Multiplexed functional complement assays

Abstract

The present disclosure relates to methods of analyzing the complement system.

Description

[0001] APL0074 Provisional (2008575-0816)

[0002] MULTIPLEXED FUNCTIONAL COMPLEMENT ASSAYS

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 730,385, filed December 10, 2024, which is hereby incorporated by reference herein in its entirety.

[0005] FIELD OF THE DISCLOSURE

[0006] The present disclosure relates to methods of analyzing the complement system.

[0007] BACKGROUND OF THE DISCLOSURE

[0008] The complement system is a highly complex and dynamic biological cascade that mediates numerous effector functions, including significant roles in both innate and adaptive immunity. The proteins of the complement system act in a series of enzymatic cascades through a variety of protein interactions and cleavage events. Complement activation occurs via three main pathways: the antibodydependent classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway. (See Figure 1).

[0009] Traditional thinking about the complement system would imply that C5 activation can be used as a proxy for C3 activation, via either the classical or alternative pathways. Data detailed herein, however, demonstrates that this is over simplistic. The link between C3 activation and C5 activation, and the associated effector functions, is not necessarily straightforward, and both need to be evaluated, simultaneously, for a more comprehensive functional understanding of the complement system in a particular sample.

[0010] Current complement assays provide simplistic snapshots of various aspects of the system, but fail to provide an assessment of more than one effector function across more than one activation pathway. There is a need in the art for a more comprehensive functional complement assay.

[0011] SUMMARY OF THE DISCLOSURE

[0012] One aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Adding a biological sample to at least one well that has been prepared to activate the classical complement pathway; (b) Incubating the biological sample in the at least one well under conditions suitable for activating both the classical and alternative complement

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[0014] 13147300V1 APL0074 Provisional (2008575-0816) pathways; (c) Washing the at least one well to remove soluble protein; (d) Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; and (e) Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of both classical and alternative complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of both classical and alternative complement pathway mediated membrane attack complex (MAC). In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article.

[0015] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Adding a biological sample to at least one well that has been coated with IgM, wherein the biological sample comprises serum or CSF; (b) Incubating the biological sample in the at least one well under conditions suitable for activating both the classical and alternative complement pathways; (c) Washing the at least one well to remove soluble protein; (d) Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; and (e) Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of both classical and alternative complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of both classical and alternative complement pathway mediated membrane attack complex (MAC). In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article. In some embodiments of this aspect, the fluorophore is FITC. In some embodiments of this aspect, the chemiluminescent agent is HRP.

[0016] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Adding a biological sample to at least one well that has been prepared to activate the classical complement pathway, wherein the biological sample comprises an inhibitor of the alternative complement pathway; (b) Incubating the biological sample in the at least one well under conditions suitable for activating the classical pathway; (c) Washing the at least one well

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[0018] 13147300V1 APL0074 Provisional (2008575-0816) to remove soluble protein; (d) Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; and (e) Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of the classical complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of the classical complement pathway mediated membrane attack complex (MAC). In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article.

[0019] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Adding a biological sample to at least one well that has been coated with IgM, wherein the biological sample comprises serum or CSF, and wherein the biological sample comprises an inhibitor of the alternative complement pathway; (b) Incubating the biological sample in the at least one well under conditions suitable for activating the classical complement pathway; (c) Washing the at least one well to remove soluble protein; (d) Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; and (e) Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of classical complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC). In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article. In some embodiments of this aspect, the fluorophore is FITC. In some embodiments of this aspect, the chemiluminescent agent is HRP.

[0020] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Adding a biological sample to at least one well that has been prepared to activate the alternative complement pathway, wherein the biological sample comprises an inhibitor of the classical complement pathway; (b) Incubating the biological sample in the at least one well under conditions suitable for activating the alternative complement pathway; (c) Washing the at least one well to remove soluble protein; (d) Adding at least two detecting agents to the

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[0022] 13147300V1 APL0074 Provisional (2008575-0816) at least one well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; and (e) Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of alternative complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of alternative complement pathway mediated membrane attack complex (MAC). In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article.

[0023] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Adding a biological sample to at least one well that has been coated with zymosan, wherein the biological sample comprises serum or CSF, and wherein the biological sample comprises an inhibitor of the classical complement pathway; (b) Incubating the biological sample in the at least one well under conditions suitable for activating the alternative complement pathway; (c) Washing the at least one well to remove soluble protein; (d) Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; and (e) Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of alternative complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of alternative complement pathway mediated membrane attack complex (MAC). In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article. In some embodiments of this aspect, the fluorophore is FITC. In some embodiments of this aspect, the chemiluminescent agent is HRP.

[0024] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Preparing a first well and a second well to activate the classical complement pathway; (b) Pre-incubating a portion of a biological sample with an alternative pathway inhibitor, adding the portion of the biological sample with the inhibitor to the first well, and adding an equivalent portion without an alternative pathway inhibitor to the second well; (c) Incubating the biological sample in the first and second well under conditions suitable for activating both the classical and alternative complement pathways; (d) Washing the first and second well to remove soluble

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[0026] 13147300V1 APL0074 Provisional (2008575-0816) protein; (e) Adding at least two detecting agents to both the first and second well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; and (f) Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the first well and in the second well, wherein the signal from the C3b detecting agent is representative of classical complement pathway opsonization in the first well, and is representative of both classical and alternative complement pathway mediated opsonization in the second well, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC) in the first well, and is representative of both classical and alternative complement pathway mediated MAC in the second well. In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article.

[0027] Another aspect of the present disclosure encompasses a method for a multiplexed functional complement assay. The method comprises: (a) Preparing a first well and a second well to activate the classical complement pathway by coating the well with IgM; (b) Pre-incubating a portion of a biological sample comprising serum or CSF with an alternative pathway inhibitor, adding the portion of the biological sample with the inhibitor to the first well, and adding an equivalent portion of the biological sample without an alternative pathway inhibitor to the second well; (c) Incubating the biological sample in the first and second well under conditions suitable for activating both the classical and alternative complement pathways; (d) Washing the first and second well to remove soluble protein; (e) Adding at least two detecting agents to both the first well and the second well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; and (f) Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the first well and in the second well, wherein the signal from the C3b detecting agent is representative of classical complement pathway opsonization in the first well, and is representative of both classical and alternative complement pathway mediated opsonization in the second well, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC) in the first well, and is representative of both classical and alternative complement pathway mediated MAC in the second well. In some embodiments of this aspect, the biological sample is serum. In some embodiments of this aspect, the biological sample is CSF. In some embodiments of this aspect, the biological sample comprises a test article. In some embodiments of this aspect, the fluorophore is FITC. In some embodiments of this aspect, the chemiluminescent agent is HRP.

