.34 ⁰ - ⁸⁵ ₀ < ₉ ° ₃ ^{77 tO} 0.81 (0.74 to 0.92) 1.4 3.089 (0.049 to 0.128) 0.003 (-0.022 to 0.028) lnBM9 0.47 0.36 ° ' ⁸⁶ ₀ ⁽ 9 ₄ ^{78 tO} °- ⁸² (°- ^{75 to} °- ⁹³ ) 1.2 3.094 (0.053 to 0.135) 0.006 (-0.022 to 0.034) sqrtBM9 0.46 0.34 ^tO 0.81 (0.75 to 0.92) 1.1 3.091 (0.051 to 0.132) 0.005 (-0.022 to 0.031)

CD91 on

BiomarkerlO intermediate 0.44 0.33 -2.2 0.072 (0.04 to 0.104) -0.001 (-0.021 to 0.02 es ^TO °’ ⁹ ) monocyt ¹⁾

InBMIO 0.44 0.33 -1.8 3.073 (0.041 to 0.104) 0 (-0.021 to 0.021) ^TO °' ⁹²⁾ sqrtBMIO 0.44 0.34 -2 0.072 (0.04 to 0.104) 0 (-0.021 to 0.021) ^TO °' ⁹¹⁾

% of lymphocytes

Biomarkerll in whole 0.46 0.36 ^to °- 6.1 3.049 (0.022 to 0.075) 0.015 (0.001 to 0.029) ⁹² ) leukocytes

InBMll 0.46 0.35 ^to °- ⁹ 6.2 3.049 (0.023 to 0.074) 0.015 (0.001 to 0.03) ² ) sqrtBMll 0.46 0.37 0.83 (0.76 to 0.91) 6.3 3.049 (0.023 to 0.075) 0.015 (0.001 to 0.03)

% of classical

Biomarker 12 monocytes in 0.47 0.36 ° ' ⁸⁶ ₀ ⁽ 9 ₄ ^{78 tO} °- ⁸² (°- ^{75 to} °- ⁹² ) 2.9 0.09 (0.048 to 0.132) 0.011 (-0.015 to 0.035) total monocytes lnBM12 0.47 0.36 0.016 to 0.035) sqrtBM12 0.47 0.35 0.015 to 0.035)

Psy is Neuropsych. SimpleClassification. ^a . A Hosmer-Lemeshow goodness of fit was used to test calibration of the model.

Ln is natural logarithm of the biomarker and sqrt is square root of the biomarker.

Underscores are best performance biomarkers.

Baseline2 is the baseline of Phase III: Model 2.

New2 is a proposed panel of biomarkers for Phase III: Model 2. Only one leukocyte biomarker, i.e. lnBM8 can be added into baseline due to the limited size of the model.

[00141] Table 3.3 Phase III: Model 3 biomarker performance.

. ,, Goodness of Goodness of , _IT „ . „„ AUC.corrected . Brier IDLevents IDLnonevents (95%markers Descnptives _{fit (original)a fit (corrected)} AUC.ong (95% CI) _{(95% CI)} improve- _{(95% CI) C} I)

Demographics aseline3 (Age, Sex, Ed, 0.67 0.64 0.91 (0.86 to 0.96) 0.9 (0.85 to 0.95) NA NA NA

Psy) markerl ^^ells" ^ °- ⁶⁷ 0.63 0.91 (0.87 to 0.96) 0.9 (0.85 to 0.95) -3.5 0.019 (0.01 to 0.029) -0.023 (-0.033 to -0.013)nBMl 0.67 0.63 0.91 (0.87 to 0.96) 0.9 (0.84 to 0.96) -5.1 0.019 (0.01 to 0.029) -0.023 (-0.033 to -0.014) rtBMl 0.67 0.64 0.91 (0.87 to 0.96) 0.9 (0.85 to 0.95) -8 0.019 (0.01 to 0.029) -0.023 (-0.033 to -0.013)

CD lie on non-marker2 classical 0.67 0.63 0.91 (0.87 to 0.96) 0.9 (0.85 to 0.95) -5.8 0.022 (0.009 to 0.034) -0.021 (-0.033 to -0.01) monocytes nBM2 0.67 0.64 0.91 (0.87 to 0.96) 0.9 (0.85 to 0.95) -8.4 0.021 (0.009 to 0.033) -0.022 (-0.033 to -0.01) rtBM2 0.67 0.64 0.91 (0.87 to 0.96) 0.9 (0.85 to 0.96) -4.4 0.022 (0.009 to 0.034) -0.021 (-0.033 to -0.01)

CD1 1c on marker3 neutrophils °- ^{67 0-64} 0.92 (0.87 to 0.96) 0.9 (0.85 to 0.95) -4.4 0.022 (0.009 to 0.034) -0.021 (-0.034 to -0.008)nBM3 0.67 0.63 0.92 (0.87 to 0.96) 0.9 (0.86 to 0.96) -5.5 0.021 (0.009 to 0.033) -0.022 (-0.034 to -0.009) rtBM3 0.67 0.64 0.92 (0.87 to 0.96) 0.9 (0.85 to 0.95) -5.1 0.021 (0.009 to 0.034) -0.021 (-0.034 to -0.009)marker4 CD59 on T&B ₀ _ _{78 0} .75 0.96 (0.93 to 0.99) 0.95 (0.92 to 0.99) 22 0.035 (0.003 to 0.067) 0.024 (0.007 to 0.041) lymphocytes nBM4 0.78 0.74 0.96 (0.92 to 0.99) 0.95 (0.91 to 0.98) 18.5 0.031 (0.003 to 0.059) 0.02 (0.002 to 0.038) rtBM4 0.78 0.75 0.96 (0.92 to 0.99) 0.95 (0.91 to 0.98) 19.9 0.033 (0.003 to 0.063) 0.022 (0.004 to 0.04)

CD59 on non-marker5 classical 0.76 0.72 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.98) 15.6 0.021 (0.005 to 0.038) 0.011 (-0.004 to 0.027) monocytes nBM5 0.76 0.73 0.95 (0.91 to 0.98) 0.94 (0.9 to 0.98) 15.7 0.021 (0.005 to 0.038) 0.011 (-0.005 to 0.027) rtBM5 0.76 0.73 0.95 (0.91 to 0.98) 0.94 (0.9 to 0.98) 15.8 0.021 (0.005 to 0.038) 0.011 (-0.004 to 0.027)

CD59 on g n

Biomarker6 0.025 (0.006 to 0.044) 0.014 (-0.003 to 0.032) neutrophils 0.73 0.95 (0.91 to 0.99) 0.94 (0.91 to 0.98) 16.3 lnBM6 0.76 0.73 0.95 (0.91 to 0.99) 0.94 (0.89 to 0.98) 14.4 0.022 (0.006 to 0.038) 0.012 (-0.005 to 0.029) sqrtBM6 0.76 0.73 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.98) 17.9 0.023 (0.006 to 0.041) 0.013 (-0.004 to 0.03)

CD163 on

Biomarker7 classical 0.77 0.73 0.95 (0.92 to 0.99) 0.94 (0.9 to 0.98) 18 0.027 (0.005 to 0.048) 0.016 (-0.002 to 0.034) monocytes lnBM7 0.77 0.74 0.95 (0.92 to 0.99) 0.94 (0.9 to 0.98) 18.1 0.024 (0.004 to 0.044) 0.014 (-0.001 to 0.028) sqrtBM7 0.74 0.95 (0.92 to 0.99) 0.94 (0.9 to 0.98) 19.6 0.026 (0.005 to 0.046) 0.015 (-0.001 to 0.031)

CD163 on Biomarker8 neutrophils 0.75 0.96 (0.93 to 0.99) 0.95 (0.92 to 0.99) 22.5 0.045 (0.011 to 0.079) 0.032 (0.006 to 0.058) lnBM8 0.80 0.76 0.96 (0.93 to 0.99) 0.95 (0.92 to 0.99) 24.4 0.049 (0.013 to 0.085) 0.035 (0.009 to 0.062) sqrtBM8 0.80 0.76 0.96 (0.93 to 0.99) 0.95 (0.92 to 0.99) 23.3 0.047 (0.012 to 0.082) 0.034 (0.008 to 0.06)

CD91 on NK Biomarker9 0. 0.034 (0.011 to 0.057) -0.005 (-0.032 to 0.022) cells 71 0.95 (0.9 to 0.99) 0.93 (0.89 to 0.98) 9.1 lnBM9 0.75 0.71 0.95 (0.9 to 0.99) 0.94 (0.88 to 0.98) 8.8 0.035 (0.01 to 0.059) -0.004 (-0.031 to 0.023) sqrtBM9 0.75 0.71 0.95 (0.9 to 0.99) 0.93 (0.88 to 0.98) 9 0.034 (0.011 to 0.058) -0.004 (-0.031 to 0.023)

CD91 on BiomarkerlO intermediate 0.76 0.70 0.95 (0.9 to 0.99) 0.93 (0.88 to 0.98) 10.4 0.035 (0.009 to 0.06) -0.003 (-0.028 to 0.022) monocytes

InBMIO 0.75 0.71 0.95 (0.9 to 0.99) 0.93 (0.89 to 0.99) 10 0.033 (0.009 to 0.057) -0.004 (-0.029 to 0.02) sqrtBMIO 0.75 0.71 0.95 (0.9 to 0.99) 0.93 (0.88 to 0.98) 10.4 0.034 (0.009 to 0.059) -0.004 (-0.028 to 0.021)

% of lymphocytes Biomarkerll in whole 0.76 0.74 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.98) 17.8 0.025 (0.009 to 0.041) 0.015 (-0.003 to 0.032) leukocytes

InBMll 0.76 0.73 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.98) 19.5 0.024 (0.008 to 0.04) 0.014 (-0.003 to 0.03) sqrtBMll 0.76 0.73 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.98) 19.5 0.025 (0.009 to 0.04) 0.014 (-0.003 to 0.031)

% of classical marker 12 monocytes in 0.77 0.72 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.99) 13.1 0.04 (0.015 to 0.065) 0.003 (-0.032 to 0.039) total monocytes lnBM12 0.77 0.73 0.95 (0.92 to 0.99) 0.94 (0.9 to 0.98) 15.3 0.041 (0.015 to 0.067) 0.005 (-0.032 to 0.041) rtBM12 0.77 0.73 0.95 (0.91 to 0.99) 0.94 (0.9 to 0.98) 14.1 0.041 (0.015 to 0.066) 0.004 (-0.032 to 0.04)

Age, Sex, Ed,

Psy, lnBM8,

New3 0.77 0.97 (0.94 to 1) 0.96 (0.93 to 0.99) 28.7 0.065 (0.018 to 0.112) sqrtBM4, 0.049 (0.024 to 0.075) sqrtBMll _ sy is Neuropsych. SimpleClassification. A Hosmer-Lemeshow goodness of fit was used to test calibration of the model. n is natural logarithm of the biomarker and sqrt is square root of the biomarker. nderscores are best performance biomarkers. aseline3 is the baseline of Phase III: Model 3. ew3 is a proposed panel of biomarkers for Phase III: Model 3.

[00142] Table 3.4. Phase III: Model 4 biomarker performance.