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[0029] 13147300V1 APL0074 Provisional (2008575-0816)

[0030] Other aspects and iterations of the disclosure are detailed below.

[0031] BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1 illustrates, in a simplified fashion, the activation cascades and effector functions of the complement system.

[0033] Figure 2 depicts graphs of results from the dual classical and alternative pathway assay. Figure 2A shows results using either a Cis inhibitor or a C3 inhibitor, in the presence or absence of an alternative pathway inhibitor. Figure 2B depicts the data from panel A where C3b and C5b-9 deposition without an alternative pathway inhibitor are shown on the same graph for both a Cis inhibitor and a C3 inhibitor.

[0034] Figure 3 depicts graphs of results from the alternative pathway assay. Figure 3A shows the results when APL-1, a C3 inhibitor, is used, and Figure 3B shows the results when a clinical stage alternative pathway inhibitor is used. The shift in the C3b curve and C5b-9 curve highlighted by the arrow in Figure 3B suggests that this clinical stage alternative pathway inhibitor has lower potency when inhibiting opsonization (C3b deposition) compared to lysis (C5b-9). This assay measures simultaneously deposition of C3b and C9 as a result of alternative pathway activation.

[0035] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0036] The methods and assays described herein allow for detection and measurement of more than one functional complement endpoint in a single biological sample. Furthermore, methods and assays described herein allow for dual activation of both the classical and alternative complement pathways simultaneously.

[0037] I. Definitions

[0038] As used herein, the phrase "alternative complement pathway" refers to one of three extracellular complement activation pathways. The alternative pathway is initiated by and amplified at, e.g., microbial surfaces and various complex polysaccharides. In this pathway, hydrolysis of C3 to C3(H2O), which occurs spontaneously at a low level, leads to binding of factor B, which is cleaved by factor D, generating a fluid phase C3 convertase that activates complement by cleaving C3 into C3a and C3b. C3b binds to targets such as cell surfaces and forms a complex with factor B, which is later cleaved

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[0040] 13147300V1 APL0074 Provisional (2008575-0816) by factor D, resulting in a C3 convertase. Surface-bound C3 convertases cleave and activate additional C3 molecules, resulting in rapid C3b deposition in close proximity to the site of activation and leading to formation of additional C3 convertase, which in turn generates additional C3b. This process results in a cycle of C3 cleavage and C3 convertase formation that significantly amplifies the response. Cleavage of C3 and binding of another molecule of C3b to the C3 convertase gives rise to a C5 convertase. The C5 convertase cleaves C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzes polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The MAC inserts itself into target cell membranes and causes cell lysis. Small amounts of MAC on the membrane of cells may have a variety of consequences other than cell death.

[0041] As used herein, the term "antibody" refers to an immunoglobulin or a derivative thereof containing an an immunoglobulin domain capable of binding to an antigen. The antibody can be of any species, e.g., human, rodent, rabbit, goat, chicken, etc. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE, or subclasses thereof such as IgG 1, lgG2, etc. In various embodiments of the invention the antibody is a fragment such as an Fab', F(ab')2, scFv (single-chain variable) or other fragment that retains an antigen binding site, or a recombinantly produced scFv fragment, including recombinantly produced fragments. See, e.g., Allen, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references therein. The antibody can be monovalent, bivalent or multivalent. The antibody may be a chimeric or "humanized" antibody in which, for example, a variable domain of rodent origin is fused to a constant domain of human origin, thus retaining the specificity of the rodent antibody. The domain of human origin need not originate directly from a human in the sense that it is first synthesized in a human being. Instead, "human" domains may be generated in rodents whose genome incorporates human immunoglobulin genes. See, e.g., Vaughan, et al., (1998), Nature Biotechnology, 16: 535-539. The antibody may be partially or completely humanized. An antibody may be polyclonal or monoclonal, though for purposes of the present invention monoclonal antibodies are generally preferred. Methods for producing antibodies that specifically bind to virtually any molecule of interest are known in the art. For example, monoclonal or polyclonal antibodies can be purified from blood or ascites fluid of an animal that produces the antibody (e.g., following natural exposure to or immunization with the molecule or an antigenic fragment thereof), can be produced using recombinant techniques in cell culture or transgenic organisms, or can be made at least in part by chemical synthesis.

[0042] As used herein, the terms "approximately" or "about" in reference to a number generally include numbers that fall within +10%, in some embodiments ±5%, in some embodiments +1%, in some

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[0044] 13147300V1 APL0074 Provisional (2008575-0816) embodiments ±0.5% of the number unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value).

[0045] As used herein, the phrase "chemiluminescent agent" refers to a compound or enzyme involved in a chemiluminescent reaction. In some embodiments, a chemiluminescent agent is a catalyst in a chemiluminescent reaction.

[0046] As used herein, the phrase "classical complement pathway" refers to one of three extracellular complement activation pathways. The classical pathway is usually triggered by binding of a complex of antigen and IgM or IgG antibody to Cl (though certain other activators can also initiate the pathway). Activated Cl cleaves C4 and C2 to produce C4a and C4b, in addition to C2a and C2b. C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3a and C3b. Binding of C3b to C3 convertase produces C5 convertase, which cleaves C5 into C5a and C5b. The C5 convertases produced in both pathways cleave C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzes polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The MAC inserts itself into target cell membranes and causes cell lysis. Small amounts of MAC on the membrane of cells may have a variety of consequences other than cell death.

[0047] As used herein, the phrase "complement system" refers to an arm of the innate immune system that plays multiple roles, including an important role in defending the body against infectious agents. The complement system comprises more than 30 serum and cellular proteins that are involved in three major extracellular activation pathways, known as the classical, alternative, and lectin pathways. Further details are found, e.g., in Kuby Immunology, 6th ed., 2006; Paul, W. E., Fundamental Immunology, Lippincott Williams & Wilkins; 6th ed., 2008; and Walport M J., Complement. First of two parts. N Engl J Med., 344(14):1058-66, 2001.

[0048] As used herein, the phrase "functional complement" refers to the ability of the complement system in a sample to activate and perform particular effector functions, such as opsonization and MAC formation. This is in contrast to a sample where complement components may be present, but they lack the ability to activate properly (e.g. the sample may contain a chelator that inhibits the complement cascade).

[0049] As used herein, the phrase "lectin complement pathway " refers to one of three extracellular complement activation pathways. The lectin pathway is initiated by binding of mannosebinding lectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein localized in the intermediate region between the endoplasmic reticulum and the Golgi. The MBL-2 gene encodes the soluble

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[0051] 13147300V1 APL0074 Provisional (2008575-0816) mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leading to a C3 convertase described above.

[0052] As used herein, the term "opsonization" refers to the deposition of C3b on a surface. In some embodiments, opsonization may be mediated by the alternative complement pathway. In some embodiments, opsonization may be mediated by the classical complement pathway.

[0053] As used herein, the phrase "room temperature" refers to a temperature of about 20C to about 28C. In some embodiments, room temperature may refer to a temperature of about 20, 21, 22, 23, 24, 25, 26, 1 or 28C. In some embodiments, room temperature may refer to a temperature between about 20-22C, 22-24C, 24-26C, or 26-28C. In some embodiments, room temperature may refer to a temperature between about 23C to about 28C.

[0054] As used herein, the term "well" refers to any concavity suitable for biological assays, such as those found on microplates or microtiter plates. Wells may be arranged on plates, and typical plates may include 24 wells, 96 wells, 394 wells, or 1536 wells. Wells are typically made from a plastic, such as polystyrene. Wells may have flat or rounded bottoms. Wells may be transparent, or pigmented, such as white or black.

[0055] II. Dual Complement Pathway Multiplexed Functional Complement Assay

[0056] One aspect of the present disclosure is a method for a dual complement pathway multiplexed functional complement assay. Generally speaking, a dual complement pathway assay disclosed herein comprises preparing at least one well to activate the complement system, adding a biological sample to the at least one well, incubating the biological sample in the at least one well under conditions suitable for activating complement, washing the at least one well to remove soluble protein, adding detection agents to the at least one well, and detecting and measuring signal from the detecting agent. Each of these steps is discussed in more detail herein.

[0057] (a) preparing at least one well to activate the complement system

[0058] A dual complement pathway assay detailed herein comprises a step of preparing at least one well to activate the complement system. In an embodiment of a dual complement pathway assay, at least one well is prepared in a manner to activate the classical complement pathway. For instance, in some embodiments, at least one well is coated with immunoglobulin. In some embodiments, at least one well is coated with IgG. In some embodiments, at least one well is coated with IgGl or lgG3. In some

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[0060] 13147300V1 APL0074 Provisional (2008575-0816) embodiments, at least one well is coated with IgM. Such coating may be performed by incubating at least one well with a solution of an immunoglobulin under conditions designed to adhere the immunoglubulin to the well. One of skill in the art would understand that the incubation length will be impacted by the temperature the incubation is performed at, and the concentration of immunoglobulin in the solution. In some embodiments, at least one well may be incubated with an immunoglobulin solution at room temperature for a duration sufficient to coat the well with immunoglobulin at a density sufficient to activate the classical complement cascade. In some embodiments, at least one well may be incubated with an immunoglobulin solution at room temperature for at least 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 minutes. In some embodiments, at least one well may be incubated with an immunoglobulin solution at room temperature for at least about 50 to about 70 minutes. In particular embodiments, at least one well may be incubated with an immunoglobulin solution as detailed in Example 1.

[0061] (b) adding a biological sample to the at least one well

[0062] A dual complement pathway assay detailed herein comprises a step of adding a biological sample to the at least one well. Such a biological sample may comprise complement proteins. Nonlimiting examples of biological samples may include serum, cerebral spinal fluid (CSF), aqueous humor, vitreous humor, whole blood, or plasma. Such samples may be collected from a human, a non-human primate, or a rodent. In some embodiments, a suitable biological sample may be collected from a human. In some embodiments a suitable biological sample may be collected from a male human. In some embodiments a suitable biological sample may be collected from a female human. In some embodiments a suitable biological sample may be collected from adult, e.g., a human at least 18 years of age, e.g., between 18 and 100 years of age. In some embodiments, a suitable biological sample may be collected from a human at least 12 years of age. In some embodiments a suitable biological sample may be collected from a human at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age. In some embodiments a suitable biological sample may be collected from a child, e.g., a human between 0 and 4 years of age, or between 5 and 11 years of age. In some embodiments, a suitable biological sample may be collected from a non-human primate. In each of the above examples, one of skill in the art would understand how to collect a biological sample in a manner designed to minimize complement activation during collection and storage of the sample.

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[0064] 13147300V1 APL0074 Provisional (2008575-0816)

[0065] In some embodiments, a suitable biological sample is a serum sample. For instance, a suitable biological sample may comprise about 5, 6, 7, 8, 9, 10, or more than 10% serum. In some embodiments, a suitable biological sample may comprise at least 10% serum. In some embodiments, a suitable biological sample may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more than 95% serum.

[0066] In some embodiments, a suitable biological sample is a CSF sample. For instance, a suitable biological sample may comprise about 5, 6, 7, 8, 9, 10, or more than 10% CSF. In some embodiments, a suitable biological sample may comprise at least 10% CSF. In some embodiments, a suitable biological sample may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more than 95% CSF.

[0067] In some embodiments, a suitable biological sample may be cell culture media that comprises complement proteins. When a suitable biological sample is a cell culture media sample, the complement proteins may be of human, non-human primate, or rodent origin.

[0068] In some embodiments, a suitable biological sample may be contacted by one or more compounds. In these embodiments, a suitable biological sample may be incubated with one or more compounds before the biological sample is added to at least one well, e.g. "pre-incubated". In some embodiments, a suitable biological sample is pre-incubated with a compound comprising a complement inhibitor. In some embodiments, the complement inhibitor may inhibit the alternative complement pathway. In some embodiments, the complement inhibitor may inhibit the classical complement pathway. In some embodiments, the complement inhibitor may inhibit the lectin complement pathway. In some embodiments, the complement inhibitor may inhibit all complement pathways. In some embodiments, inhibitors may be used as experimental controls. In some embodiments, inhibitors may be used to inhibit a particular complement pathway, allowing activation through other pathways in later steps of a dual complement pathway assay detailed herein. For instance, in a dual complement pathway assay detailed herein, a suitable biological sample may be incubated with an alternative complement pathway inhibitor. In some embodiments, the alternative complement pathway inhibitor is iptacopan.