Brier

Goodness of Goodness of AUC.corrected improveme IDLevents (95% IDLnone vents

Biomarkers Descriptives fit (original) fit (corrected) AUC.orig (95% CI) (95% CI) nt CI) (95% CI)

Base model4 Demographics (Age, 0.43 0.28 0.86 (0.79 to 0.93) 0.81 (0.76 to 0.92) NA NA NA

Sex, Ed, Apoe)

Biomarkerl MFI of CDllc on NK 0.43 0.26 0.85 (0.76 to 0.94) 0.8 (0.72 to 0.93) -5.6 0.05 (0.03 to -0.02 (-0.03 to -

0.07) 0.01)

InBMl 0.43 0.25 0.85 (0.76 to 0.92) 0.79 (0.71 to 0.91) -5.7 0.05 (0.03 to -0.02 (-0.03 to -

0.07) 0.01) sqrtBMl 0.43 0.25 0.86 (0.77 to 0.92) 0.79 (0.71 to 0.91) -5.6 0.05 (0.03 to -0.02 (-0.03 to -

0.07) 0.01)

Biomarker2 MFI of CDllc on 0.46 0.26 0.87 (0.78 to 0.95) 0.8 (0.75 to 0.93) -1.4 0.07 (0 to 0.11) -0.01 (-0.03 to

CD14 CD16 ⁺ Mono 0) lnBM2 0.46 0.26 0.86 (0.77 to 0.94) 0.8 (0.74 to 0.93) -1.3 0.06 (0.02 to 0.1) -0.01 (-0.03 to

0) sqrtBM2 0.46 0.26 0.87 (0.77 to 0.93) 0.8 (0.73 to 0.96) -1.1 0.06 (0.01 to 0.1) -0.01 (-0.03 to

0)

Biomarker3 MFI of CDllc on 0.45 0.27 0.86 (0.77 to 0.93) 0.8 (0.73 to 0.92) -2.7 0.06 (0.02 to -0.02 (-0.03 to

CD14 Neu 0.09) 0) lnBM3 0.45 0.25 0.86 (0.77 to 0.93) 0.79 (0.73 to 0.93) -2.9 0.05 (0.02 to -0.02 (-0.03 to

0.09) 0) sqrtBM3 0.45 0.25 0.86 (0.78 to 0.93) 0.79 (0.75 to 0.92) -2.8 0.05 (0.02 to -0.02 (-0.03 to

0.09) 0)

Biomarker4 MFI of CD59 on T&B 0.56 0.42 0.9 (0.83 to 0.96) 0.86 (0.79 to 0.96) 18.8 0.07 (0.02 to 0.01 (-0.01 to

0.12) 0.03) lnBM4 0.56 0.40 0.91 (0.82 to 0.96) 0.86 (0.8 to 0.97) 18.5 0.07 (0.03 to 0.01 (-0.01 to

0.12) 0.03) sqrtBM4 0.56 0.41 0.91 (0.82 to 0.97) 0.86 (0.81 to 0.96) 18.6 0.07 (0.02 to 0.01 (-0.01 to

0.12) 0.03)

Biomarker5 MFI of CD59 on 0.56 0.40 0.91 (0.82 to 0.97) 0.85 (0.8 to 0.96) 19.2 0.07 (0.03 to 0.01 (-0.01 to

CD14 CD16 ⁺ Mono 0.12) 0.03) lnBM5 0.56 0.40 0.91 (0.82 to 0.96) 0.85 (0.79 to 0.97) 18.9 0.07 (0.03 to 0.01 (-0.01 to

0.12) 0.03) sqrtBM5 0.56 0.40 0.91 (0.83 to 0.97) 0.85 (0.78 to 0.98) 19.1 0.07 (0.03 to 0.01 (-0.01 to

0.12) 0.03)

Biomarker6 MFI of CD59 on 0.56 0.42 0.91 (0.82 to 0.97) 0.86 (0.79 to 0.96) 20.5 0.08 (0.03 to 0.01 (-0.01 to

CD14 CD16 ⁺ Neu 0.12) 0.03)

lnBM6 0.56 0.40 0.91 (0.83 to 0.96) 0.85 (0.8 to 0.97) 19.6 0.07 (0.03 to 0.01 (-0.01 to

0.12) 0.03) sqrtBM6 0.56 0.40 0.91 (0.84 to 0.97) 0.85 (0.82 to 0.96) 20 0.07 (0.03 to 0.01 (0 to 0.03)

0.12) Biomarker7 MFI of CD163 on 0.57 0.43 0.91 (0.83 to 0.96) 0.86 (0.79 to 0.97) 20.1 0.08 (0.03 to 0.01 (-0.01 to

CD14+CD16' Mono 0.14) 0.03) lnBM7 0.57 0.43 0.91 (0.83 to 0.96) 0.86 (0.82 to 0.96) 19.4 0.07 (0.02 to 0.01 (-0.01 to

0.13) 0.03) sqrtBM7 0.57 0.43 0.91 (0.83 to 0.96) 0.86 (0.81 to 0.96) 19.7 0.08 (0.02 to 0.01 (-0.01 to

0.15) 0.03)

Biomarker8 MFI of CD163 on 0.63 0.49 0.93 (0.87 to 0.97) 0.88 (0.86 to 0.97) 28.3 0.12 (0.04 to 0.2) 0.03 (0 to 0.06)

CD14- Neu lnBM8 0.64 0.51 0.94 (0.86 to 0.98) 0.89 (0.82 to 0.97) 29.8 0.13 (0.05 to 0.2) 0.03 (0 to 0.06) sqrtBM8 0.63 0.50 0.93 (0.85 to 0.98) 0.89 (0.86 to 0.98) 29.1 0.13 (0.04 to 0.03 (0 to 0.06)

0.21) Biomarker9 MFI of CD91 on NK 0.59 0.41 0.91 (0.82 to 0.97) 0.85 (0.8 to 0.96) 13.4 0.1 (0.03 to 0.16) 0 (-0.03 to 0.04) lnBM9 0.59 0.40 0.91 (0.82 to 0.97) 0.85 (0.76 to 0.97) 13.3 0.1 (0.03 to 0.17) 0 (-0.04 to 0.04) sqrtBM9 0.59 0.40 0.92 (0.82 to 0.98) 0.85 (0.81 to 0.97) 13.4 0.1 (0.03 to 0.16) 0 (-0.03 to 0.04)

BiomarkerlO MFI of CD91 on 0.57 0.38 0.92 (0.82 to 0.98) 0.85 (0.72 to 0.96) 13.4 0.09 (0.04 to 0 (-0.03 to 0.03)

CD14 ⁺ CD16 ⁺ Mono 0.15)

InBMIO 0.58 0.40 0.92 (0.84 to 0.98) 0.86 (0.8 to 0.95) 13.1 0.09 (0.04 to 0 (-0.04 to 0.04)

0.14) sqrtBMIO 0.57 0.40 0.92 (0.83 to 0.97) 0.86 (0.74 to 0.95) 13.3 0.09 (0.04 to 0 (-0.04 to 0.03)

0.15) Biomarkerll Absolute % Lym 0.59 0.45 0.92 (0.85 to 0.98) 0.88 (0.83 to 0.97) 28.1 0.11 (0.07 to 0.02 (0 to 0.05)

0.16)

InBMll 0.58 0.44 0.92 (0.84 to 0.97) 0.88 (0.82 to 0.98) 26.4 0.1 (0.06 to 0.14) 0.02 (0 to 0.05) sqrtBMll 0.58 0.46 0.92 (0.84 to 0.97) 0.88 (0.84 to 0.97) 27.3 0.1 (0.06 to 0.14) 0.02 (0 to 0.05)

Biomarkerl2 Relative % 0.61 0.43 0.92 (0.85 to 0.98) 0.87 (0.81 to 0.96) 20 0.12 (0.06 to 0.01 (-0.03 to

CD14+CD16' Mono 0.18) 0.05) lnBM12 0.61 0.44 0.93 (0.86 to 0.98) 0.87 (0.77 to 0.98) 20.3 0.12 (0.06 to 0.01 (-0.03 to

0.18) 0.05) sqrtBM12 0.61 0.44 0.93 (0.85 to 0.99) 0.87 (0.79 to 0.97) 20.2 0.12 (0.06 to 0.01 (-0.03 to

0.18) 0.05)

New4 Age, Sex, Ed, Apoe, 0.64 0.51 0.94 (0.86 to 0.98) 0.89 (0.82 to 0.97) 29.8 0.13 (0.05 to 0.2) 0.03 (0 to 0.06) lnBM8

Model 4 only included cognitively normal subjects alongside their PET Ap status to stratify study cohort into healthy controls (CN & <25CL), and preclinical (CN & >25CL). Base model 4 is the baseline of Stage III: Model 4, which has age, sex, years of education and genotype of ApoE. Model 4 has 30 cases. In order to prove the concept, one biomarker, MFI of CD163 on CD14' neutrophils (lnBM8), is added to Base model 4 to compose New 4. In the paired-sample design ROC analysis by using SPSS (DeLong test), AUC is improved from Base model 4: 0.87 (Cl: 0.81 to 0.96) to New 4: 0.94 (Cl: 0.86 to 0.98) with P value of 0.063.

[00143] Table 3.5. Phase IV: Validation cohort.

Goodness

Goodness of of fit Brier IDI.events IDI.nonevent

Biomarkers Descriptives fit (original) (corrected) AUC.orig (95% CI) AUC.corrected (95% CI) improvement (95% CI) s (95% CI)

Demographics

(Age, Sex, Ed, Base model ApoE) 0.40 0.25 0.81 (CI: 0.72 to 0.9) 0.74 (CI: 0.26 to 0.84) NA NA NA

MFI of CD11c in 0.05 (CI: 0 (CI: -0.02 to

BM1 lym 0.49 0.30 0.85 (CI: 0.72 to 0.95) 0.78 (CI: 0.37 to 0.87) 9.9 0.02 to 0.08) 0.03)

MFI of CD1 lo in 0.06 (CI: 0 (CI: -0.02 to

BM2 CD14- mono 0.49 0.33 0.84 (CI: 0.71 to 0.94) 0.78 (CI: 0.33 to 0.87) 13.3 0.02 to 0.09) 0.03)

MFI of CD59 in 0.07 (CI: - 3.02 (CI: -0.03

BM3 CD14 ⁺ mono 0.48 0.29 0.87 (CI: 0.74 to 0.96) 0.79 (CI: 0.31 to 0.87) 7.7 0.01 to 0.13) to 0.07)

MFI of CD59 in 0.01 (CI: - 3.03 (CI: -0.01

BM4 CD14- mono 0.45 0.23 0.85 (CI: 0.73 to 0.93) 0.77 (CI: 0.43 to 0.88) 7.5 0.04 to 0.06) to 0.06)

MFI of CD91 in

CD14 ⁺ CD59 ^dim 0 (CI: -0.03 0.01 (CI: 0.01

BM5 mono 0.44 0.23 0.83 (CI: 0.7 to 0.93) 0.75 (CI: 0.34 to 0.87) 3.2 to 0.03) to 0.02)

MFI of CD91 in 0 (CI: -0.02 0.01 (CI: 0.01

BM6 neutro 0.43 0.23 0.82 (CI: 0.69 to 0.94) 0.74 (CI: 0.39 to 0.89) 4.6 to 0.01) to 0.02)

MFI of CD163 in 0.02 (CI: 0 0 (CI: -0.02 to

BM7 lym 0.44 0.22 0.83 (CI: 0.7 to 0.93) 0.74 (CI: 0.33 to 0.86) 4.2 to 0.04) 0.02)

MFI of CD163 in 0.02 (CI: - 3.01 (CI: -0.01

BM8 CD14- neutro 0.43 0.21 0.83 (CI: 0.68 to 0.94) 0.73 (CI: 0.21 to 0.82) 4.5 0.01 to 0.04) to 0.03)

Age, Sex, Ed, ApoE, BM2, 0.13 (CI: 3.03 (CI: -0.04

VI BM3 0.58 0.37 0.9 (CI: 0.78 to 0.98) 0.83 (CI: 0.5 to 0.92) 22.6 0.06 to 0.2) to 0.09)

Age, Sex, Ed,

ApoE, BM3, 0.11 (CI: 0.06 (CI: 0.02

V2 BM6 0.59 0.38 0.91 (CI: 0.81 to 0.99) 0.84 (CI: 0.51 to 0.88) 25.1 0.01 to 0.21) to 0.11)

Age, Sex, Ed,

ApoE, BM2, 0.18 (CI: 0.07 (CI: 0.01

V3 BM3, BM6 0.68 0.45 0.95 (CI: 0.86 to 1) 0.86 (CI: 0.6 to 0.96) 39.8 0.11 to 0.26) to 0.14)

Here in validation cohort we defined disease state simply by centiloids >25CL for an “overall” assessment. Base model is the baseline of Stage III: Validation cohort, which has age, sex, years of education and genotype of ApoE. Validation cohort has 49 cases. A pair of biomarkers, MFI of CDl lc in CD14' monocytes (BM2) and MFI of CD59 in CD14 ⁺ monocytes (BM3), are added to the Base model to compose Validation composite 1 (VI). In the paired-sample design ROC analysis by using SPSS (DeEong test), AUC is improved by 5.2% from the Base model: 0.81 (CI: 0.69 to 0.89) to VI: 0.85 (CI: 0.74 to 0.91) with P value of 0.112. The other performed pair, BM3 and MFI of CD91 in neutrophils (BM6), are added to the Base model to compose V2, which improves AUC by 7% from the Base model: 0.8 (CI: 0.68 to 0.88) to V2: 0.85 (CI: 0.74 to 0.92) with P value of 0.072. Further, if adds three performed biomarkers, BM2, BM3, and BM6 to the baseline, this V3 significantly improves AUC by 7.1% from the Base model: 0.82 (CI: 0.70 to 0.89) to V2: 0.88 (CI: 0.77 to 0.94) with P value of 0.05. However, this model of V3 is overfitted due to not enough cases have been included.