[0069] In some embodiments, a suitable biological sample may be contacted by one or more test articles. In these embodiments, a suitable biological sample may be pre-incubated with one or more test articles before the sample is added to at least one well. Non-limiting examples of test articles include small molecule compounds, pre-clinical compounds, clinical compounds, biologic compounds including antibodies, etc.

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[0071] 13147300V1 APL0074 Provisional (2008575-0816)

[0072] (c) incubating the biological sample in the at least one well under conditions suitable for activating complement

[0073] A dual complement pathway assay detailed herein comprises a step of incubating a biological sample in at least one well under conditions suitable for activating complement. Generally speaking, conditions for activating complement may include incubation at about room temperature to about 40C, for a length of time sufficient to activate the complement cascade. In some embodiments, a biological sample in at least one well is incubated at about 35C to about 40C for about 10 to about 20 min, for instance, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In some embodiments, a biological sample in at least one well is incubated at about 35C to about 40C for about 10 to about 15, about 13 to about 17, or about 15 to about 20 minutes.

[0074] In some embodiments, an incubation to activate complement may take place in the presence of one or more inhibitors, as detailed in section (b) above.

[0075] In some embodiments, the complement activation is stopped by the addition of a chelator to the at least one well. In some embodiments, EDTA is used to stop the complement activation.

[0076] (d) washing the at least one well to remove soluble protein

[0077] A dual complement pathway assay detailed herein comprises a step of washing the at least one well after complement activation to remove soluble protein from the well, leaving complement fragments that have been deposited on the well surface. In some embodiments, such washing is done with a buffer, such as PBS. In some embodiments, such washing may be done with buffers comprising a surfactant, such as Tween.

[0078] (e) adding detection agents to the at least one well

[0079] A dual complement pathway assay detailed herein comprises a step of adding detection agents to the at least one well. In typical embodiments, at least two types of detection agents are added to the at least one well. For instance, one detection agent may recognize opsonization, while another detection agent may recognize MAC formation. In some embodiments, more than two detection agents may be added to the at least one well. In some embodiments, one detection agent may recognize C3b, while another detection agent may recognize the MAC complex (e.g., may recognize C6, C7, C8, C9, or a combination thereof). This allows for multiplex analysis of both the functional endpoint of opsonization, and the functional endpoint of MAC complex formation. In some embodiments, the detection agent is

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[0081] 13147300V1 APL0074 Provisional (2008575-0816) an antibody, e.g. a monoclonal antibody. In some embodiments, the detection agent that recognizes C3b is labelled with a fluorophore. In some embodiments, the detection agent that recognizes the MAC complex (e.g., may recognize C6, C7, C8, C9, or a combination thereof) is labelled with a chemiluminescent agent. Generally speaking, the emission spectrum for the C3b detection agent and the MAC complex detection agent should have as little overlap as possible so that the C3b signal does not bleed into the MAC complex signal. For instance, in some embodiments, the C3b detection agent is labelled with a fluorophore that has an emission spectrum similar to FITC, for example, Alexa Fluor 488, and the MAC complex detection agent is labelled with a chemiluminescent agent that has an emission spectrum similar to HRP. In some embodiments, the C3b detection agent is labelled with FITC, and the MAC complex detection agent is labelled with HRP.

[0082] In particular embodiments, at least two detecting agents are added to the at least one well, wherein at least one detecting agent is a fluorophore labelled monoclonal anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled monoclonal antibody specific for C6, C7, C8, C9, or a combination thereof. In further particular embodiments, at least two detecting agents are added to the at least one well, wherein at least one detecting agent is a FITC labelled monoclonal anti- C3b antibody, and at least one detecting agent is an HRP labelled monoclonal antibody specific for C6, C7, C8, C9, or a combination thereof. By way of non-limiting example only, a suitable FITC labelled anti- C3b antibody may be the 1H8 monoclonal antibody, and a suitable HRP labelled anti-C5b-C9 antibody may be the aEll monoclonal antibody.

[0083] (f) detecting and measuring signal from the detecting agent

[0084] A dual complement pathway assay detailed herein comprises a step of detecting and measuring the signal from each detecting agent used. In some embodiments, a detection agent comprises a fluorophore, and the fluorescence is detected and measured using equipment known in the art for detecting and measuring fluorescence. For instance, in some embodiments, a detection agent comprises FITC, and the fluorescence is detected and measured using equipment known in the art for detecting and measuring FITC fluorescence.

[0085] In some embodiments, a detection agent comprises a chemiluminescent agent, and the chemiluminescence is detected using equipment known in the art for detecting and measuring chemiluminescence. For instance, in some embodiments, a detection agent comprises HRP as a

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[0087] 13147300V1 APL0074 Provisional (2008575-0816) chemiluminescent agent, and the HRP chemiluminescent signal is detected using equipment known in the art for detecting and measuring HRP chemiluminescence.

[0088] In a dual complement pathway assay detailed herein, the signal detected and measured from the C3b detecting agent is representative of C3b opsonization, and the signal from the MAC complex detecting agent is representative of membrane attack complex (MAC) formation and deposition.

[0089] In some embodiments, a dual complement pathway assay may comprise (a) preparing a first well and a second well to activate the classical complement pathway; (b) pre-incubating a portion of a biological sample with an alternative pathway inhibitor, adding the portion of the biological sample with the inhibitor to the first well, and adding an equivalent portion without an alternative pathway inhibitor to the second well; (c) incubating the biological sample in the first and second well under conditions suitable for activating both the classical and alternative complement pathways; (d) washing the first and second well to remove soluble protein; (e) adding at least two detecting agents to both the first and second well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; and (f) detecting and measuring the signal from the C3b detecting agent and the C9 detecting agent in the first well and in the second well, wherein the signal from the C3b detecting agent is representative of classical complement pathway opsonization in the first well, and is representative of both classical and alternative complement pathway mediated opsonization in the second well, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC) in the first well, and is representative of both classical and alternative complement pathway mediated MAC in the second well.