[00144] T able A.6.1 Phase III Model 1 biomarker skewness and normality statistics

Kolmogorov- Shapiro- Std. Smirnova ^a Wilk

Descriptives Skewness Error (Sig) (Sig.)

Baseline3.1 0.118 0.224 0.000 0.000

Biomarkerl -0.784 0.224 0.048 0.001

InBMl -0.179 0.224 .200* 0.647 sqrtBMl 0.288 0.224 .200* 0.426

Biomarker2 -1.096 0.224 0.039 0.000 lnBM2 -0.327 0.224 .200* 0.422 sqrtBM2 0.381 0.224 .200* 0.307

Biomarker3 -1.323 0.224 0.000 0.000 lnBM3 0.452 0.224 0.154 0.100 sqrtBM3 0.880 0.224 0.002 0.000

Biomarker4 1.385 0.224 0.000 0.000 lnBM4 0.740 0.224 0.000 0.000 sqrtBM4 1.056 0.224 0.000 0.000

Biomarker5 1.293 0.224 0.000 0.000 lnBM5 0.323 0.224 0.015 0.046 sqrtBM5 0.825 0.224 0.000 0.000

Biomarker6 1.079 0.224 0.000 0.000 lnBM6 0.553 0.224 0.000 0.001 sqrtBM6 0.808 0.224 0.000 0.000

Biomarker7 0.322 0.224 .200* 0.499 lnBM7 -2.041 0.224 0.000 0.000 sqrtBM7 -0.987 0.224 0.020 0.000

Biomarker8 -0.392 0.224 .200* 0.107 lnBM8 -0.080 0.224 .200* 0.665 sqrtBM8 0.155 0.224 .200* 0.533

Biomarker9 1.922 0.224 0.000 0.000 lnBM9 0.715 0.224 0.001 0.000 sqrtBM9 1.308 0.224 0.000 0.000

BiomarkerlO 1.064 0.224 0.000 0.000

InBMIO 0.257 0.224 0.018 0.046 sqrtBMIO 0.656 0.224 0.000 0.000

Biomarkerll -1.037 0.224 0.036 0.000

InBMll 0.005 0.224 .200* 0.267 sqrtBMll 0.492 0.224 .200* 0.023

Biomarkerl2 -0.976 0.224 0.000 0.000 lnBM12 -3.034 0.224 0.000 0.000 sqrtBM12 -1.895 0.224 0.000 0.000 ^a . Lilliefors Significance Correction.

*. This is a lower bound of the true significance.

[00145] Table A.6.2 Phase III Model 2 biomarker skewness and normality statistics Kolmogorov- Std. Smirnova* Shapiro-

Descriptives Skewness Error (Sig.) Wilk (Sig.)

Baseline3.2 0.574 0.224 0.094 0.000

Biomarkerl -0.784 0.224 0.048 0.001

InBMl -0.184 0.224 .200* 0.638 sqrtBMl 0.288 0.224 .200* 0.433

Biomarker2 -1.096 0.224 0.039 0.000 lnBM2 -0.333 0.224 .200* 0.408 sqrtBM2 0.380 0.224 .200* 0.310

Biomarker3 -1.323 0.224 0.000 0.000 lnBM3 0.449 0.224 0.083 0.102 sqrtBM3 0.882 0.224 0.002 0.000

Biomarker4 1.385 0.224 0.000 0.000 lnBM4 0.739 0.224 0.000 0.000 sqrtBM4 1.056 0.224 0.000 0.000

Biomarker5 1.293 0.224 0.000 0.000 lnBM5 0.327 0.224 0.014 0.044 sqrtBM5 0.825 0.224 0.000 0.000

Biomarker6 1.079 0.224 0.000 0.000 lnBM6 0.548 0.224 0.000 0.001 sqrtBM6 0.810 0.224 0.000 0.000

Biomarker7 0.322 0.224 .200* 0.499 lnBM7 -2.041 0.224 0.000 0.000 sqrtBM7 -0.987 0.224 0.020 0.000

Biomarker8 -0.392 0.224 .200* 0.107 lnBM8 -0.080 0.224 .200* 0.665 sqrtBM8 0.155 0.224 .200* 0.533

Biomarker9 1.922 0.224 0.000 0.000 lnBM9 0.711 0.224 0.001 0.000 sqrtBM9 1.305 0.224 0.000 0.000

BiomarkerlO 1.064 0.224 0.000 0.000

InBMIO 0.256 0.224 0.024 0.049 sqrtBMIO 0.657 0.224 0.000 0.000

Biomarkerll -1.037 0.224 0.036 0.000

InBMll 0.004 0.224 .200* 0.268 sqrtBMll 0.522 0.224 .200* 0.013

Biomarkerl2 -0.976 0.224 0.000 0.000 lnBM12 -3.032 0.224 0.000 0.000 sqrtBM12 -1.899 0.224 0.000 0.000 ^a . Lilliefors Significance Correction.

*. This is a lower bound of the true significance. T able A.6.3 Phase III Model 3 biomarker skewness and normality statistics

Kolmogorov- Shapiro- Std. Smirnova ^a Wilk

Biomarkers Skewness Error (Sig.) (Sig.)

Baseline 0.375 0.254 0.000 0.000

Biomarkerl -0.816 0.254 .200* 0.010

InBMl -0.315 0.254 .200* 0.637 sqrtBMl 0.261 0.254 .200* 0.833

Biomarker2 -0.658 0.254 .200* 0.052 lnBM2 -0.585 0.254 .200* 0.111 sqrtBM2 0.056 0.254 .200* 0.991

Biomarker3 -1.303 0.254 0.000 0.000 lnBM3 0.365 0.254 0.164 0.441 sqrtBM3 0.833 0.254 0.004 0.003

Biomarker4 1.608 0.254 0.000 0.000 lnBM4 0.000 0.000 sqrtBM4 1.305 0.254 0.000 0.000

Biomarker5 1.436 0.254 0.000 0.000 lnBM5 0.380 0.254 0.049 0.064 sqrtBM5 0.926 0.254 0.001 0.001

Biomarker6 1.283 0.254 0.000 0.000 lnBM6 0.665 0.254 0.001 0.004 sqrtBM6 0.960 0.254 0.000 0.000

Biomarker7 0.598 0.254 0.026 0.050 lnBM7 -2.293 0.254 0.000 0.000 sqrtBM7 -1.285 0.254 0.000 0.000

Biomarker8 -0.309 0.254 .200* 0.174 lnBM8 -0.141 0.254 .200* 0.409 sqrtBM8 0.082 0.254 .200* 0.444

Biomarker9 1.904 0.254 0.000 0.000 lnBM9 0.506 0.254 0.003 0.013 sqrtBM9 1.123 0.254 0.000 0.000

BiomarkerlO 1.057 0.254 0.000 0.000

InBMIO 0.314 0.254 0.059 0.082 sqrtBMIO 0.694 0.254 0.000 0.001

Biomarkerll -0.963 0.254 0.043 0.001

InBMll 0.039 0.254 .200* 0.759 sqrtBMll 0.518 0.254 0.177 0.077

Biomarkerl2 -1.259 0.254 0.000 0.000 lnBM12 -3.054 0.254 0.000 0.000 sqrtBM12 -2.118 0.254 0.000 0.000 ^a . Lilliefors Significance Correction.

*. This is a lower bound of the true significance. [00146] Table A.7.1. Phase III variables entered/removed ^a

Model Variables

Variables Entered Removed Method

1 Biomarker3, Biomarker2, Enter

B iomarker l ^b

2 Biomarker5, Biomarker4, Enter

Biomarker6 ^b

3 Biomarker7, B iomarker 8 ^b Enter

4 Biomarker9, B iomarker 10 ^b Enter

5 B iomarker 11 , B iomarker 12 ^b Enter ^a . Dependent Variable: Image. PET. Centiloid. ^b . All requested variables entered.

[00147] Table A.7.2. Phase III collinearity Statistics

Division of

Biomarkers Tolerance VIE biomarkers

Biomarker 1 0.60 1.68 CD 11c

Biomarker2 0.70 1.43

CDl lc

Biomarker3 0.58 1.73

CDl lc

Biomarker4 0.31 3.20 CD59

Biomarker5 0.47 2.14 CD59

Biomarker6 0.26 3.92 CD59

Biomarker7 0.80 1.25

CD to J

Biomarker8 0.62 1.62 CD163

Biomarker9 0.70 1.43 CD91

BiomarkerlO 0.47 2.15 CD91

Biomarkerll 0.86 1.17 ,

%Lym

Biomarkerl2 0.69 1.45

%Mono

[00148] In model 1 (Table 3.1), the baseline 1 discriminated between <25CL and >25CL with an AUC of 0.85 (0.79 to 0.91). The MFI of CD163 on neutrophils (lnBM8) had the best improved AUC of 0.89 (0.84 to 0.94); Brier improvement of 16%; IDI with events of 0.06 (0.024 to 0.096) and without events of 0.041 (0.015 to 0.067). Two other performing biomarkers, MFI of CD59 on CD14-CD16+ monocytes (lnBM5) and MFI of CDl lc on total neutrophils (lnBM3), were included to form a panel of biomarkers: New 1. New 1 had an AUC of 0.91 (0.86 to 0.95), Brier improvement 17.8%, IDI with and without events of 0.085 (0.049 to 0.12) and 0.050 (0.019 to 0.081), respectively. New 1 had sensitivity of 89.9% (95%CI 80.2% to 95.8%), specificity of 76.9% (66.0% to 85.7%) and overall accuracy 83.0% at the Youden index cut-off point (Table A.8.1).

[00149] Table A.8.1 ROC metrics for new models in Phase III (in composite with base models). AUC

New Cut-off Sensitivity % Specificity % Accuracy % NPV panels’* values (95% CI) (95% CI) % (95%

„ 86.7 (75.4 to 85.7 (73.8 to (88.7

New ! Cut-off 1 > 0.49 0.867 0.857

94 '.1) 93 ' .6 ,,) 86.2 to

97.3) n Cut .-off 2 > 0.20 95 (86.1 to 60.7 (46.8 to 78.5 0.722 0.919

90.4

_M New _o 2 r C’ut.-ofiTf 1i >M0n.3o9 77.5 (61.6 to 88.9 (80 to 85.1 (84.6 ov.Z) v4.o) to 0.775 0.889

96.2) n Cut.-o mff 2 > > n0.1i 1i 95 ₉ (8 ₉ 3 _4.) 1 to 56.8 ₆₇ (4 ₈ 5 ₎ .3 to 6 ,9.4 0.521 0.958

90.1

M New Q 3 r Cu.t-ofiTf 1i > . H0.A68Q 72.9 ( '55 _{1 A} .9 to 93.2 ( '8 ,1,.3 to 82.6 (84 to 0.921 0.759 oJ. l) VO.O)

96.1) n . . ,, , z 95.8 (85.8 to 50 (34.6 to

Cut-off 2 > 0.16 _{99 5) 65 4)} 73.9 0.677 0.917

94.1

N NTew 4 Cut-of iTf 1i > . n 0.2o5^ 95.2 ₉₉ (76.2 to 83.3 ₉ ( j72.1 to 86 ,.2 (86.3 o 0. 0.982 t 645

98.8) ^a . Best performed biomarkers adding into Base model to form panels. Cut-off 1: cut-off value at the Youden index. Cut-off 2: cut-off value at the 95% sensitivity. PPV and NPV:

Positive and negative predictive values.