[0090] In some embodiments, a dual complement pathway assay may comprise (a) preparing a first well and a second well to activate the classical complement pathway by coating the well with IgM; (b) pre-incubating a portion of a biological sample comprising serum or CSF with an alternative pathway inhibitor, adding the portion of the biological sample with the inhibitor to the first well, and adding an equivalent portion of the biological sample without an alternative pathway inhibitor to the second well; (c) incubating the biological sample in the first and second well under conditions suitable for activating both the classical and alternative complement pathways; (d) washing the first and second well to remove soluble protein; (e) adding at least two detecting agents to both the first well and the second well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; (f) detecting and measuring the signal from the C3b detecting agent and the C9

[0091] Page 14 of 29

[0092] 13147300V1 APL0074 Provisional (2008575-0816) detecting agent in the first well and in the second well, wherein the signal from the C3b detecting agent is representative of classical complement pathway opsonization in the first well, and is representative of both classical and alternative complement pathway mediated opsonization in the second well, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC) in the first well, and is representative of both classical and alternative complement pathway mediated MAC in the second well.

[0093] III. Alternative Complement Pathway Only Multiplexed Functional Complement Assay

[0094] One aspect of the present disclosure is a method for an alternative pathway only multiplexed functional complement assay. Generally speaking, an alternative pathway only assay disclosed herein comprises preparing at least one well to activate the complement system, adding a biological sample to the at least one well, incubating the biological sample in the at least one well under conditions suitable for activating complement, washing the at least one well to remove soluble protein, adding detection agents to the at least one well, and detecting and measuring signal from the detecting agent. Each of these steps is discussed in more detail herein.

[0095] (a) preparing at least one well to activate the complement system

[0096] An alternative pathway only assay detailed herein comprises a step of preparing at least one well to activate the complement system. In an embodiment of an alternative pathway only assay, at least one well is prepared in a manner to activate the alternative complement pathway. For instance, in some embodiments, at least one well is coated with zymosan. In some embodiments, at least one well is coated with bacterial components, such as LPS.

[0097] Such coating may be performed by incubating at least one well with a solution of an zymosan. One of skill in the art would understand that the incubation length will be impacted by the temperature the incubation is performed at, and the concentration of zymosan in the solution. In some embodiments, at least one well may be incubated with a zymosan solution at room temperature for a duration sufficient to coat the well with zymosan at a density sufficient to support the activation of the alternative complement cascade. In some embodiments, at least one well may be incubated with a zymosan solution at room temperature for at least 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 minutes. In some embodiments, at least one well may be incubated with a zymosan solution at room temperature

[0098] Page 15 of 29

[0099] 13147300V1 APL0074 Provisional (2008575-0816) for at least about 50 to about 70 minutes. In particular embodiments, at least one well may be incubated with a zymosan solution as detailed in Example 2.

[0100] (b) adding a biological sample to the at least one well

[0101] An alternative pathway only assay detailed herein comprises a step of adding a biological sample to the at least one well. Such a biological sample may comprise complement proteins. Nonlimiting examples of biological samples may include serum, cerebral spinal fluid (CSF), aqueous humor, vitreous humor, whole blood, or plasma. Such samples may be collected from a human, a non-human primate, or a rodent. In some embodiments, a suitable biological sample may be collected from a human. In some embodiments a suitable biological sample may be collected from a male human. In some embodiments a suitable biological sample may be collected from a female human. In some embodiments a suitable biological sample may be collected from adult, e.g., a human at least 18 years of age, e.g., between 18 and 100 years of age. In some embodiments, a suitable biological sample may be collected from a human at least 12 years of age. In some embodiments a suitable biological sample may be collected from a human at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age. In some embodiments a suitable biological sample may be collected from a child, e.g., a human between 0 and 4 years of age, or between 5 and 11 years of age. In some embodiments, a suitable biological sample may be collected from a non-human primate. In each of the above examples, one of skill in the art would understand how to collect a biological sample in a manner designed to minimize complement activation during collection and storage of the sample.

[0102] In some embodiments, a suitable biological sample is a serum sample. For instance, a suitable biological sample may comprise about 5, 6, 7, 8, 9, 10, or more than 10% serum. In some embodiments, a suitable biological sample may comprise at least 10% serum. In some embodiments, a suitable biological sample may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more than 95% serum.

[0103] In some embodiments, a suitable biological sample is a CSF sample. For instance, a suitable biological sample may comprise about 5, 6, 7, 8, 9, 10, or more than 10% CSF. In some embodiments, a suitable biological sample may comprise at least 10% CSF. In some embodiments, a suitable biological sample may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more than 95% CSF.

[0104] Page 16 of 29

[0105] 13147300V1 APL0074 Provisional (2008575-0816)

[0106] In some embodiments, a suitable biological sample may be cell culture media that comprises complement proteins. When a suitable biological sample is a cell culture media sample, the complement proteins may be of human, non-human primate, or rodent origin.

[0107] In some embodiments, a suitable biological sample may be contacted by one or more compounds. In these embodiments, a suitable biological sample may be incubated with one or more compounds before the biological sample is added to at least one well, e.g. "pre-incubated". In some embodiments, a suitable biological sample is pre-incubated with a compound comprising a complement inhibitor. In some embodiments, the complement inhibitor may inhibit the alternative complement pathway. In some embodiments, the complement inhibitor may inhibit the classical complement pathway. In some embodiments, the complement inhibitor may inhibit the lectin complement pathway. In some embodiments, the complement inhibitor may inhibit all complement pathways. In some embodiments, inhibitors may be used as experimental controls. In some embodiments, inhibitors may be used to inhibit a particular complement pathway. For instance, in an alternative pathway only assay detailed herein, a suitable biological sample may be incubated with a classical complement pathway inhibitor and / or a lectin pathway inhibitor, such as a chelator. Suitable chelators are known in the art, and may include EGTA. In some embodiments, a classical pathway complement inhibitor and / or a lectin pathway inhibitor may be GVB / MgEGTA.

[0108] In some embodiments, a suitable biological sample may be contacted by one or more test articles. In these embodiments, a suitable biological sample may be pre-incubated with one or more test articles before the biological sample is added to at least one well. Non-limiting examples of test articles include small molecule compounds, pre-clinical compounds, clinical compounds, biologic compounds including antibodies, etc.