New 1: Added MFI of CD59 on T&B (BM4), MFI of CD163 on CD14’ neutrophils (BM8), MFI of CD91 on CD14 ⁺ CD16 ⁺ monocytes (BM10) and MFI of CDl lc on CD14 CD16 ⁺ monocytes (lnBM2) to the Base model; New 2: Added MFI of CD163 on CD14" neutrophils (BM8) to the Base model; and New 3: Added MFI of CD59 on CD14 CD16 ⁺ monocytes (BM5), MFI of CD163 on CD14’ neutrophils (lnBM8), MFI of CD91 on CD14 ⁺ CD16 ⁺ monocytes (BM10) and MFI of CDl lc on CD14 CD16 ⁺ monocytes (sqrtBM2) to the Base model.

[00150] In model 2 (Table 3.2 above), the baseline 2 discriminated between <25CL and >25CL & <100CL with an AUC of 0.84 (0.77 to 0.91). The MFI of CD163 on neutrophils (lnBM8) had the best improved AUC of 0.90 (0.84 to 0.96); Brier improvement of 20%; IDI with events of 0.106 (0.050 to 0.162) and without events of 0.051 (0.024 to 0.077). Notably, model 2 only had 52 cases and only one biomarker was added so as to not overfit the model, to form New 2. New 2 had sensitivity of 90.0% (76.3% to 97.2%), specificity of 76.5% (65.8% to 85.3%) and overall accuracy of 81.0% at the Youden index cut-off point (Table A.8.1).

[00151] In model 3 (Table 3.3 above), the baseline 3 was overexaggerated because all MCI subjects had been classified as cases. The MFI of CD163 on neutrophils (lnBM8) had the best improved AUC of 0.96 (0.93 to 0.99); Brier improvement of 24.4%; IDI with events of 0.049 (0.013 to 0.085) and without events of 0.035 (0.009 to 0.062). Two other performing biomarkers, MFI of CD59 on CD14-CD16- lymphocytes (sqrtBM4) and absolute percentage of CD14- lymphocyte in whole blood (sqrtBMl l), were also included to form a panel of biomarkers: New3. New 3 improved the AUC from the baseline by 6%. New 3 had sensitivity of 91.2% (80.7% to 97.1%), specificity of 93.9% (85.2% to 98.3%) and overall accuracy of 92.7% at the Youden index cut-off point (Table A.8.1).

[00152] Overall, different biomarkers, composites or panels were proposed in different models. Whereas three candidate biomarkers: CDl lc, CD59 and CD163 performed very well, CD163 was always had the best performance. Model cut-off values, sensitivity, specificity and accuracy for each biomarker can be found in Table A.8.2-4.

[00153] Table A.8.2 Phase III Model 1 Cut-off values for test biomarkers alone.

Standalone c _u t-off _v . Sensitivity % (95% Specificity % (95% Accuracy AUC %

. bi .omar ,kers _b values ^{Va ue} CI) CI) % (95% CI)

BM1 Cut-off 1 < 11.00 52.5 (39.1 to 65.7) 75 (61.6 to 85.6) 63.5 ^tO

Cut-off 2 < 20.30 96.6 (88.3 to 99.6) 7.1 (2 to 17.3) 53.0

BM2 Cut-off 1 < 38.70 49.2 (35.9 to 62.5) 85.7 (73.8 to 93.6) 67.0 ^tO

Cut-off 2 < 93.50 94.9 (85.9 to 98.9) 5.4 (1.1 to 14.9) 51.3

BM3 Cut-off 1 < 9.82 66.1 (52.6 to 77.9) 71.4 (57.8 to 82.7) 68.7 Cut-off 2 < 14.70 94.9 (85.9 to 98.9) 26.8 (15.8 to 40.3) 61.7

BM4 Cut-off 1 > 12.20 47.5 (34.3 to 60.9) 91.1 (80.4 to 97) 68.7 ^tO

Cut-off 2 > 5.84 94.9 (85.9 to 98.9) 3.6 (0.4 to 12.3) 50.4

BM5 Cut-off 1 > 13.90 49.2 (35.9 to 62.5) 91.1 (80.4 to 97) 69.6 ^69-8 ^ ^{8 t} °

/o.j) Cut-off 2 > 8.45 94.9 (85.9 to 98.9) 23.2 (13 to 36.4) 60.0

BM6 Cut-off 1 > 20.00 50.9 (37.5 to 64.1) 87.5 (75.9 to 94.8) 68.7 56 (54.5 to 75)

Cut-off 2 > 12.20 94.9 (85.9 to 98.9) 1.8 (0.1 to 9.6) 49.6

BM7 Cut-off 1 < 157.00 67.8 (54.4 to 79.4) 71.4 (57.8 to 82.7) 69.6 ⁷³ - ² (62.2 t° ol.3) Cut-off 2 < 207.00 94.9 (85.9 to 98.9) 14.3 (6.4 to 26.2) 55.7

BM8 Cut-off 1 < 12.70 72.9 (59.7 to 83.6) 69.6 (55.9 to 81.2) 71.3 ⁶⁸ -9 J ⁷ ’ ⁷ J to

Cut-off 2 < 16.10 94.9 (85.9 to 98.9) 8.9 (3 to 19.6) 53.0

BM9 Cut-off 1 > 3.63 69.5 (56.1 to 80.8) 62.5 (48.6 to 75.1) 66.1

Cut-off 2 > 2.43 94.9 (85.9 to 98.9) 5.4 (1.1 to 14.9) 51.3

BM10 Cut-off 1 > 241.00 50.9 (37.5 to 64.1) 83.9 (71.7 to 92.4) 67.0 ⁶⁵ ' ₇ ( ⁵³ j ^{8 t} °

Cut-off 2 > 109.00 94.9 (85.9 to 98.9) 12.5 (5.2 to 24.1) 54.8

BM11 Cut-off 1 < 0.27 69.5 (56.1 to 80.8) 64.3 (50.4 to 76.6) 67.0 ⁶⁴ ^^ ² to

Cut-off 2 < 0.36 94.9 (85.9 to 98.9) 10.7 (4 to 21.9) 53.9

BM12 Cut-off 1 > 0.61 71.2 (57.9 to 82.2) 53.6 (39.7 to 67) 62.6 65.9 (54 ⁸ to

Cut-off 2 > 0.52 94.9 (85.9 to 98.9) 23.2 (13 to 36.4) 60.0 ^b . Test biomarkers alone without adding into baseline.

Cut-off 1: cut-off value at the Youden index.

Cut-off 2: cut-off value at the 95% sensitivity. Table A.8.3 Phase III Model 2 Cut-off values for test biomarkers alone.

Standalone c _u t- _o ff Specificity % Accuracy AUC % biomarkeC ^valueS Sensitivity % (95% CI) ^P _{(95% CI} ) % (95% CI)

BM1 Cut-off 1 < 10.90 47.2 (30.4 to 64.5) 75 (61.6 to 85.6) 64.1

Cut-off 2 < 18.20 94.4 (81.3 to 99.3) 17.9 (8.9 to 30.4) 47.8

BM2 Cut-off 1 < 38.70 47.2 (30.4 to 64.5) 85.7 (73.8 to 93.6) 70.7

Cut-off 2 < 69.60 94.4 (81.3 to 99.3) 25 (14.4 to 38.4) 52.2

BM3 Cut-off 1 < 12.50 91.7 (77.5 to 98.3) 42.9 (29.7 to 56.8) 62.0

Cut-off 2 < 13.90 94.4 (81.3 to 99.3) 28.6 (17.3 to 42.2) 54.4

BM4 Cut-off 1 > 12.20 36.1 (20.8 to 53.8) 91.1 (80.4 to 97) 69.6 56.5 (41 5 to

OO.J) Cut-off 2 > 5.64 94.4 (81.3 to 99.3) 0 (0 to 6.4) 37.0

BM5 Cut-off 1 > 13.90 44.4 (27.9 to 61.9) 91.1 (80.4 to 97) 72.8

Cut-off 2 > 8.45 94.4 (81.3 to 99.3) 23.2 (13 to 36.4) 51.1

BM6 Cut-off 1 > 20.00 41.7 (25.5 to 59.2) 87.5 (75.9 to 94.8) 69.6 ^tO

Cut-off 2 > 12.00 94.4 (81.3 to 99.3) 0 (0 to 6.4) 37.0

BM7 Cut-off 1 < 161.00 66.7 (49 to 81.4) 69.6 (55.9 to 81.2) 68.5 6 ⁹ .8H56/I to

Cut-off 2 < 207.00 94.4 (81.3 to 99.3) 14.3 (6.4 to 26.2) 45.7

BM8 Cut-off 1 < 12.70 75 (57.8 to 87.9) 69.6 (55.9 to 81.2) 71.7 ⁷ °’ ⁶ o _O nU ⁵ .ZZ;) ^{6 t} ° Cut-off 2 < 16.10 94.4 (81.3 to 99.3) 8.9 (3 to 19.6) 42.4

BM9 Cut-off 1 > 4.06 58.3 (40.8 to 74.5) 69.6 (55.9 to 81.2) 65.2

Cut-off 2 > 2.43 94.4 (81.3 to 99.3) 5.4 (1.1 to 14.9) 40.2

BM10 Cut-off 1 > 241.00 38.9 (23.1 to 56.5) 83.9 (71.7 to 92.4) 66.3 ⁵⁷ ' V?'? ^{5 L} °

Cut-off 2 > 106.00 94.4 (81.3 to 99.3) 10.7 (4 to 21.9) 43.5

BM11 Cut-off 1 < 0.27 77.8 (60.9 to 89.9) 62.5 (48.6 to 75.1) 68.5 65.7 052.3 to

Cut-off 2 < 0.36 94.4 (81.3 to 99.3) 10.7 (4 to 21.9) 43.5

BM12 Cut-off 1 > 0.59 80.6 (64 to 91.8) 46.4 (33 to 60.3) 59.8 65.1 (52.4 to

/ J)

Cut-off 2 > 0.53 94.4 (81.3 to 99.3) 25 (14.4 to 38.4) 52.2 ^b . Test biomarkers alone without adding into baseline.

Cut-off 1: cut-off value at the Youden index.

Cut-off 2: cut-off value at the 95% sensitivity. A.8.4 Phase III Model 3 Cut-off values for test biomarkers alone.

Standalone Cut-off _v . Sensitivity % (95% Specificity % (95% Accuracy \UC % (95% biomar ,kers _a values ^{Va ue} CI) CI) % CI)

BM1 Cut-off 1 < 11.00 44.7 (30.2 to 59.9) 81.8 (67.3 to 91.8) 62.6 ^to

Cut-off 2 < 18.80 95.7 (85.5 to 99.5) 18.2 (8.2 to 32.7) 58.2

BM2 Cut-off 1 < 38.70 42.6 (28.3 to 57.8) 86.4 (72.7 to 94.8) 63.7 55 (52.1 to 75)

Cut-off 2 < 78.40 95.7 (85.5 to 99.5) 18.2 (8.2 to 32.7) 58.2

BM3 Cut-off 1 < 9.68 59.6 (44.3 to 73.6) 77.3 (62.2 to 88.5) 68.1 68.5 (55.8 to

/o) Cut-off 2 < 17.00 95.7 (85.5 to 99.5) 15.9 (6.6 to 30.1) 57.1

BM4 Cut-off 1 > 12.20 40.4 (26.4 to 55.7) 100 (92 to 100) 69.2

Cut-off 2 > 5.64 95.7 (85.5 to 99.5) 0 (0 to 8) 49.5

BM5 Cut-off 1 > 11.70 57.5 (42.2 to 71.7) 84.1 (69.9 to 93.4) 70.3 ^{74 ?} <>t ⁶ ? ³ '°

82.9) Cut-off 2 > 8.45 95.7 (85.5 to 99.5) 29.6 (16.8 to 45.2) 63.7

BM6 Cut-off 1 > 20.00 44.7 (30.2 to 59.9) 95.5 (84.5 to 99.4) 69.2 Cut-off 2 > 12.00 95.7 (85.5 to 99.5) 0 (0 to 8) 49.5

BM7 Cut-off 1 < 142.00 55.3 (40.1 to 69.8) 90.9 (78.3 to 97.5) 72.5 ⁷¹ ' ⁷ _o ??; ^{7 t} °

81.1) Cut-off 2 < 232.00 95.7 (85.5 to 99.5) 2.3 (0.1 to 12) 50.6

BM8 Cut-off 1 < 12.70 68.1 (52.9 to 80.9) 72.7 (57.2 to 85) 70.3 ⁶⁷ ' ⁵ _? ( ⁵⁴ ' ^{6 tO}

Cut-off 2 < 18.30 95.7 (85.5 to 99.5) 4.6 (0.6 to 15.5) 51.7

BM9 Cut-off 1 > 3.83 63.8 (48.5 to 77.3) 68.2 (52.4 to 81.4) 65.9 62.7^(49.5 to

Cut-off 2 > 2.41 95.7 (85.5 to 99.5) 4.6 (0.6 to 15.5) 51.7

BM10 Cut-off 1 > 246.00 40.4 (26.4 to 55.7) 93.2 (81.3 to 98.6) 65.9 ^6L2 ₇ ⁽⁴⁷ ' ^{9 tO}

Cut-off 2 > 95.30 95.7 (85.5 to 99.5) 4.6 (0.6 to 15.5) 51.7

BM11 Cut-off 1 < 0.27 59.6 (44.3 to 73.6) 61.4 (45.5 to 75.6) 60.4 ⁵⁷ ' ⁷ oJ8 ⁴ .4 ⁴ )' ^{7 L<>}

Cut-off 2 < 0.36 95.7 (85.5 to 99.5) 13.6 (5.2 to 27.4) 56.0

BM12 Cut-off 1 > 0.59 78.7 (64.3 to 89.3) 50 (34.6 to 65.4) 64.8

Cut-off 2 > 0.42 95.7 (85.5 to 99.5) 9.1 (2.5 to 21.7) 53.9 b. Test biomarkers alone without adding into baseline.

Cut-off 1: cut-off value at the Youden index.

Cut-off 2: cut-off value at the 95% sensitivity.

[00154] 3.6. Results of Phase IV

[00155] Phase III identified CDllc, CD59, CD91 and CD163 as potential biomarkers to differentiate individuals who are PET Ap positive (Cl >25) versus those who are PET Ap negative (CL <25) after correction for age, gender, education and APOE s 4 allele status (Model 1, P = 0.021). Applicants then validated them in an independent cohort (Phase IV, N = 112). Several leukocyte markers showed connection to clinical diagnosis or association with Centiloids, which are consistent with Phase III. A multivariable model for the validation cohort found that a combination of CD59 & CD91 discriminated Ap status, CL >25 or CL <25, with an AUC of 0.91 (CI: 0.81 to 0.99). The AUC was 0.1 higher than for the mean base model AUC at 0.81 (CI: 0.72 to 0.9) and after correction for age, gender, education and APOE a 4 allele status (p = 0.072). A combination of CDl lc, CD59 & CD91 was tried, had an AUC after correction at 0.95 (CI: 0.86 to 1), 0.14 above base model, p = 0.05, although this value could reflect an overfitting due to the relatively small sample size (Detailed in Table 3.5).

[00156] 4. DISCUSSION:

[00157] Leukocyte surface markers exhibited differential expression when compared among groups of CN, MCI and AD, and/or associated with AP-PET data. The design of Phases I and II was maximal in variety of major CD markers that constrained its screening capacity. The new scope of leukocyte surface markers in Phase III was adjusted to reflecting the GWAS and other evidence that phagocytosis/endocytosis in the microglial/monocyte pathways may play a key role in AD pathogenesis [7, 21]. Although the exact molecular mechanism remains unclear, decreased expression of phagocytic receptors, MerTK and P2X7; scavenger receptors, CD36 and CD163; and integrins, CDl lb, CDl lc and CD18, accords closely with certain of the present inventors’ previous work [7] that infers an impaired monocyte phagocytic function in the AD patients (Table 1; Figure 1 Left). It is also notable that CD 18 is a common subunit of CDl lb/CD18 and CDl lc/CD18, which are also known as complement receptor 3 and 4 (CR3, CR4). Both CR3 and CR4 have been found to be down-regulated in AD by this study. CD59 is a main inhibitor of the membrane attack complex (MAC) [22]. What Applicants found the upregulation of CD59 in AD may prevent cell lysis and phagocytic clearance. See Figure 1 Right side. Orchestrated changes in two or more pathways represent a functional inhibition in AD associated with opsonisation, phagocytosis and clearance of pathogenic factors.

[00158] In this study, three markers, CD33, CD35/CR1 and CD91, are protein products of AD risk genes reported by GWAS meta-analysis [23, 24] or by other genetic association studies [25-27]. Both CD35 and CD91 were found up-regulated in AD by this study. Bradshaw et al. [28] found that the CD33 risk allele, rs3865444C, was associated with greater cell surface expression of CD33 in the monocytes of young and older individuals; however, it was associated with diminished internalization of A[342, accumulation of neuritic tau-positive change and increased numbers of activated human microglia. Overexpression of CD33 on monocytes in individuals carrying the rs3865444C risk genotype may be a mechanism to compensate its loss of function. CD35/CR1 is a receptor for complement factors, C3b and C4b [29], but its relevance to AD is unknown. CD91 is a receptor for ApoE, and the endocytosis and degradation of secreted amyloid precursor protein (APP) [30], suggesting that a single pathway links two molecules implicated in the pathophysiology of AD.

[00159] Besides CD91, RAGE is also a receptor for A|3. Yan et al. [31] reported that expression of RAGE increased in AD, particularly in neurons close to deposits of A[3 and to neurofibrillary tangles. In mice, Deane et al. [32] showed that RAGE mediated the transport of human A[34O and A[342 across the blood-brain barrier and resulted in the expression of proinflammatory cytokines and endothelin-1. Arancio et al. [33] showed that RAGE is a cofactor for AP-induced neuronal perturbation and is involved in microglial cell activation in transgenic mice. In the current study, expression of RAGE was increased in AD, consistent with Yan et al. [31] and it may provide a mirror to what happens in brain-resident microglia. CD36 was found to be down-regulated in AD in this study. CD36 is a receptor for oxidized low density lipoprotein (LDL) [34] and a receptor for Ap [35]. Immobilized Ap oligomers bound CD36-Fc protein showed comparable affinity to that of RAGE-Fc and TREM2-Fc [36].

[00160] Applicants’ data from blood grouping suggest there may be abnormalities in percentages of lymphocytes and monocytic subsets. Hematopoietic aging is characterized by expansion of hematopoietic stem cells (HSCs) with impaired function, that more myeloid and fewer lymphoid daughter cell are generated, which is termed as myeloid-biased haematopoiesis [37, 38]. The percentage of each type of leukocyte in the blood is largely determined by differences in stem cell activity and hence, decreased lymphocytes might be due to this myelopoiesis [39]. However, such differences do not explain the observed patterns of other monocytic subsets altered in opposite direction. It is possible that CD14+CD16+ monocytes (a highly phagocytic cells resembling tissue macrophages) exudate into sites of Ap plaques in the brain, leading to a transient decline in MCI and an incomplete recovery in AD, most likely due to failure in chemotactic function or obliterated passage, representing a U-shaped response. It is apparent from the patterns of CD14-CD16+ monocytes that these two minor monocytic subsets had similar responses, whereas the CD14-CD16+ monocytes had lesser recovery in AD. Either percentage of CD14+CD16+ or percentage of CD14-CD16+ was correlated with none of CL, EM or PACC, due to this U- shaped response. It is interesting to note that percentage of monocytes in the blood did not change, possibly due to this subset inconsistency.

[00161] Applicants and others have found that a significant percentage (30%) of CN individuals show high Ap PET and low Ap42 levels in the CSF, these people are now classified as preclinical AD. Mild Cognitive Impairment (MCI) is considered a prodromal phase of AD, with 40-60% of people meeting criteria for MCI eventually progressing to clinical AD, or about 5-25% per vear [401. Diaenostic decision making is a two-class prediction problem; hence Applicants established a binary classifier, i.e. health and disease, and the classifier boundary was determined by the logistic model. Unfortunately, diagnosis of AD is based a multidimensional criteria, including clinical considerations, cognition, behaviour, advanced imaging and CSF biomarkers, moreover, there is neither specific criterion for early detection, nor is a clear definition for preclinical stages, and, thus, Applicants used CL as a simple surrogate for identifying stages in the model 1 and 2, however, Applicants further added clinical classifications into the model 3 to reflecting the latest movements made by NIA-AA [13] and IWG [41].

[00162] Applicants’ findings may have implications for early diagnosis for individuals at increased risk from genetic factor (e.g. APOE) to watch for preclinical signs of AD without obvious changes in neuroimaging or CSF biomarkers and to initiate early intervention. The use of plasma biomarkers for AD has been demonstrated recently by Nakamura et al. and Fossati et al. [15, 42], therefore, justify the reason using blood based biomarkers for AD diagnosis, and Applicants’ novel findings could attribute to, if connected, AD proteinopathies in the blood. The role of the altered leukocyte biomarkers or blood grouping or transcriptional networks such as PU.l [21, 43] in modulating these processes remains to be determined but may contribute to molecular pathology of AD.

[00163] 4.7. In conclusion, Applicants’ results suggest that leukocyte biomarkers are objective, measurable indicator of AD progression and could be valuable for early diagnosis. Furthermore, leukocyte biomarkers are superior in terms of cost and simplicity and can be easily employed in the clinic, confirming the importance of leukocyte biomarkers for development of novel efficacious drug as well as outcome measures for all phases of clinical trials.

[00164] References:

[1] 2020 Alzheimer's disease facts and figures. Alzheimers Dement. 2020.

[2] Scheltens P, Blennow K, Breteler MMB, de Strooper B, Frisoni GB, Salloway S, et al. Alzheimer's disease. The Lancet. 2016;388:505-17.

[3] Wang J, Gu BJ, Masters CL, Wang YJ. A systemic view of Alzheimer disease - insights from amyloid-beta metabolism beyond the brain. Nat Rev Neurol. 2017;13:612-23.

[4] Coraci IS, Husemann J, Berman JW, Hulette C, Dufour JH, Campanella GK, et al. CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta- amyloid fibrils. Am J Pathol. 2002;160:101-12.

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Clauses

[00165] Clause 1. A composition comprising: a binding agent for CD163 conjugated with a detectable moiety.

[00166] Clause 2. The composition of clause 1, wherein the binding agent for CD163 is chosen from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[00167] Clause 3. The composition of any one of clauses 1-2, wherein the detectable moiety is chosen from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[00168] Clause 4. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a fluorophore; and ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing.

[00169] Clause 5. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a fluorophore; ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing; and whole blood of a human patient.

[00170] Clause 6. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a fluorophore; and at least one anti-coagulant.

[00171] Clause 7. The composition of clause 6, wherein the at least one anti-coagulant is chosen from ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing.

[00172] Clause 8. The composition of any one of clauses 4-7, wherein the composition does not contain heparin.

[00173] Clause 9. The composition of any one of clauses 4-8, wherein the composition does not contain allophycocyanin.

[00174] Clause 10. The composition of any one of clauses 4-9, further comprising bovine serum albumin and sodium azide.

[00175] Clause 11. The composition of any one of clauses 4-10, wherein the fluorophore is chosen from fluorescein isothiocyanate, allophycocyanin, or a combination thereof.

[00176] Clause 12. The composition of any one of clauses 4-10, wherein the fluorophore is chosen from: fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR); fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR): BUV395: BUV563; BUV615; BUV661; BUV737; BUV805; BV421; BV480; BV510; BV605; BV650; BV711; BV750; BV786; FITC; PE; PE- CF594; PerCP-Cy™5.5; R718; or a combination of two or more thereof.

[00177] Clause 13. The composition of any one of clauses 4-10, wherein the fluorophore is chosen from: fluorescein, rhodamine, lanthanide phosphors, and their derivatives thereof. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine, tetramethylrhodamine, and sulforhodamine.

[00178] Clause 14. The composition of any one of clauses 4-10, wherein the fluorophore is chosen from fluorescent proteins such as GFP, YFP, RFP, eGFP, mCherry, tdtomato, FITC, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750, Pacific Blue, Coumarin, BODIPY FE, Pacific Green, Oregon Green, Cy3, Cy5, Pacific Orange, TRITC, Texas Red, R-Phycoerythrin, Allophycocyanin, or other fluorophores known in the art.

[00179] Clause 15. The composition of any one of clauses 4-14, wherein the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00180] Clause 16. The composition of any one of clauses 4-15, wherein the composition does not contain heparin.