[0109] (c) incubating the biological sample in the at least one well under conditions suitable for activating complement

[0110] An alternative pathway only assay detailed herein comprises a step of incubating a biological sample in at least one well under conditions suitable for activating complement. Generally speaking, conditions for activating complement may include incubation at about room temperature to about 40C, for a length of time sufficient to activate the complement cascade. In some embodiments, a biological sample in at least one well is incubated at about 35C to about 40C for about 10 to about 50 min, for instance, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

[0111] Page 17 of 29

[0112] 13147300V1 APL0074 Provisional (2008575-0816)

[0113] 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 minutes. In some embodiments, a biological sample in at least one well is incubated at about 35C to about 40C for about 20 to about 40 or about 25 to about 35 minutes.

[0114] In some embodiments, an incubation to activate complement may take place in the presence of one or more inhibitors, as detailed in section (b) above.

[0115] In some embodiments, the complement activation is stopped by the addition of a chelator to the at least one well. In some embodiments, EDTA is used to stop the complement activation.

[0116] (d) washing the at least one well to remove soluble protein

[0117] An alternative pathway only assay detailed herein comprises a step of washing the at least one well after complement activation to remove soluble protein from the well, leaving complement fragments that have been deposited on the well surface. In some embodiments, such washing is done with a buffer, such as PBS. In some embodiments, such washing may be done with buffers comprising a surfactant, such as Tween.

[0118] (e) adding detection agents to the at least one well

[0119] An alternative pathway only assay detailed herein comprises a step of adding detection agents to the at least one well. In typical embodiments, at least two types of detection agents are added to the at least one well. For instance, one detection agent may recognize opsonization, while another detection agent may recognize MAC formation. In some embodiments, more than two detection agents may be added to the at least one well. In some embodiments, one detection agent may recognize C3b, while another detection agent may recognize the MAC complex (e.g., may recognize C6, C7, C8, C9, or a combination thereof). This allows for multiplex analysis of both the functional endpoint of opsonization, and the functional endpoint of MAC complex formation. In some embodiments, the detection agent is an antibody, e.g. a monoclonal antibody. In some embodiments, the detection agent that recognizes C3b is labelled with a fluorophore. In some embodiments, the detection agent that recognizes the MAC complex (e.g., may recognize C6, C7, C8, C9, or a combination thereof) is labelled with a chemiluminescent agent. Generally speaking, the emission spectrum for the C3b detection agent and the MAC complex detection agent should have as little overlap as possible so that the C3b signal does not bleed into the MAC complex signal. For instance, in some embodiments, the C3b detection agent is labelled with a fluorophore that has an emission spectrum similar to FITC, for example, Alexa Fluor 488, and the MAC complex detection agent is labelled with a chemiluminescent agent that has an emission

[0120] Page 18 of 29

[0121] 13147300V1 APL0074 Provisional (2008575-0816) spectrum similar to HRP. For instance, in some embodiments, the C3b detection agent is labelled with FITC, and the MAC complex detection agent is labelled with HRP.

[0122] In particular embodiments, at least two detecting agents are added to the at least one well, wherein at least one detecting agent is a fluorophore labelled monoclonal anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled monoclonal antibody specific for C6, C7, C8, C9, or a combination thereof. In further particular embodiments, at least two detecting agents are added to the at least one well, wherein at least one detecting agent is a FITC labelled monoclonal anti- C3b antibody, and at least one detecting agent is an HRP labelled monoclonal antibody specific for C6, C7, C8, C9, or a combination thereof. By way of non-limiting example only, a suitable FITC labelled anti- C3b antibody may be the 1H8 monoclonal antibody, and a suitable HRP labelled anti-C5b-C9 antibody may be the aEll monoclonal antibody.

[0123] (f) detecting and measuring signal from the detecting agent

[0124] An alternative pathway only assay detailed herein comprises a step of detecting and measuring the signal from each detecting agent used. In some embodiments, a detection agent comprises a fluorophore, and the fluorescence is detected and measured using equipment known in the art for detecting and measuring fluorescence. For instance, in some embodiments, a detection agent comprises FITC, and the fluorescence is detected and measured using equipment known in the art for detecting and measuring FITC fluorescence.

[0125] In some embodiments, a detection agent comprises a chemiluminescent agent, and the chemiluminescence is detected using equipment known in the art for detecting and measuring chemiluminescence. For instance, in some embodiments, a detection agent comprises HRP as a chemiluminescent agent, and the HRP chemiluminescent signal is detected using equipment known in the art for detecting and measuring HRP chemiluminescence.

[0126] In an alternative pathway only assay detailed herein, the signal detected and measured from the C3b detecting agent is representative of C3b opsonization, and the signal from the MAC complex detecting agent is representative of membrane attack complex (MAC) formation and deposition.

[0127] IV. Non-clinical methods

[0128] One aspect of the present disclosure encompasses non-clinical use of the assays detailed herein. For instance, in some embodiments, an assay detailed herein may be used to analyze how a test article

[0129] Page 19 of 29

[0130] 13147300V1 APL0074 Provisional (2008575-0816) impacts both the classical pathway and the alternative pathway of complement activation, using only a single biological sample. Such analysis may be used in the development of compounds that inhibit, enhance, or otherwise impact the complement system. By way of non-limiting example, an assay detailed herein may be used to develop an antibody, aptamer, small molecule, or other type of complement inhibitor.

[0131] V. Clinical methods

[0132] One aspect of the present disclosure encompasses clinical use of the assays detailed herein. For instance, in some embodiments, an assay detailed herein may be used to help calculate a clinical dose of a compound that impacts the complement system, and unlike an assay that relies on hemolysis only, an assay detailed herein would provide analysis of how a compound impacts both C3b and C5b-9.

[0133] In some embodiments, an assay detailed herein may be used to predict a patient's response to a clinical compound or treatment. In some embodiments, such a clinical compound or treatment may be a complement inhibitor. In some embodiments, an assay detailed herein may be used to track a patient's response to administration of a compound or treatment, or to stratify patients into groups based on the patient's response to a compound or treatment. The above embodiments may also be combined with an analysis of disease specific biomarkers.