[00181] Clause 17. The composition of any one of clauses 1-16, wherein the composition further comprises a second binding agent for for one or more of scavenger receptors and their regulators conjugated with a second detectable moiety.

[00182] Clause 18. The composition of clause 17, wherein the second binding agent is chosen from a single chain variable fragment, an antibody mimetic, an antibody fragment, an antibody, a monoclonal antibody, and combinations thereof.

[00183] Clause 19. The composition of any one of clauses 17-18, wherein the second detectable moiety is chosen from a radioisotope, a stable isotope, a fluorophore, and combinations thereof.

[00184] Clause 20. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a fluorophore to obtain bound CD 163: measuring the concentration of bound CD 163 in the blood sample by fluorescence of the fluorophore; comparing the concentration with a concentration range of CD 163 in healthy humans; observing a decreased concentration of CD 163 in the blood sample; thereby diagnosing AD in the human patient.

[00185] Clause 21. The method of clause 20, wherein the measuring the concentration of bound CD 163 comprises measuring the mean fluorescence intensity of bound CD 163 on leukocytes.

[00186] Clause 22. The method of any one of clauses 20-21, wherein the comparing the concentration comprises comparing the mean fluorescence intensity of bound CD 163 for the human patient with the mean fluorescence intensity of bound CD 163 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00187] Clause 23. The method of any one of clauses 20-22, wherein the observing a decreased concentration of CD 163 in the blood sample comprises measuring a decrease in mean fluorescence intensity of at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, or at least about 19% compared to a mean fluorescence intensity of bound CD163 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00188] Clause 24. The method of any one of clauses 20-23, wherein the mouse antihuman IgG monoclonal antibody for CD 163 conjugated with a fluorophore comprises mouse anti-human CD 163 antigen conjugated with fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00189] Clause 25. A method for determining the efficacy of an AD therapeutic candidate in a human patient, comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a fluorophore to obtain first bound CD 163; measuring a first concentration of first bound CD 163 in the first blood sample by fluorescence of the fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with the mouse anti-human IgG monoclonal antibody for CD 163 conjugated with the fluorophore to obtain second bound CD 163; measuring a second concentration of second bound CD 163 in the second blood sample by fluorescence of the fluorophore;

(D) observing increase from the first concentration of first bound CD 163 to the second concentration of second bound CD 163;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[00190] Clause 26. The method of clause 25, wherein the AD therapeutic candidate is a phagocytosis-promoting agent.

[00191] Clause 27. The method of clause 25, wherein the AD therapeutic candidate is a scavenger receptor agonist.

[00192] Clause 28. The method of any one of clauses 25-27, wherein the measuring the first concentration comprises measuring the first concentration on leukocytes, and the measuring the second concentration comprises measuring the second concentration on leukocytes.

[00193] Clause 29. A composition comprising a binding agent for CD91 conjugated with a detectable moiety.

[00194] Clause 30. The composition of clause 29, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00195] Clause 31. The composition of any one of clauses 29-30, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00196] Clause 32. A use of a binding agent for CD163 for the diagnosis of AD in a human patient in need thereof.

[00197] Clause 33. The use of clause 32, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00198] Clause 34. The use of any one of clauses 32-33, wherein the binding agent is conjugated with a detectable moiety.

[00199] Clause 35. The use of clause 34, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof. [00200] Clause 36. A binding agent for CD163 for use in the diagnosis of AD in a human patient in need thereof.

[00201] Clause 37. The binding agent for CD163 of clause 36, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00202] Clause 38. The binding agent for CD163 of any one of clauses 36-37, wherein the binding agent is conjugated with a detectable moiety.

[00203] Clause 39. The binding agent for CD163 of clause 38, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00204] Clause 40. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for CD91 conjugated with a fluorophore to obtain bound CD91; measuring the concentration of bound CD91 in the blood sample by fluorescence of the fluorophore; comparing the concentration with a concentration range of CD91 in healthy humans; observing an increased concentration of CD91 in the blood sample; thereby diagnosing AD in the human patient.

[00205] Clause 41. The method of clause 40, wherein the measuring the concentration of bound CD91 comprises measuring the mean fluorescence intensity of bound CD91 on leukocytes.

[00206] Clause 42. The method of any one of clauses 40-41, wherein the comparing the concentration comprises comparing the mean fluorescence intensity of bound CD91 for the human patient with the mean fluorescence intensity of bound CD91 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00207] Clause 43. The method of any one of clauses 40-42, wherein the observing an increased concentration of CD91 in the blood sample comprises measuring an increase in mean fluorescence intensity of at least about 44%, at least about 50%, at least about 55%, or at least about 59% compared to a mean fluorescence intensity of bound CD91 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00208] Clause 44. A method for determining the efficacy of an AD therapeutic candidate in a human patient, comprising:

(A) obtaining a first blood sample from the human oatient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a mouse anti-human IgG monoclonal antibody for CD91 conjugated with a fluorophore to obtain first bound CD91; measuring a first concentration of first bound CD91 in the first blood sample by fluorescence of the fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with the mouse anti-human IgG monoclonal antibody for CD91 conjugated with the fluorophore to obtain second bound CD91; measuring a second concentration of second bound CD91 in the second blood sample by fluorescence of the fluorophore;

(D) observing a decrease from the first concentration of first bound CD91 to the second concentration of second bound CD91;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[00209] Clause 45. The method of clause 44, wherein the AD therapeutic candidate is a phagocytosis-promoting agent.

[00210] Clause 46. The method of clause 44, wherein the AD therapeutic candidate is a scavenger receptor agonist.

[00211] Clause 47. The method of any one of clauses 44-46, wherein the measuring the first concentration comprises measuring the first concentration on leukocytes, and the measuring the second concentration comprises measuring the second concentration on leukocytes.

[00212] Clause 48. A use of a binding agent for CD91 for the diagnosis of AD in a human patient in need thereof.

[00213] Clause 49. The use of clause 48, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00214] Clause 50. The use of any one of clauses 48-49, wherein the binding agent is conjugated with a detectable moietv. [00215] Clause 51. The use of clause 50, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00216] Clause 52. A binding agent for CD91 for use in the diagnosis of AD in a human patient in need thereof.

[00217] Clause 53. The binding agent for CD91 of clause 52, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00218] Clause 54. The binding agent for CD91 of any one of clauses 51-53, wherein the binding agent is conjugated with a detectable moiety.

[00219] Clause 55. The binding agent for CD91 of clause 54, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00220] Clause 56. A composition comprising a binding agent for MerTK conjugated with a detectable moiety.

[00221] Clause 57. The composition of clause 56, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00222] Clause 58. The composition of any one of clauses 56-57, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00223] Clause 59. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for MerTK conjugated with a fluorophore to obtain bound MerTK; measuring the concentration of bound MerTK in the blood sample by fluorescence of the fluorophore; comparing the concentration with a concentration range of MerTK in healthy humans; observing a decreased concentration of MerTK in the blood sample; thereby diagnosing AD in the human patient.

[00224] Clause 60. The method of clause 59, wherein the measuring the concentration of bound MerTK comprises measuring the mean fluorescence intensity of bound MerTK on leukocytes.

[00225] Clause 61. The method of any one of clauses 59-60, wherein the comparing the concentration comprises comparine the mean fluorescence intensity of bound MerTK for the human patient with the mean fluorescence intensity of bound MerTK for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00226] Clause 62. The method of any one of clauses 59-61, wherein the observing a decreased concentration of MerTK in the blood sample comprises measuring a decrease in mean fluorescence intensity of at least about 12% compared to a mean fluorescence intensity of bound MerTK for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00227] Clause 63. A method for determining the efficacy of an AD therapeutic candidate in a human patient, comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a mouse anti-human IgG monoclonal antibody for MerTK conjugated with a fluorophore to obtain first bound MerTK; measuring a first concentration of first bound MerTK in the first blood sample by fluorescence of the fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with the mouse anti-human IgG monoclonal antibody for MerTK conjugated with the fluorophore to obtain second bound MerTK; measuring a second concentration of second bound MerTK in the second blood sample by fluorescence of the fluorophore;

(D) observing an increase from the first concentration of first bound MerTK to the second concentration of second bound MerTK;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[00228] Clause 64. The method of clause 63, wherein the AD therapeutic candidate is a phagocytosis-promoting agent.

[00229] Clause 65. The method of clause 63, wherein the AD therapeutic candidate is a scavenger receptor agonist. [00230] Clause 66. The method of any one of clauses 63-65, wherein the measuring the first concentration comprises measuring the first concentration on leukocytes, and the measuring the second concentration comprises measuring the second concentration on leukocytes.

[00231] Clause 67. A use of a binding agent for MerTK for the diagnosis of AD in a human patient in need thereof.

[00232] Clause 68. The use of clause 67, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00233] Clause 69. The use of any one of clauses 67-68, wherein the binding agent is conjugated with a detectable moiety.

[00234] Clause 70. The use of clause 69, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00235] Clause 71. A binding agent for MerTK for use in the diagnosis of AD in a human patient in need thereof.

[00236] Clause 72. The binding agent for MerTK of clause 71, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00237] Clause 73. The binding agent for MerTK of any one of clauses 84-85, wherein the binding agent is conjugated with a detectable moiety.

[00238] Clause 74. The binding agent for MerTK of clause 73, wherein the detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00239] Clause 75. A composition comprising: a first binding agent for CD59 conjugated with a first detectable moiety; and a second binding agent for CD163 conjugated with a second detectable moiety.

[00240] Clause 76. The composition of clause 75, wherein the first binding agent and second binding agent are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00241] Clause 77. The composition of any one of clauses 75-76, wherein the first detectable moiety, second detectable moiety, and third detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00242] Clause 78. A composition comprising: a monoclonal antibody for CD59 conjugated with a first fluorophore; and a monoclonal antibody for CD 163 coniueated with a second fluorophore. [00243] Clause 79. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00244] Clause 80. The composition of any one of clauses 78-79, further comprising at least one anticoagulant.

[00245] Clause 81. The composition of any one of clauses78-80, wherein the at least one anticoagulant comprises ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing.

[00246] Clause 82. The composition of any one of clauses 78-81, wherein the composition does not contain heparin.

[00247] Clause 83. The composition of any one of clauses 78-82, wherein the composition does not contain allophycocyanin.

[00248] Clause 84. The composition of any one of clause 78-83, wherein the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00249] Clause 85. The composition of any one of clauses 78-84, further comprising bovine serum albumin and sodium azide.

[00250] Clause 86. The composition of any one of clauses 78-85, wherein the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00251] Clause 87. The composition of any one of clauses 78-86, wherein the first fluorophore is R-phycoerythrin.

[00252] Clause 88. The composition of any one of clauses 78-87, wherein the second fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00253] Clause 89. The composition of any one of clauses 78-88, further comprising whole blood from a human patient.

[00254] Clause 90. A kit comprising: a first binding agent for CD59 conjugated with a first detectable moiety; and a second binding agent for CD163 conjugated with a second detectable moiety.

[00255] Clause 91. The kit of clause 90, wherein the first binding agent and second binding agent, are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof. [00256] Clause 92. The kit of any one of clauses 90-91, wherein the first detectable moiety and second detectable moiety, are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00257] Clause 93. A kit comprising: a monoclonal antibody for CD59 conjugated with a first fluorophore; and a monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00258] Clause 94. A kit comprising: a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00259] Clause 95. The kit of any one of clauses 93-94, wherein the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00260] Clause 96. The kit of any one of clauses 93-95, wherein the first fluorophore is R-phy coery thrin .

[00261] Clause 97. The kit of any one of clauses 93-96, wherein the second fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00262] Clause 98. A method of measuring relative expression of CD59 and CD163 in a human patient, comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a first binding agent for CD59 conjugated with a first detectable moiety; and a second binding agent for CD 163 conjugated with a second detectable moiety, to form bound CD59, and bound CD 163; measuring the concentration of the first detectable moiety and the second detectable moiety, thereby determining the relative expression of CD59 and CD163.

[00263] Clause 99. The method of clause 98, wherein the measuring the concentration comprises measuring the concentration on leukocytes.

[00264] Clause 100. A method of measuring relative expression of CD59 and CD163 in a human patient, comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a monoclonal antibody for CD59 conjugated with a first fluorophore; and a monoclonal antibody for CD 163 conjugated with a second fluorophore, to form bound CD59 and bound CD 163; measuring fluorescence of the first fluorophore and the second fluorophore, thereby determining the relative expression of CD59 and CD163.

[00265] Clause 101. A method of measuring relative expression of CD59 and CD163 in a human patient, comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore, to form bound CD59 and bound CD 163; measuring fluorescence of the first fluorophore and the second fluorophore, thereby determining the relative expression of CD59 and CD163.

[00266] Clause 102. The methods of any one of clauses 100-101, wherein the relative expression of CD59 and CD163 is the relative expression of CD59 and CD163 on leukocytes.

[00267] Clause 103. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore to obtain bound CD59; a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore to obtain bound CD 163; measuring a concentration of bound CD59 and a concentration of bound CD 163 in the blood sample by fluorescence of the first fluorophore and the second fluorophore; observing an increase in the concentration of bound CD59 relative to a concentration range of CD59 in healthy humans; observing a decrease in the concentration of bound CD 163 relative to a concentration range of CD 163 in healthy humans; thereby diagnosing AD in the human patient.

[00268] Clause 104. The method of clause 103, wherein the measuring the concentration comprises measuring the concentration on leukocytes. [00269] Clause 105. The method of any one of clauses 103-104, wherein the measuring the concentration of bound CD59 comprises measuring the mean fluorescence intensity of bound CD59.

[00270] Clause 106. The method of any one of clauses 103-105, wherein the measuring the concentration of bound CD 163 comprises measuring the mean fluorescence intensity of bound CD 163.

[00271] Clause 107. The method of any one of clauses 103-106, wherein the observing an increase in the concentration of bound CD59 comprises comparing a mean fluorescence intensity of bound CD59 for the human patient with a mean fluorescence intensity of bound CD59 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00272] Clause 108. The method of clause 107, wherein the observing an increase in the concentration of bound CD59 comprises measuring an increase in mean fluorescence intensity of at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 78% compared to a mean fluorescence intensity of bound CD59 for the pool of human patients who have <15CL as measured by amyloid PET imaging.

[00273] Clause 109. The method of any one of clauses 103-108, wherein the observing a decrease in the concentration of bound CD 163 comprises comparing a mean fluorescence intensity of bound CD 163 for the human patient with a mean fluorescence intensity of bound CD 163 for a pool of human participants who have <25CL as measured by amyloid PET imaging.

[00274] Clause 110. The method of clause 109, wherein the observing a decrease in the concentration of bound CD 163 comprises measuring a decrease in mean fluorescence intensity of at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, or at least about 19% compared to a mean fluorescence intensity of bound CD 163 for the pool of human patients who have <15CL as measured by amyloid PET imaging.

[00275] Clause 111. A method for determining the efficacy of an AD therapeutic candidate in a human patient, comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore to obtain first bound CD59; a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore to obtain first bound CD 163; measuring a first concentration of first bound CD59 and a first concentration of first bound CD 163 in the first blood sample by fluorescence of the first fluorophore and second fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with the mouse anti-human IgG monoclonal antibody for CD59 conjugated with the first fluorophore to obtain second bound CD59; the mouse anti-human IgG monoclonal antibody for CD 163 conjugated with the second fluorophore to obtain second bound CD 163; measuring a second concentration of second bound CD59 and a second concentration of second bound CD 163 in the second blood sample by fluorescence of the first fluorophore and second fluorophore;

(D) observing a decrease from the first concentration of first bound CD59 to the second concentration of second bound CD59 in the human patient; observing an increase from the first concentration of first bound CD 163 to the second concentration of second bound CD 163;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[00276] Clause 112. The method of clause 111, wherein the AD therapeutic candidate is a phagocytosis-promoting agent.

[00277] Clause 113. The method of clause 111, wherein the AD therapeutic candidate is a scavenger receptor agonist.

[00278] Clause 114. The method of any one of clauses 111-113, wherein the measuring the first concentration comprises measuring the first concentration on leukocytes, and the measuring the second concentration comprises measuring the second concentration on leukocytes.

[00279] Clause 115. A use of a binding agent for CD59 and a binding agent for CD163 for the diagnosis of AD in a human patient in need thereof. [00280] Clause 116. The use of clause 115, wherein the binding agents are chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00281] Clause 117. The use of any one of clauses 115-116, wherein the binding agent for CD59 is conjugated with a first detectable moiety, and the binding agent for CD163 is conjugated with a second detectable moiety.

[00282] Clause 118. The use of clause 117, wherein the first detectable moiety and the second detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00283] Clause 119. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for a scavenger receptor conjugated with a fluorophore to obtain bound scavenger receptor; measuring a concentration of bound scavenger receptor in the blood sample by fluorescence of the fluorophore; observing a decrease in the concentration of bound scavenger receptor relative to a concentration range of scavenger receptor in healthy humans; thereby diagnosing AD in the human patient.

[00284] Clause 120. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a plurality of antibody-fluorophore conjugates, each antibody-fluorophore conjugate in the plurality comprising a mouse anti-human IgG monoclonal antibody for a scavenger receptor conjugated with a fluorophore; thereby forming bound scavenger receptors; measuring concentrations of the bound scavenger receptors in the blood sample by fluorescence of the fluorophores; observing a decrease in the concentration of at least one bound scavenger receptor relative to a concentration range of a corresponding scavenger receptor in healthy humans; thereby diagnosing AD in the human patient. [00285] Clause 121. The method of any one of clauses 119-120, wherein the scavenger receptor is chosen from CD163, MerTK, CD18, and combinations thereof.

[00286] Clause 122. The method of any one of clauses 119-121, wherein the scavenger receptor is expressed on leukocytes.

[00287] Clause 123. A method for determining the efficacy of an AD therapeutic candidate in a human patient, comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a first aliquot of a plurality of antibody-fluorophore conjugates, each antibody-fluorophore conjugate in the plurality comprising a mouse anti-human IgG monoclonal antibody for a scavenger receptor conjugated with a fluorophore; thereby forming first bound scavenger receptors; measuring first concentrations of the first bound scavenger receptors in the first blood sample by fluorescence of the fluorophores;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with a second aliquot of the plurality of antibody-fluorophore conjugates; thereby forming second bound scavenger receptors; measuring second concentrations of the second bound scavenger receptors in the second blood sample by fluorescence of the fluorophores;

(D) observing an increase from the first concentration of first bound scavenger receptors to the second concentration of second bound scavenger receptors in the human patient;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[00288] Clause 124. The method of clause 123, wherein the AD therapeutic candidate is a phagocytosis-promoting agent.

[00289] Clause 125. The method of clause 123, wherein the AD therapeutic candidate is a scavenger receptor agonist. [00290] Clause 126. The method of any one of clauses 123-125, wherein the measuring first concentrations comprises measuring first concentrations on leukocytes, and the measuring second concentrations comprises measuring second concentrations on leukocytes. [00291] Clause 127. The method of any one of clauses 123-126, wherein the scavenger receptor is chosen from CD163, MerTK, CD18, and combinations thereof.

Clause 128. A composition comprising: a first binding agent for CD 18 conjugated with a first detectable moiety.

[00292] Clause 129. The composition of clause 128, wherein the first binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof. [00293] Clause 130. The composition of any one of clauses 128-129, wherein the first detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00294] Clause 131. A composition comprising: a monoclonal antibody for CD 18 conjugated with a first fluorophore.

[00295] Clause 132. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD 18 conjugated with a first fluorophore. [00296] Clause 133. The composition of any one of clauses 131-132, further comprising whole blood from a human patient.

[00297] Clause 134. A kit comprising: a first binding agent for CD 18 conjugated with a first detectable moiety.

[00298] Clause 135. The kit of clause 134, wherein the first binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof. [00299] Clause 136. The kit of any one of clauses 134-135, wherein the first detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00300] Clause 137. A kit comprising: a monoclonal antibody for CD 18 conjugated with a first fluorophore.

[00301] Clause 138. A kit comprising: a mouse anti-human IgG monoclonal antibody for CD 18 conjugated with a first fluorophore. [00302] Clause 139. A method of measuring relative expression of CD18 in a human patient, comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a monoclonal antibody for CD 18 coniueated with a first fluorophore, to form bound CD 18; measuring fluorescence of the first fluorophore, thereby determining the relative expression of CD18.

[00303] Clause 140. A method of measuring relative expression of CD 18 in a human patient, comprising: obtaining a sample of whole blood from the human patient; contacting the sample with a mouse anti-human IgG monoclonal antibody for CD 18 conjugated with a first fluorophore, to form bound CD 18; measuring fluorescence of the first fluorophore, thereby determining the relative expression of CD18.

[00304] Clause 141. The methods of any one of clauses 139-140, wherein the relative expression of CD 18 is the relative expression of CD 18 on leukocytes.

[00305] Clause 142. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for CD 18 conjugated with a first fluorophore to obtain bound CD 18; measuring a concentration of bound CD 18 in the blood sample by fluorescence of the first fluorophore; observing a decrease in the concentration of bound CD 18 relative to a concentration range of CD 18 in healthy humans; thereby diagnosing AD in the human patient.

[00306] Clause 143. The method of clause 142, wherein the measuring the concentration comprises measuring the concentration on leukocytes.

[00307] Clause 144. A use of a binding agent for CD18 for the diagnosis of AD in a human patient in need thereof.

[00308] Clause 145. The use of clause 144, wherein the binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00309] Clause 146. The use of any one of clauses 144-145, wherein the binding agent for CD 18 is conjugated with a first detectable moiety.

[00310] Clause 147. The use of clause 146, wherein the first detectable moiety is chosen from radioisotopes, stable isotopes, fluoroohores. and combinations thereof. [00311] Clause 148. A method for determining the efficacy of an AD therapeutic candidate in a human patient, comprising:

(A) obtaining a first blood sample from the human patient; optionally storing the first blood sample in ice; optionally testing the first blood sample within three hours of obtaining the first blood sample; reacting the first blood sample with a mouse anti-human IgG monoclonal antibody for CD 18 conjugated with a first fluorophore to obtain first bound CD 18; measuring a first concentration of first bound CD 18 in the first blood sample by fluorescence of the first fluorophore;

(B) administering the AD therapeutic candidate to the human patient;

(C) obtaining a second blood sample from the human patient; optionally storing the second blood sample in ice; optionally testing the second blood sample within three hours of obtaining the second blood sample; reacting the second blood sample with the mouse anti-human IgG monoclonal antibody for CD 18 conjugated with the first fluorophore to obtain second bound CD 18; measuring a second concentration of second bound CD 18 in the second blood sample by fluorescence of the first fluorophore;

(D) o observing an increase from the first concentration of first bound CD 18 to the second concentration of second bound CD 18;

(E) thereby determining the efficacy of the AD therapeutic candidate in the human patient.

[00312] Clause 149. The method of clause 148, wherein the AD therapeutic candidate is a phagocytosis-promoting agent.

[00313] Clause 150. The method of clause 148, wherein the AD therapeutic candidate is a scavenger receptor agonist.

[00314] Clause 151. The method of any one of clauses 148-150, wherein the measuring the first concentrations comprises measuring the first concentrations on leukocytes, and the measuring the second concentrations comprises measuring the second concentrations on leukocytes.

[00315] Clause 152. A composition comprising: one or more first binding agents for one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE conjugated with one or more first detectable moieties; and one or more second binding agents for one or more of CD 18, CD 163, and MerTK conjugated with one or more second detectable moieties.

[00316] Clause 153. A composition comprising: one or more of monoclonal antibodies for one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE conjugated with one or more first fluorophores; and one or more monoclonal antibodies for one or more of CD 18, CD 163, and MerTK conjugated with one or more second fluorophores.

[00317] Clause 154. The composition of any one of clauses 152-153, further comprising at least one anticoagulant.

[00318] Clause 155. The composition of any one of clauses 152-154, further comprising whole blood from a human patient.

[00319] Clause 156. A kit comprising: one or more first binding agents for one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE conjugated with one or more first detectable moieties; and one or more second binding agents for one or more of CD 18, CD 163, and MerTK conjugated with one or more second detectable moieties.

[00320] Clause 157. A kit comprising: one or more of monoclonal antibodies for one or more of CD33, CD35, CD36, CD59, CD91, P2X7, and RAGE conjugated with one or more first fluorophores; and one or more monoclonal antibodies for one or more of CD 18, CD 163, and MerTK conjugated with one or more second fluorophores.