[0134] In some embodiments, an assay detailed herein may reveal an abnormality or dysfunction of a patient's complement system.

[0135] VI. Kits

[0136] One aspect of the present disclosure is a kit comprising one or more components of an assay detailed herein. For instance, in some embodiments, a kit may comprise one or more plates, where the wells of each plate have been coated with a complement activating material. In some embodiments, a kit may comprise one or more plates comprising wells, and the components for coating the wells with a complement activating material (e.g. immunoglobulin, zymosan, etc). In some embodiments, a kit may further comprise complement inhibitors, detection reagents, wash buffers, or any combination thereof.

[0137] Page 20 of 29

[0138] 13147300V1 APL0074 Provisional (2008575-0816)

[0139] EXAMPLES

[0140] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.

[0141] Example 1: Dual Assay

[0142] Unless otherwise noted, the steps detailed below were performed in a high-binding 384 well Optiplate™ (Perkin Elmer). Wells were coated with 25pL IgM in a carbonate / bicarbonate buffer (1:20) for 60 min at room temperature. Afterwards, wells were washed twice with PBS (50 pL). Wells were then blocked with (50pL) 2% BSA in PBS for between about 90 to about 120 minutes at room temperature. Wells were subsequently washed three times with PBS (50 p.L).

[0143] Biological samples (25pL) comprising diluted serum (10% serum) in GVB / Mg+EGTA+ buffer were then added to one or more wells and incubated for about 15 minutes at 37C to allow for complement activation. If desired, in a separate polypropylene plate, serum (1.2x; 25 pL) may be preincubated with one or more compounds (e.g. inhibitors) (6x; 5 |1L) for about 15 to about 20 minutes at room temperature, and then 25 |1L transferred to the coated / blocked well as detailed above. Complement activation is stopped with 50 nM EDTA (2.8 pL), and wells washed three times with PBS-0.05% Tween (50 pL).

[0144] Primary antibody (25 pL), diluted in blocking buffer (2% BSA in PBS), is added to the wells and allowed to incubate for about 60 minutes at room temperature. The anti-C3b antibody (clone 1H8) is FITC labelled, and the anti-C5b-9 antibody (clone aEll) is labelled with HRP. Wells are then washed twice with PBS-0.05% Tween (50 pL) and six times with PBS (50 pL). The FITC signal is detected and measured. Then 25 pL of HRP substrate is added to the well at room temperature, followed by the HRP- stop solution. The HRP signal is then detected and measured over time.

[0145] Figure 2A depicts graphs of results from the dual assay, using either a Cis inhibitor or a C3 inhibitor, and either in the presence or absence of an alternative pathway inhibitor. Under conditions where there is no alternative pathway inhibitor, the assay measures deposited C3b and MAC complex stemming from both the classical pathway and the alternative pathway amplification loop which is triggered by C3b generated from classical pathway activation. Under conditions where an alternative pathway inhibitor is present, the assay measures deposited C3b and C5b-9 (MAC) complex from only the classical pathway. Panel B depicts graphs of combined C3b and C5b-9 deposition. It is apparent from the

[0146] Page 21 of 29

[0147] 13147300V1 APL0074 Provisional (2008575-0816) figure that C3b deposition is more sensitive to C3 inhibitor compared to C5b-9 which is reflected in lower IC50 value. The data for Cis inhibitor is the opposite, with C5b-9 deposition being more sensitive to Cis inhibition compared to C3b deposition, thus underscoring mechanistic differences between the two inhibitors. The assay can measure simultaneously C3b and C5b-9 deposition as a result of classical and alternative pathway of complement activation.

[0148] Example 2: Alternative Pathway only assay

[0149] Unless otherwise noted, the steps detailed below were performed in a high-binding 384 well Optiplate™ (Perkin Elmer). Wells were coated with 25pL zymosan in carbonate / bicarbonate buffer (1:20; 500 pg / mL) for 60 min at room temperature. Afterwards, wells were washed three times with PBS- 0.05% Tween (50 pL). Wells were then blocked with (50pL) StartingBlock™ T20 (PBS) for between about 90 to about 120 minutes at room temperature. Wells were subsequently washed three times with PBS- 0.05% Tween (50 pL).

[0150] Biological samples (25pL) comprising serum (10%) diluted in GVB / MgEGTA were then added to one or more wells and incubated for about 30 minutes at 37C to allow for complement activation. If desired, in a separate polypropylene plate, serum (1.2x; 25 pL) may be preincubated with one or more compounds (e.g. inhibitors) (6x; 5 pL) for about 15 to about 20 minutes at room temperature, and then 25 pL transferred to the coated / blocked well as detailed above. Complement activation is stopped with 50 nM EDTA (2.8 pL), and wells washed three times with PBS-0.05% Tween (50 pL).

[0151] Primary antibody (25 pL), diluted in StartingBlock™ T20 (PBS) is added to the wells and allowed to incubate for about 60 minutes at room temperature. The anti-C3b antibody (1H8) is FITC labelled, and the anti-C5b-9 antibody (aEll) is labelled with HRP. Well are then washed twice with PBS-0.05% Tween (50 pL) and six times with PBS (50 pL). The FITC signal is detected and measured. Then 25 pL of HRP substrate is added to the well at room temperature, followed by the HRP-stop solution. The HRP signal is then detected and measured over time.

[0152] Figure 3 depicts graphs of results from the alternative pathway only assay. Figure 3A shows the results when APL-1, a C3 inhibitor, is used, and Figure 3B shows the results when a clinical stage alternative pathway inhibitor is used. The shift in the C3b curve and C9 curve highlighted by the arrow in Figure 3B suggests that this clinical stage alternative pathway inhibitor has lower potency when inhibiting opsonization (C3b signal) compared to lysis (MAC signal).

[0153] Page 22 of 29

[0154] 13147300V1 APL0074 Provisional (2008575-0816)

[0155] EQUIVALENTS

[0156] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

[0157] Page 23 of 29

[0158] 13147300V1

Claims

APL0074 Provisional (2008575-0816)CLAIMSWhat is claimed is:

1. A method for a multiplexed functional complement assay, the method comprising: a. Adding a biological sample to at least one well that has been prepared to activate the classical complement pathway; b. Incubating the biological sample in the at least one well under conditions suitable for activating both the classical and alternative complement pathways; c. Washing the at least one well to remove soluble protein; d. Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; e. Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of both classical and alternative complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of both classical and alternative complement pathway mediated membrane attack complex (MAC).

2. A method for a multiplexed functional complement assay, the method comprising: a. Adding a biological sample to at least one well that has been coated with IgM, wherein the biological sample comprises serum or CSF; b. Incubating the biological sample in the at least one well under conditions suitable for activating both the classical and alternative complement pathways; c. Washing the at least one well to remove soluble protein; d. Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; e. Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of both classical and alternative complement pathway mediated opsonization, and the signalPage 24 of 2913147300V1APL0074 Provisional (2008575-0816) from the C9 detecting agent is representative of both classical and alternative complement pathway mediated membrane attack complex (MAC).

3. A method for a multiplexed functional complement assay, the method comprising: a. Adding a biological sample to at least one well that has been prepared to activate the classical complement pathway, wherein the biological sample comprises an inhibitor of the alternative complement pathway; b. Incubating the biological sample in the at least one well under conditions suitable for activating the classical pathway; c. Washing the at least one well to remove soluble protein; d. Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; e. Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of the classical complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of the classical complement pathway mediated membrane attack complex (MAC).

4. A method for a multiplexed functional complement assay, the method comprising: a. Adding a biological sample to at least one well that has been coated with IgM, wherein the biological sample comprises serum or CSF, and wherein the biological sample comprises an inhibitor of the alternative complement pathway; b. Incubating the biological sample in the at least one well under conditions suitable for activating the classical complement pathway; c. Washing the at least one well to remove soluble protein; d. Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; e. Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of classical complement pathway mediated opsonization, and the signal from the C9Page 25 of 2913147300V1APL0074 Provisional (2008575-0816) detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC).

5. A method for a multiplexed functional complement assay, the method comprising: a. Adding a biological sample to at least one well that has been prepared to activate the alternative complement pathway, wherein the biological sample comprises an inhibitor of the classical complement pathway; b. Incubating the biological sample in the at least one well under conditions suitable for activating the alternative complement pathway; c. Washing the at least one well to remove soluble protein; d. Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; e. Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of alternative complement pathway mediated opsonization, and the signal from the C9 detecting agent is representative of alternative complement pathway mediated membrane attack complex (MAC).

6. A method for a multiplexed functional complement assay, the method comprising: a. Adding a biological sample to at least one well that has been coated with zymosan, wherein the biological sample comprises serum or CSF, and wherein the biological sample comprises an inhibitor of the classical complement pathway; b. Incubating the biological sample in the at least one well under conditions suitable for activating the alternative complement pathway; c. Washing the at least one well to remove soluble protein; d. Adding at least two detecting agents to the at least one well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; e. Measuring the signal from the C3b detecting agent and the C9 detecting agent in the at least one well, wherein the signal from the C3b detecting agent is representative of alternative complement pathway mediated opsonization, and the signal from the C9Page 26 of 2913147300V1APL0074 Provisional (2008575-0816) detecting agent is representative of alternative complement pathway mediated membrane attack complex (MAC).

7. A method for a multiplexed functional complement assay, the method comprising: a. Preparing a first well and a second well to activate the classical complement pathway; b. Pre-incubating a portion of a biological sample with an alternative pathway inhibitor, adding the portion of the biological sample with the inhibitor to the first well, and adding an equivalent portion without an alternative pathway inhibitor to the second well; c. Incubating the biological sample in the first and second well under conditions suitable for activating both the classical and alternative complement pathways; d. Washing the first and second well to remove soluble protein; e. Adding at least two detecting agents to both the first and second well, wherein at least one detecting agent is directed to C3b, and at least one detecting agent is directed to C6, C7, C8, C9, or a combination thereof; f. Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the first well and in the second well, wherein the signal from the C3b detecting agent is representative of classical complement pathway opsonization in the first well, and is representative of both classical and alternative complement pathway mediated opsonization in the second well, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC) in the first well, and is representative of both classical and alternative complement pathway mediated MAC in the second well.

8. A method for a multiplexed functional complement assay, the method comprising: a. Preparing a first well and a second well to activate the classical complement pathway by coating the well with IgM; b. Pre-incubating a portion of a biological sample comprising serum or CSF with an alternative pathway inhibitor, adding the portion of the biological sample with the inhibitor to the first well, and adding an equivalent portion of the biological sample without an alternative pathway inhibitor to the second well; c. Incubating the biological sample in the first and second well under conditions suitable for activating both the classical and alternative complement pathways; d. Washing the first and second well to remove soluble protein;Page 27 of 2913147300V1APL0074 Provisional (2008575-0816) e. Adding at least two detecting agents to both the first well and the second well, wherein at least one detecting agent is a fluorophore labelled anti-C3b antibody, and at least one detecting agent is a chemiluminescent agent labelled antibody specific for C6, C7, C8, C9, or a combination thereof; f. Detecting and Measuring the signal from the C3b detecting agent and the C9 detecting agent in the first well and in the second well, wherein the signal from the C3b detecting agent is representative of classical complement pathway opsonization in the first well, and is representative of both classical and alternative complement pathway mediated opsonization in the second well, and the signal from the C9 detecting agent is representative of classical complement pathway mediated membrane attack complex (MAC) in the first well, and is representative of both classical and alternative complement pathway mediated MAC in the second well.

9. The method of any of claims 1-8, wherein the biological sample is serum.

10. The method of any of claims 1-8, wherein the biological sample is CSF.

11. The method of claim 6 or claim 8, wherein the alternative pathway inhibitor is iptacopan.

12. The method of any of claims 1-8, wherein the biological sample comprises a test article.

13. The method of any of claims 1-6, wherein the biological sample comprises a complement inhibitor.

14. The method of any of claims 1-6, wherein the biological sample comprises both a complement inhibitor and a test article.

15. The method of any of claims 2, 4, 6, or 8, wherein the fluorophore is FITC.

16. The method of any of claims 2, 4, 6, or 8, wherein the chemiluminescent agent is HRP.

17. The method of any of claims 2, 4, 6, or 8, wherein the fluorophore is FITC and the chemiluminescent agent is HRP.

18. The method of any of claims 1-8, wherein the biological sample is a human sample.

19. The method of any of claims 1-8, wherein the biological sample is a non-human primate sample.Page 28 of 2913147300V1

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