[00321] Clause 158. A composition comprising: one or more of monoclonal antibodies for one or more of scavenger receptors and their regulators CD163, CD91, P2X7, MerTK CD59, CD18 conjugated with different fluorophores, and the fluorophores have different but compatible emission and excitation spectrum in multiplecolour flow cytometry analysis.

[00322] Clause 159. A composition comprising: one or more first binding agents for one or more of scavenger receptors and their regulators CD163, CD91, P2X7, MerTK CD59, CD18 conjugated with different first detectable moieties.

[00323] Clause 160. A kit comprising: one or more of monoclonal antibodies for one or more of CD 18 and CD59 conjugated with one or more first fluorophores.

[00324] Clause 161. A kit comprising: one or more first binding agents for one or more of, CD 18 and CD59, conjugated with one or more first detectable moieties.

[00325] Clause 162. A composition comprising: one or more of monoclonal antibodies for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first fluorophores.

[00326] Clause 163. A composition comprising: one or more first binding agents for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first detectable moieties.

[00327] Clause 164. A kit comprising: one or more of monoclonal antibodies for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first fluorophores.

[00328] Clause 165. A kit comprising: one or more first binding agents for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first detectable moieties.

[00329] Clause 166. A composition comprising: one or more of monoclonal antibodies for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first fluorophores; one or more of monoclonal antibodies for one or more of CD 18 and CD59 conjugated with one or more second fluorophores.

[00330] Clause 167. A composition comprising: one or more first binding agents for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first detectable moieties; one or more of second binding agents for one or more of CD 18 and CD59 conjugated with one or more second detectable moieties.

[00331] Clause 168. A kit comprising: one or more of monoclonal antibodies for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first fluorophores; one or more of monoclonal antibodies for one or more of CD 18 and CD59, conjugated with one or more second fluorophores.

[00332] Clause 169. A kit comprising: one or more first binding agents for one or more of CD91, CD 163, P2X7, and MerTK conjugated with one or more first detectable moieties; one or more of second binding agents for one or more of CD 18 and CD59 conjugated with one or more second detectable moieties.

[00333] Clause 170. A kit comprising: a first binding agent for one or more of CD 163. CD91. and MerTK, conjugated with a first detectable moiety.

[00334] Clause 171. The kit of clause 170, wherein the wherein the first binding agent is chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00335] Clause 172. The kit of any one of clauses 170-171, wherein the first detectable moiety is chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00336] Clause 173. A composition comprising: a first binding agent for CD59 conjugated with a first detectable moiety; and a second binding agent for CD163 conjugated with a second detectable moiety.

[00337] Clause 174. The composition of clause 173, wherein the first binding agent and second binding agent are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00338] Clause 175. The composition of any one of clauses 173-174, wherein the first detectable moiety and second detectable moiety are chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00339] Clause 176. A composition comprising: a monoclonal antibody for CD59 conjugated with a first fluorophore; and a monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00340] Clause 177. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00341] Clause 178. The composition of any one of clauses 176-177, further comprising at least one anticoagulant.

[00342] Clause 179. The composition of any one of clauses 176-178, wherein the at least one anticoagulant comprises ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing.

[00343] Clause 180. The composition of any one of clausesl76-179, further comprising bovine serum albumin and sodium azide.

[00344] Clause 181. The composition of any one of clauses 176-180, further comprising phosphate buffered saline, gelatin, and sodium azide. [00345] Clause 182. The composition of any one of clauses 176-181, wherein the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00346] Clause 183. The composition of any one of clauses 176-182, wherein the first fluorophore is R-phycoerythrin.

[00347] Clause 184. The composition of any one of clauses 176-183, wherein the second fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00348] Clause 185. The composition of any one of clauses 176-184, further comprising whole blood from a human patient.

[00349] Clause 186. A kit comprising: a first binding agent for CD59 conjugated with a first detectable moiety; and a second binding agent for CD163 conjugated with a second detectable moiety.

[00350] Clause 187. The kit of clause 186, wherein the first binding agent and second binding agent are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00351] Clause 188. The kit of any one of clauses 186-187, wherein the first detectable moiety and second detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00352] Clause 189. A kit comprising: a monoclonal antibody for CD59 conjugated with a first fluorophore; and a monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00353] Clause 190. A kit comprising: a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a first fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a second fluorophore.

[00354] Clause 191. The kit of any one of clauses 189-190, wherein the kit does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00355] Clause 192. The kit of any one of clauses 189- 191, wherein the first fluorophore is R-phycoerythrin.

[00356] Clause 193. The kit of any one of clauses 189-192, wherein the second fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR). [00357] Clause 194. A use of a binding agent for CD59 and a binding agent for CD163 for the diagnosis of AD in a human patient in need thereof.

[00358] Clause 195. The use of clause 194, wherein the binding agents are chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00359] Clause 196. The use of any one of clauses 194-195, wherein the binding agent for CD59 is conjugated with a first detectable moiety, and the binding agent for CD163 is conjugated with a second detectable moiety.

[00360] Clause 197. The use of clause 196, wherein the first detectable moiety and the second detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00361] Clause 198. A composition comprising: a first binding agent for CD91 conjugated with a first detectable moiety; a second binding agent for CD59 conjugated with a second detectable moiety; and a third binding agent for CD 163 conjugated with a third detectable moiety.

[00362] Clause 199. The composition of clause 198, wherein the first binding agent, second binding agent, and third binding agent, are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00363] Clause 200. The composition of any one of clauses 198-199, wherein the first detectable moiety, second detectable moiety, and third detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00364] Clause 201. A composition comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore.

[00365] Clause 202. A composition comprising: a mouse anti-human IgG monoclonal antibody for CD91 conjugated with a first fluorophore; a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a second fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a third fluorophore.

[00366] Clause 203. The composition of any one of clauses 201-202, further comprising at least one anticoagulant. [00367] Clause 204. The composition of any one of clauses 201-203, wherein the at least one anticoagulant comprises ethylene diamine tetraacetic acid, at least one salt thereof, citric acid, at least one salt thereof, or a combination of any of the foregoing.

[00368] Clause 205. The composition of any one of clauses 201-204, further comprising bovine serum albumin and sodium azide.

[00369] Clause 206. The composition of any one of clauses 201-205, further comprising phosphate buffered saline, gelatin, and sodium azide.

[00370] Clause 207. The composition of any one of clauses 201-206, wherein the first fluorophore is fluorescein isothiocyanate.

[00371] Clause 208. The composition of any one of clauses 201-207, wherein the composition does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00372] Clause 209. The composition of any one of clauses 201-208, wherein the second fluorophore is R-phycoerythrin.

[00373] Clause 210. The composition of any one of clauses 201-209, wherein the third fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00374] Clause 211. The composition of any one of clauses 201-210, further comprising whole blood from a human patient.

[00375] Clause 212. A kit comprising: a first binding agent for CD91 conjugated with a first detectable moiety; a second binding agent for CD59 conjugated with a second detectable moiety; and a third binding agent for CD 163 conjugated with a third detectable moiety.

[00376] Clause 213. The kit of clause 212, wherein the first binding agent, second binding agent, and third binding agent are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00377] Clause 214. The kit of any one of clauses 212-213, wherein the first detectable moiety, second detectable moiety, and third detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00378] Clause 215. A kit comprising: a monoclonal antibody for CD91 conjugated with a first fluorophore; a monoclonal antibody for CD59 conjugated with a second fluorophore; and a monoclonal antibody for CD 163 conjugated with a third fluorophore.

[00379] Clause 216. A kit comprising: a mouse anti-human IgG monoclonal antibodv for CD91 conjugated with a first fluorophore; a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a second fluorophore; and a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a third fluorophore.

[00380] Clause 217. The kit of any one of clauses 215-216, wherein the first fluorophore is fluorescein isothiocyanate.

[00381] Clause 218. The kit of any one of clauses 215-217, wherein the kit does not contain fluorescent dye sold under the trademark Alexa Fluor® 488 (Molecular Probes, Inc., Eugene OR).

[00382] Clause 219. The kit of any one of clauses 215-218, wherein the second fluorophore is R-phycoerythrin.

[00383] Clause 220. The kit of any one of clauses 215-219, wherein the third fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00384] Clause 221. A use of a binding agent for CD91, a binding agent for CD59, and a binding agent for CD 163 for the diagnosis of AD in a human patient in need thereof.

[00385] Clause 222. The use of clause 221, wherein the binding agents are chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof.

[00386] Clause 223. The use of any one of clauses 221-222, wherein the binding agent for CD91 is conjugated with a first detectable moiety, the binding agent for CD59 is conjugated with a second detectable moiety, and the binding agent for CD 163 is conjugated with a third detectable moiety.

[00387] Clause 224. The use of clause 223, wherein the first detectable moiety, the second detectable moiety, and the third detectable moiety, are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00388] Clause 225. A composition comprising: a first binding agent for CD91 conjugated with a first detectable moiety; a second binding agent for CD59 conjugated with a second detectable moiety; and a third binding agent for CD 163 conjugated with a third detectable moiety.

[00389] Clause 226. The composition of clause 225, wherein the first binding agent, second binding agent, and third binding agent are independently chosen from single chain variable fragments, antibody mimetics, antibody fragments, antibodies, monoclonal antibodies, and combinations thereof. [00390] Clause 227. The composition of any one of clauses 225-226, wherein the first detectable moiety, second detectable moiety, and third detectable moiety are independently chosen from radioisotopes, stable isotopes, fluorophores, and combinations thereof.

[00391] Clause 228. The composition of clause 225, wherein the first binding agent for CD91 is a monoclonal antibody for CD91, and the first detectable moiety is a first fluorophore; the second binding agent for CD59 is a monoclonal antibody for CD59, and the second detectable moiety is a second fluorophore; and the third binding agent for CD 163 is a monoclonal antibody for CD 163, and the third detectable moiety is a third fluorophore.

[00392] Clause 229. The composition of clause 228, wherein the monoclonal antibody for CD91 is a mouse anti-human IgG monoclonal antibody for CD91; the monoclonal antibody for CD59 is a mouse anti-human IgG monoclonal antibody for CD59; and the monoclonal antibody for CD 163 is a mouse anti-human IgG monoclonal antibody for CD163.

[00393] Clause 230. The composition of any one of clauses 228-229, wherein the first fluorophore is fluorescein isothiocyanate.

[00394] Clause 231. The composition of any one of clauses 228-230, wherein the second fluorophore is R-phycoerythrin.

[00395] Clause 232. The composition of any one of clauses 228-231, wherein the third fluorophore is fluorescent dye sold under the trademark Alexa Fluor® 647 (Molecular Probes, Inc., Eugene OR).

[00396] Clause 233. The composition of any one of clauses 225-232, further comprising whole blood from a human patient.

[00397] Clause 234. The composition of any one of clauses 225-233 further comprising at least one anticoagulant.

[00398] Clause 235. A method for diagnosing AD in a human patient, comprising: obtaining a blood sample from the human patient; optionally storing the blood sample in ice; optionally testing the blood sample within three hours of obtaining the blood sample; reacting the blood sample with a mouse anti-human IgG monoclonal antibody for CD91 conjugated with a first fluorophore to obtain bound CD91; a mouse anti-human IgG monoclonal antibody for CD59 conjugated with a second fluorophore to obtain bound CD59; a mouse anti-human IgG monoclonal antibody for CD 163 conjugated with a third fluorophore to obtain bound CD 163; measuring a concentration of bound CD91, a concentration of bound CD59, and a concentration of bound CD 163 in the blood sample by fluorescence of the first fluorophore, the second fluorophore, and the third fluorophore; observing an increase in the concentration of bound CD91 relative to a concentration range of CD91 in healthy humans; observing an increase in the concentration of bound CD59 relative to a concentration range of CD59 in healthy humans; observing a decrease in the concentration of bound CD 163 relative to a concentration range of CD 163 in healthy humans; thereby diagnosing AD in the human patient.

[00399] As previously stated, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. It will be appreciated that many modifications and other variations stand within the intended scope of this invention as claimed below. Furthermore, the foregoing description of various embodiments does not necessarily imply exclusion. For example, “some” embodiments may include all or part of “other” and “further” embodiments within the scope of this invention. In addition, “a” does not mean “one and only one;” “a” can mean “one and more than one.”